Corrigendum to Commission Directive 2004/73/EC of 29 April 2004 adapting to technical progress for the 29th 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 (Official Journal of the European Union L 152 of 30 April 2004)
Date: 2004-06-16
Institutions: European Commission
Subjects: approximation of laws, classification, dangerous substance, labelling, packaging

16.6.2004

EN

Official Journal of the European Union

L 216/3

CORRIGENDA

Corrigendum to Commission Directive 2004/73/EC of 29 April 2004 adapting to technical progress for the 29th
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

(‘Official Journal of the European Union’ L 152 of 30 April 2004)

Directive 2004/73/EC should read as follows:

‘COMMISSION DIRECTIVE 2004/73/EC

of 29 April 2004

adapting to technical progress
the 29th 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
Community,

establishing the European

the

approximation

Having regard to Council Directive 67/548/EEC of 27 June 1967
on
and
administrative provisions relating to the classification, packaging
and labelling of dangerous substances (1), and in particular
Article 28 thereof,

regulations

laws,

the

of

Whereas:

(1)

(2)

and further

existing substances

Annex I
to Directive 67/548/EEC contains a list of
dangerous substances, together with particulars of the
classification and labelling of each substance. That list
needs to be updated to include further notified new
substances
as well
adapting the existing entries to technical progress such as
setting environmental concentration limits for certain
substances. Accordingly it
is also necessary to delete
entries for certain substances and to split some entries
because the classification no longer applies to all the
substances under
labelling of
substances containing 1,3-butadiene should be changed
in order to reflect that that substance will be classified as
a mutagen by the present Directive.

entries. The

those

Annex V to Directive 67/548/EEC lays down the
methods for the determination of the physicochemical
properties,
toxicity and ecotoxicity of substances and
preparations. It is appropriate to amend that Annex in
order to obtain a reduction to a minimum of the number
of animals used for experimental purposes, in accordance
with Council Directive 86/609/EEC of 24 November
1986 on the approximation of
laws, regulations and
administrative provisions of the Member States regarding

the protection of animals used for experimental and
other scientific purposes (2). The methods for subchronic
oral toxicity in Chapters B.1, B.4, B.5, B.31 and B.35
should be revised accordingly. Furthermore, Chapter B.42
should be added to Annex V in order to make available a
refined method on subchronic oral
toxicity. Finally,
Chapter A.21 on physico-chemical properties, Chapter
B.43 on subchronic oral toxicity and Chapters C.21 to
C.24 on environmental toxicity should be added in order
to allow for the determination of properties which are
not yet sufficiently covered by the methods in Annex V.

(3)

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

HAS ADOPTED THIS DIRECTIVE:

Article 1

Directive 67/548/EEC is amended as follows:

1. Annex I is amended as follows:

(a) note K in the foreword is replaced by the text set out in

Annex 1A;

(1) OJ 196, 16.8.1967, p. 1. Directive as last amended by Regulation

(EC) No 807/2003 (OJ L 122, 16.5.2003, p. 36).

(2) OJ L 358, 18.12.1986, p. 1. Directive as last amended by Directive
2003/65/EC of the European Parliament and of the Council (OJ L
230, 16.9.2003, p. 32).

L 216/4

EN

Official Journal of the European Union

16.6.2004

(b) the entries corresponding to the entries set out

in
Annex 1B to this Directive are replaced by the text set
out in that Annex;

(c)

the entries set out in Annex 1C to this Directive are
inserted in accordance with the order of the entries set
out in Annex I to Directive 67/548/EEC;

(d) the entries with index numbers 604-050-00-X, 607-
050-00-8, 607-171-00-6 and 613-130-00-3 are deleted;

(e)

(f)

the entry with index number 048-002-00-0 is replaced
by the entries with index numbers 048-002-00-0 and
048-011-00-X set out in Annex 1D to this Directive;

the entry with index number 609-006-00-3 is replaced
by the entries with index numbers 609-006-00-3 and
609-065-00-5 set out in Annex 1D to this Directive;

(g) the entry with index number 612-039-00-6 is replaced
by the entries with index numbers 612-039-00-6 and
612-207-00-9 as set out in Annex 1D.

2. Annex V is amended as follows:

(a)

the text set out in Annex 2A to this Directive is added
as Chapter A.21;

(b) chapter B.1 bis is replaced by the text set out in Annex

2B to this Directive;

(c) chapter B.1 tris is replaced by the the text set out in

Annex 2C to this Directive;

(d) chapter B.4 is replaced by the text set out in Annex 2D

to this Directive;

(h) the text set out in Annex 2H to this Directive is added

as Chapter B.42 and B.43;

(i)

the text set out in Annex 2I to this Directive is added as
Chapter C.21 to C.24.

Article 2

1. Member States shall bring into force the laws, regulations
and administrative provisions necessary to comply with this
Directive by 31 October 2005 at the latest. They shall forthwith
communicate to the Commission the text of those provisions
and a correlation table between those provisions and this
Directive. When Member States adopt those provisions, 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.

Article 3

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

Article 4

(e) chapter B.5 is replaced by the text set out in Annex 2E

This Directive is addressed to the Member States.

to this Directive;

(f) chapter B.31 is replaced by the text set out in Annex 2F

to this Directive;

(g) chapter B.35 is replaced by the text set out in Annex 2G

to this Directive;

Done at Brussels, 29 April 2004.

For the Commission

Margot WALLSTRÖM

Member of the Commission

16.6.2004

EN

Official Journal of the European Union

L 216/5

‘Note K:

ANNEX 1A

The classification as a carcinogen or mutagen need not apply if it can be shown that the substance contains less than
0.1 % w/w 1,3-butadiene (Einecs No 203-450-8). If the substance is not classified as a carcinogen or mutagen, at least the
S-phrases (2-)9-16 should apply. This note applies to certain complex oil-derived substances in Annex I.’

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

ANNEX 1B

‘006-005-00-4

thiram
tetramethylthiuram disulphide

205-286-2

137-26-8

Xn; R20/22-48/22
Xi; R36/38
R43
N; R50-53

Xn; N
R: 20/22-36/38-43-48/
22-50/53
S: (2-)26-36/37-60-61

006-006-01-7

hydrogen cyanide …%
hydrocyanic acid …%

B

200-821-6

74-90-8

T+; R26/27/28
N; R50-53

T+; N
R: 26/27/28-50/53
S: (1/2-)7/9-16-36/37-
38-45-60-61

006-012-00-2

ziram (ISO)
zinc bis
dimethyldithiocarbamate

205-288-3

137-30-4

T+; R26
Xn; R22-48/22
Xi; R37-41
R43
N; R50-53

T+; N
R: 22-26-37-41-43-48/
22-50/53
S: (1/2-)22-26-28-36/37/
39-45-60-61

C ≥ 25 %: Xn, N; R20/22-36/38-43-48/
22-50/53
20 % ≤ C < 25 %: Xn, N; R36/38-43-48/
22-50/53
10 % ≤ C < 20 %: Xn, N; R43-48/22-50/
53
2,5 % ≤ C < 10 %: Xi, N; R43-50/53
1 % ≤ C < 2,5 %: Xi, N; R43-51/53
0,25 % ≤ C < 1 %: N; R51/53
0,025 % ≤ C < 0,25 %: R52/53

C ≥ 25 %: T+, N; R26/27/28-50-53
7 % ≤ C < 25 %: T+, N; R26/27/28-51-53
2,5 % ≤ C < 7 %: T, N; R23/24/25-51-53
1 % ≤ C < 2,5 %: T, N; R23/24/25-52-53
0,25 % ≤ C < 1 %: Xn; R20/21/22-52-53
0,1 % ≤ C < 0,25 %: Xn; R20/21/22

C ≥ 25 %: T+, N; R22-26-37-41-43-48/
22-50-53
20 % ≤ C < 25 %: T+, N; R26-37-41-43-
48/22-50-53
10 % ≤ C < 20 %: T+, N; R26-41-43-48/
22-50-53
7 % ≤ C < 10 %: T+, N; R26-36-43-50-53
5 % ≤ C < 7 %: T, N; R23-36-43-50-53
1 % ≤ C < 5 %: T, N; R23-43-50-53
0,25 % ≤ C < 1 %: Xn, N; R20-50-53
0,1 % ≤ C < 0,25 %: Xn, N; R20-51-53
0,025 % ≤ C < 0,1 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

006-021-00-1

linuron (ISO)
3-(3,4-dichlorophenyl)-1-
methoxy-1-methylurea

E

206-356-5

330-55-2

006-044-00-7

isoproturon
3-(4-isopropylphenyl)-1,1-
dimethylurea

251-835-4

34123-59-6

Repr. Cat. 2; R61
Repr. Cat. 3; R62
Carc. Cat. 3; R40
Xn; R22-48/22
N; R50-53

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

T; N
R: 61-22-40-48/22-62-
50/53
S: 53-45-60-61

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

C ≥ 2,5 %: Xn, N; R40-50-53
1 % ≤ C < 2,5 %: Xn, N; R40-51-53
0,25 % ≤ C < 1 %: N; R51-53
0,025 % ≤ C < 0,25 %: R52-53

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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

006-072-00-X

S-benzyl N,N-
dipropylthiocarbamate
prosulfocarb

401-730-6

52888-80-9

Xn; R22
R43
N; R51-53

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

006-089-00-2

chlorine dioxide

233-162-8

10049-04-4

006-089-01-X

chlorine dioxide … %

B

233-162-8

10049-04-4

007-001-00-5

ammonia, anhydrous

231-635-3

7664-41-7

007-008-00-3

hydrazine

E

206-114-9

302-01-2

007-010-00-4

sodium nitrite

231-555-9

7632-00-0

007-011-00-X

potassium nitrite

231-832-4

7758-09-0

007-013-00-0

1,2-dimethylhydrazine

E

—

540-73-8

O; R8
R6
T+; R26
C; R34
N; R50

T; R25
C; R34
N; R50

R10
T; R23
C; R34
N; R50

R10
Carc. Cat. 2; R45
T; R23/24/25
C; R34
R43
N; R50-53

O; T+; N
R: 6-8-26-34-50
S: (1/2-)23-26-28-36/37/
39-38-45-61

C ≥ 5 %: T+; N; R26-34-50
1 % ≤ C < 5 %: T+; N; R26-36/37/38-50
0,5 % ≤ C < 1 %: T; N; R23-36/37/38-50
0,2 % ≤ C < 0,5 %: T; N; R23-50
0,02 % ≤ C < 0,2 %: Xn; N; R20-50

T; N
R: 25-34-50
S: (1/2-)23-26-28-36/37/
39-45-61

C ≥ 25 %: T; N; R25-34-50
10 % ≤ C < 25 %: C, N; R22-34-50
3 % ≤ C < 10 %: Xn; N; R22-36/37/38-50
0,3 % ≤ C < 3 %: Xi; R36

T; N
R: 10-23-34-50
S: (1/2-)9-16-26-36/37/
39-45-61

T; N
R: 45-10-23/24/25-34-
43-50/53
S: 53-45-60-61

C ≥ 25 %: T, N; R23-34-50
5 % ≤ C < 25 %: T; R23-34
0,5 % ≤ C < 5 %: Xn; R20-36/37/38

C ≥ 25 %: T, N; R45-23/24/25-34-43-50/
53
10 % ≤ C < 25 %: T, N; R45-20/21/22-
34-43-51/53
3 % ≤ C < 10 %: T, N; R45-20/21/22-36/
38-43-51/53
2,5 % ≤ C < 3 %: T, N; R45-43-51/53
1 % ≤ C < 2,5 %: T; R45-43-52/53
0,25 % ≤ C < 1 %: T; R45-52/53
0,1 % ≤ C < 0,25 %: T; R45

O; R8
T; R25
N; R50

O; R8
T; R25
N; R50

O; T; N
R: 8-25-50
S: (1/2-)45-61

O; T; N
R: 8-25-50
S: (1/2-)45-61

C ≥ 25 %: T, N; R25-50
5 % ≤ C < 25 %: T; R25
1 % ≤ C < 5 %: Xn; R22

C ≥ 25 %: T, N; R25-50
5 % ≤ C < 25 %: T; R25
1 % ≤ C < 5 %: Xn; R22

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

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

C ≥ 25 %: T, N; R45-23/24/25-51/53
3 % ≤ C < 25 %: T; R45-20/21/22-52/53
2,5 % ≤ C < 3 %: T; R45-52/53
0,01 % ≤ C < 2,5 %: T; R45

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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

007-017-00-2

isobutyl nitrite

E

208-819-7

542-56-3

007-027-00-7

1,6-bis(3,3-bis((1-methylpentyli-
denimino)propyl)ureido)hexane

420-190-2

—

008-003-00-9

hydrogen peroxide solution … % B

231-765-0

7722-84-1

009-015-00-7

sulphuryl difluoride

220-281-5

2699-79-8

015-002-00-7

red phosphorus

231-768-7

7723-14-0

015-014-00-2

tributyl phosphate

204-800-2

126-73-8

F; R11
Xn; R20/22
Carc. Cat. 2; R45
Muta. Cat. 3; R68

F; T
R: 11-20/22-45-68
S: 53-45

Xn; R21/22-48/21
C; R34
R43
N; R50-53

C; N
R: 21/22-34-43-48/21-
50/53
S: (1/2-)7-26-36/37/39-
45-60-61

R5
O; R8
C; R35
Xn; R20/22

O; C
R: 5-8-20/22-35
S: (1/2-)17-26-28-36/37/
39-45

T; R23
Xn; R48/20
N; R50

T; N
R: 23-48/20-50
S: (1/2-)45-63-60-61

F; R11
R16
R52-53

F
R: 11-16-52/53
S: (2-)7-43-61

Carc.Cat.3; R40
Xn; R22
Xi; R38

Xn
R: 22-38-40
S: (2-)36/37-46

C ≥ 70 %: C; R20/22-35
50 % ≤ C < 70 %: C; R20/22-34
35 % ≤ C < 50 %: Xn; R22-37/38-41
8 % ≤ C < 35 %: Xn; R22-41
5 % ≤ C < 8 %: Xi; R36
Footnote:
C ≥ 70 %: R5, O;R8
50 % ≤ C < 70 %: O; R8

015-015-00-8

tricresyl phosphate
tritolyl phosphate
o-o-o, o-o-m, o-o-p, o-m-m,
o-m-p, o-p-p

C

201-103-5

78-30-8

T; R39/23/24/25
N; R51-53

T; N
R: 39/23/24/25-51/53
S: (1/2-)20/21-28-45-61

C ≥ 25 %: T, N; R39/23/24/25-51/53
2,5 % ≤ C < 25 %: T; R39/23/24/25-52/
53
1 % ≤ C < 2,5 %: T; R39/23/24/25
0,2 % ≤ C < 1 %: Xn; R68/20/21/22

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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

015-016-00-3

tricresyl phosphate
tritolyl phosphate
m-m-m, m-m-p, m-p-p, p-p-p

C

201-105-6

78-32-0

015-020-00-5 mevinphos (ISO)

232-095-1

7786-34-7

2-methoxycarbonyl-1-methylvi-
nyl dimethyl phosphate

Xn; R21/22
N; R51-53

T+; R27/28
N; R50-53

Xn; N
R: 21/22-51/53
S: (2-)28-61

C ≥ 25 %: Xn, N; R21/22-51/53
5 % ≤ C < 25 %: Xn; R21/22-52/53
2,5 % ≤ C < 5 %: R52/53

T+; N
R: 27/28-50/53
S: (1/2-)23-28-36/37-45-
60-61

C ≥ 7 %: T+, N; R27/28-50-53
1 % ≤ C < 7 %: T, N; R24/25-50-53
0,1 % ≤ C < 1 %: Xn, N; R21/22-50-53
0,0025 % ≤ C < 0,1 %: N; R50-53
0,00025 % ≤ C < 0,0025 %: N; R51-53
0,000025 % ≤ C < 0,00025 %: R52-53

015-021-00-0

trichlorfon (ISO)
dimethyl 2,2,2-trichloro-1-
hydroxyethylphosphonate

200-149-3

52-68-6

Xn; R22
R43
N; R50-53

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

C ≥ 25 %: Xn, N; R22-43-50-53
1 % ≤ C < 25 %: Xi, N; R43-50-53
0,025 % ≤ C < 1 %: N; R50-53
0,0025 % ≤ C < 0,025 %: N; R51-53
0,00025 % ≤ C < 0,0025 %: R52-53

015-027-00-3

sulfotep (ISO)
O,O,O,O-tetraethyl dithiopyro-
phosphate

222-995-2

3689-24-5

T+; R27/28
N; R50-53

T+; N
R: 27/28-50/53
S: (1/2-)23-28-36/37-45-
60-61

C ≥ 7 %: T+, N; R27/28-50-53
1 % ≤ C < 7 %: T, N; R24/25-50-53
0,1 % ≤ C < 1 %: Xn, N; R21/22-50-53
0,025 % ≤ C < 0,1 %: N; R50-53
0,0025 % ≤ C < 0,025 %: N; R51-53
0,00025 % ≤ C < 0,0025 %: R52-53

015-032-00-0

prothoate (ISO)
O,O-diethyl isopropylcarbamoyl-
methyl phosphorodithioate

218-893-2

2275-18-5

T+; R27/28
R52-53

T+
R: 27/28-52/53
S: (1/2-)28-36/37-45-61

015-033-00-6

phorate (ISO)
O,O-diethyl ethylthiomethyl
phosphorodithioate

206-052-2

298-02-2

T+; R27/28
N; R50-53

T+; N
R: 27/28-50/53
S: (1/2-)28-36/37-45-60-
61

C ≥ 7 %: T+, N; R27/28-50-53
1 % ≤ C < 7 %: T, N; R24/25-50-53
0,1 % ≤ C < 1 %: Xn, N; R21/22-50-53
0,025 % ≤ C < 0,1 %: N; R50-53
0,0025 % ≤ C < 0,025 %: N; R51-53
0,00025 % ≤ C < 0,0025 %: R52-53

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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

015-034-00-1

parathion (ISO)
O,O-diethyl O-4-nitrophenyl
phosphorothioate

200-271-7

56-38-2

T+; R26/28
T; 24-48/25
N; R50-53

T+; N
R: 24-26/28-48/25-50/
53
S: (1/2-)28-36/37-45-60-
61

015-035-00-7

parathion - methyl (ISO)
O,O-dimethyl O-4-nitrophenyl
phosphorothioate

206-050-1

298-00-0

R5
R10
T+; R26/28
T; R24
Xn; R48/22
N; R50-53

T+; N
R: 5-10-24-26/28-48/
22-50/53
S: (1/2-)28-36/37-45-60-
61

C ≥ 25 %: T+, N; R24-26/28-48/25-50-53
10 % ≤ C < 25 %: T+, N; R21-26/28-48/
25-50-53
7 % ≤ C < 10 %: T+, N; R21-26/28-48/
22-50-53
3 % ≤ C < 7 %: T, N; R21-23/25-48/22-
50-53
1 % ≤ C < 3 %: T, N; R23/25-48/22-50-
53
0,25 % ≤ C < 1 %: Xn, N; R20/22-50-53
0,1 % ≤ C < 0,25 %: Xn, N; R20/22-51-53
0,025 % ≤ C < 0,1 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

C ≥ 25 %: T+, N; R24-26/28-48/22-50-53
10 % ≤ C < 25 %: T+, N; R21-26/28-48/
22-50-53
7 % ≤ C < 10 %: T+, N; R21-26/28-50-53
3 % ≤ C < 7 %: T, N; R21-23/25-50-53
1 % ≤ C < 3 %: T, N; R23/25-50-53
0,25 % ≤ C < 1 %: Xn, N; R20/22-50-53
0,1 % ≤ C < 0,25 %: Xn, N; R20/22-51-53
0,025 % ≤ C < 0,1 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

015-041-00-X malathion (ISO)

204-497-7

121-75-5

1,2-bis (ethoxycarbonyl) ethyl
O,O-dimethyl phosphorodithio-
ate

Xn; R22
N; R50-53

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

C ≥ 25 %: Xn, N; R22-50-53
0,25 % ≤ C < 25 %: N; R50-53
0,025 % ≤ C < 0,25 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

015-042-00-5

chlorthion (common name not
adopted by ISO)
O-(3-chloro-4-nitrophenyl) O,O-
dimethyl phosphorothioate

207-902-5

500-28-7

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

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

C ≥ 25 %: Xn, N; R20/21/22-50-53
0,25 % ≤ C < 25 %: N; R50-53
0,025 % ≤ C < 0,25: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

015-047-00-2

ethion (ISO)
O,O,O′,O′-tetraethyl S,S′-
methylenedi (phosphorodithio-
ate)
diethion

209-242-3

563-12-2

T; R25
Xn; R21
N; R50-53

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

C ≥ 25 %: T, N; R21-25-50-53
3 % ≤ C < 25 %: Xn, N; R22-50-53
0,0025 % ≤ C < 3 %: N; R50-53
0,00025 % ≤ C < 0,0025 %: N; R51-53
0,000025 % ≤ C < 0,00025 %: R52-53

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

015-052-00-X

fenchlorphos (ISO)
O,O-dimethyl O-2,4,5-
trichlorophenyl phosphorothio-
ate

206-082-6

299-84-3

Xn; R21/22
N; R50-53

Xn; N
R: 21/22-50/53
S: (2-)25-36/37-60-61

015-055-00-6

naled (ISO)
1,2-dibromo-2,2-dichloroethyl
dimethyl phosphate

206-098-3

300-76-5

015-063-00-X

dioxathion (ISO)
1,4-dioxan-2,3-diyl-O,O,O′,O′-
tetraethyl di(phosphorodithioate)

201-107-7

78-34-2

Xn; R21/22
Xi; R36/38
N; R50

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

Xn; N
R: 21/22-36/38-50
S: (2-)36/37-61

C ≥ 25 %: Xn, N; R21/22-36/38-50
20 % ≤ C < 25 %: Xi, N; R36/38-50
0,025 % ≤ C < 20 %: N; R50

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

C ≥ 25 %: T+, N; R24-26/28-50-53
7 % ≤ C < 25 %: T+, N; R21-26/28-50-53
3 % ≤ C < 7 %: T, N; R21-23/25-50-53
1 % ≤ C < 3 %: T, N; R23/25-50-53
0,1 % ≤ C < 1 %: Xn, N; R20/22-50-53
0,025 % ≤ C < 0,1 %: N; R50-53
0,0025 % ≤ C < 0,025 %: N; R51-53
0,00025 % ≤ C < 0,0025 %: R52-53

015-065-00-0

S-[2-(ethylsulphinyl)ethyl] O,O-
dimethyl phosphorodithioate

015-076-00-0

potasan
O,O-diethyl O-(4-
methylcoumarin-7-yl) phosphor-
othioate

—

—

2703-37-9

T+; R26/27/28
N; R51-53

T+; N
R: 26/27/28-51/53
S: (1/2-)13-28-45-61

299-45-6

T+; R26/27/28
N; R50-53

T+; N
R: 26/27/28-50/53
S: (1/2-)13-28-45-60-61

C ≥ 7 %: T+, N; R26/27/28-50-53
1 % ≤ C < 7 %: T, N; R23/24/25-50-53
0,1 % ≤ C < 1 %: Xn, N; R20/21/22-50-
53
0,025 % ≤ C < 0,1 %: N; R50-53
0,0025 % ≤ C < 0,025 %: N; R51-53
0,00025 % ≤ C < 0,0025 %: R52-53

015-078-00-1

demeton-S-methylsulphon
S-2-ethylsulphonylethyl dimethyl
phosphorothioate

241-109-5

17040-19-6

015-083-00-9

bensulide (ISO)
O,O-diisopropyl 2-
phenylsulphonylaminoethyl
phosphorodithioate

212-010-4

741-58-2

T; R25
Xn; R21
N; R51-53

Xn; R22
N; R50-53

T; N
R: 21-25-51/53
S: (1/2-)22-28-36/37-45-
61

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

.

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

015-084-00-4

chlorpyrifos (ISO)
O,O-diethyl O-3,5,6-trichloro-2-
pyridyl phosphorothioate

220-864-4

2921-88-2

T; R25
N; R50-53

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

C ≥ 25 %: T, N; R25-50-53
3 % ≤ C < 25 %: Xn, N; R22-50-53
0,0025 % ≤ C < 3 %: N; R50-53
0,00025 % ≤ C < 0,0025 %: N; R51-53
0,000025 % ≤ C < 0,00025 %: R52-53

015-095-00-4 methamidophos (ISO)

233-606-0

10265-92-6

O,S-dimethyl phosphorami-
dothioate

015-096-00-X

oxydisulfoton; O,O-diethyl S-[2-
(ethylsulphinyl)ethyl] phosphor-
odithioate

219-679-1

2497-07-6

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

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

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

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

C ≥ 25 %: T+, N; R24-28-50-53
7 % ≤ C < 25 %: T+, N; R21-28-50-53
3 % ≤ C < 7 %: T, N; R21-25-50-53
1 % ≤ C < 3 %: T, N; R25-50-53
0,25 % ≤ C < 1 %: Xn, N; R22-50-53
0,1 % ≤ C < 0,25 %: Xn, N; R22-51-53
0,025 % ≤ C < 0,1 %: R52-53

015-097-00-5

phenthoate (ISO)
ethyl 2-
(dimethoxyphosphinothioylthio)-
2-phenylacetate

015-100-00-X

phoxim (ISO)
α-(diethoxyphosphinothioyli-
mino) phenylacetonitrile

219-997-0

2597-03-7

Xn; R21/22
N; R50-53

238-887-3

14816-18-3

Xn; R22
N; R50-53

015-101-00-5

phosmet (ISO)
O,O-dimethyl phthalimido-
methyl S-phosphorodithioate

211-987-4

732-11-6

015-105-00-7

triphenyl phosphite

202-908-4

101-02-0

015-107-00-8

ethoprophos (ISO)
ethyl-S,S-dipropyl phosphoro-
dithioate

236-152-1

13194-48-4

015-108-00-3

bromophos (ISO)
O-4-bromo-2,5-dichlorophenyl
O,O-dimethyl phosphorothioate

218-277-3

2104-96-3

Xn; R21/22
N; R50-53

Xi; R36/38
N; R50-53

T+; R26/27
T; R25
R43
N; R50-53

Xn; R22
N; R50-53

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

C ≥ 25 %: Xn, N; R21/22-50-53
0,25 % ≤ C < 25 %: N; R50-53
0,025 % ≤ C < 0,25 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

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

C ≥ 25 %: Xn, N; R22-50-53
0,025 % ≤ C < 25 %: N; R50-53
0,0025 % ≤ C < 0,025 %: N; R51-53
0,00025 % ≤ C < 0,0025 %: R52-53

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

C ≥ 25 %: Xn, N; R21/22-50-53
0,25 % ≤ C < 25 %: N; R50-53
0,025 % ≤ C < 0,25 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

Xi; N
R: 36/38-50/53
S: (2-)28-60-61

C ≥ 25 %: Xi, N; R36/38-50/53
5 % ≤ C < 25 %: Xi, N; R36/38-51/53
2,5 % ≤ C < 5 %: N; R51/53
0,25 % ≤ C < 2,5 %: R52/53

T+; N
R: 25-26/27-43-50/53
S: (1/2-)27/28-36/37/39-
45-60-61

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

C ≥ 25 %: Xn, N; R22-50-53
0,25 % ≤ C < 25 %: N; R50-53
0,025 % ≤ C < 0,25: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

015-109-00-9

crotoxyphos (ISO)
1-phenylethyl 3-
(dimethoxyphosphinyloxy) iso-
crotonate

231-720-5

7700-17-6

T; R24/25
N; R50-53

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

C ≥ 25 %: T, N; R24/25-50-53
3 % ≤ C < 25 %: Xn, N; R21/22-50-53
2,5 % ≤ C < 3 %: N; R50-53
0,25 % ≤ C < 2,5 %: N; R51-53
0,025 % ≤ C < 0,25 %: R52-53

015-110-00-4

cyanofenphos (ISO)
O-4-cyanophenyl O-ethyl
phenylphosphonothioate

—

13067-93-1

015-114-00-6

chlormephos (ISO)
S-chloromethyl O,O-diethyl
phosphorodithioate

246-538-1

24934-91-6

015-115-00-1

chlorthiophos (ISO)

244-663-6

21923-23-9

015-122-00-X O-6-ethoxy-2-ethylpyrimidin-4-

253-855-9

38260-54-7

yl O,O-
dimethylphosphorothioate
etrimfos

015-123-00-5

fenamiphos (ISO)
ethyl-4-methylthio-m-tolyl iso-
propyl phosphoramidate

244-848-1

22224-92-6

T; R25-39/25
Xn; R21
Xi; R36
N; R51-53

T+; R27/28
N; R50-53

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

Xn; R22
N; R50-53

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

015-126-00-1

heptenophos (ISO)
7-chlorobicyclo(3.2.0)hepta-2,6-
dien-6-yl dimethyl phosphate

245-737-0

23560-59-0

T; R25
N; R50-53

T; N
R: 21-25-36-39/25-51/
53
S: (1/2-)36/37-45-61

T+; N
R: 27/28-50/53
S: (1/2-)28-36/37-45-60-
61

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

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

C ≥ 25 %: Xn, N; R22-50-53
2,5 % ≤ C < 25 %: N; R50-53
0,25 % ≤ C < 2,5 %: N; R51-53
0,025 % ≤ C < 0,25 %: R52-53

T+; N
R: 24-28-50/53
S: (1/2-)23-28-36/37-45-
60-61

C ≥ 25 %: T+, N; R24-28-50-53
7 % ≤ C < 25 %: T+, N; R21-28-50-53
3 % ≤ C < 7 %: T, N; R21-25-50-53
1 % ≤ C < 3 %: T, N; R25-50-53
0,25 % ≤ C < 1 %: Xn, N; R22-50-53
0,1 % ≤ C < 0,25 %: Xn, N; R22-51-53
0,025 % ≤ C < 0,25 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

T; N
R: 25-50/53
S: (1/2-)23-28-37-45-60-
61

C ≥ 25 %: T, N; R25-50-53
3 % ≤ C < 25 %: Xn, N; R22-50-53
0,25 % ≤ C < 3 %: N; R50-53
0,025 % ≤ C < 0,25 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

015-127-00-7

iprobenfos
S-benzyl diisopropyl
phosphorothioate

015-128-00-2

IPSP
S-ethylsulphinylmethyl O,O-
diisopropylphosphorodithioate

247-449-0

26087-47-8

—

5827-05-4

Xn; R22
N; R51-53

T+; R27
T; R25
N; R50-53

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

T+; N
R: 25-27-50/53
S: (1/2-)28-36/37-45-60-
61

015-129-00-8

isofenphos (ISO)
O-ethyl O-2-
isopropoxycarbonylphenyl-iso-
propylphosphoramidothioate

246-814-1

25311-71-1

T; R24/25
N; R50-53

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

015-131-00-9

isoxathion (ISO)
O,O-diethyl O-5-phenylisoxazol-
3-ylphosphorothioate

242-624-8

18854-01-8

T; R24/25
N; R50-53

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

C ≥ 25 %: T+, N; R25-27-50-53
7 % ≤ C < 25 %: T+, N; R22-27-50-53
3 % ≤ C < 7 %: T, N; R22-24-50-53
1 % ≤ C < 3 %: T, N; R24-50-53
0,25 % ≤ C < 1 %: Xn, N; R21-50-53
0,1 % ≤ C < 0,25 %: Xn, N; R21-51-53
0,025 % ≤ C < 0,1 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

C ≥ 25 %: T, N; R24/25-50-53
3 % ≤ C < 25 %: Xn, N; R21/22-50-53
0,25 % ≤ C < 3 %: N; R50-53
0,025 % ≤ C < 0,25: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

015-132-00-4

S-(chlorophenylthiomethyl) O,O-
dimethylphosphorodithioate
methylcarbophenothione

—

953-17-3

T; R24/25
N; R50-53

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

C ≥ 25 %: T, N; R24/25-50-53
3 % ≤ C < 25 %: Xn, N; R21/22-50-53
0,025 % ≤ C < 3 %: N; R50-53
0,0025 % ≤ C < 0,025 %: N; R51-53
0,00025 % ≤ C < 0,0025 %: R52-53

015-133-00-X

piperophos (ISO)
S-2-methylpiperidinocarbonyl-
methyl-O,O-dipropyl phosphor-
odithioate

015-134-00-5

pirimiphos-methyl (ISO)
O-(2-diethylamino-6-methylpyri-
midin-4-yl) O,O-dimethyl
phosphorothioate

015-135-00-0

O-(4-bromo-2-chlorophenyl) O-
ethyl S-propyl phosphorothioate
profenofos (ISO)

—

24151-93-7

Xn; R22
N; R50-53

249-528-5

29232-93-7

Xn; R22
N; R50-53

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

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

C ≥ 25 %: Xn, N; R22-50-53
2,5 % ≤ C < 25 %: N; R50-53
0,25 % ≤ C < 2,5 %: N; R51-53
0,025 % ≤ C < 0,25 %: R52-53

255-255-2

41198-08-7

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

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

C ≥ 25 %: Xn, N; R20/21/22-50-53
0,025 % ≤ C < 25 %: N; R50-53
0,0025 % ≤ C < 0,025 %: N; R51-53
0,00025 % ≤ C < 0,0025 %: R52-53

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

015-136-00-6

trans-isopropyl-3-[[(ethylamino)-
methoxyfosfinothioyl]oxy]croto-
nate; isopropyl 3-
[[(ethylamino)methoxyphosphi-
nothioyl]oxy]isocrotonate
propetamphos (ISO)

250-517-2

31218-83-4

T; R25
N; R50-53

015-138-00-7

quinalphos (ISO)
O,O-diethyl-O-quinoxalin-2-yl
phosphorothioate

237-031-6

13593-03-8

T; R25
Xn; R21
N; R50-53

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

C ≥ 25 %: T, N; R25-50-53
3 % ≤ C < 25 %: Xn, N; R22-50-53
0,25 % ≤ C < 3 %: N; R50-53
0,025 % ≤ C < 0,25 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

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

C ≥ 25 %: T, N; R21-25-50-53
3 % ≤ C < 25 %: Xn, N; R22-50-53
0,025 % ≤ C < 3 %: N; R50-53
0,0025 % ≤ C < 0,025 %: N; R51-53
0,00025 % ≤ C < 0,0025 %: R52-53

015-139-00-2

S-tert-butylthiomethyl O,O-
diethylphosphorodithioate
terbufos (ISO)

235-963-8

13071-79-9

T+; R27/28
N; R50-53

T+; N
R: 27/28-50/53
S: (1/2-)36/37-45-60-61

C ≥ 7 %: T+, N; R27/28-50-53
1 % ≤ C < 7 %: T, N; R24/25-50-53
0,1 % ≤ C < 1 %: Xn, N; R21/22-50-53
0,025 % ≤ C < 0,1 %: N; R50-53
0,0025 % ≤ C < 0,025 %: N; R51-53
0,00025 % ≤ C < 0,0025 %: R52-53

C ≥ 25 %: C; R20/21-34-52/53
10 % ≤ C < 25 %: C; R34
5 % ≤ C < 10 %: Xi; R36/37/38

015-154-00-4

2-chloroethylphosphonic acid
ethephon

240-718-3

16672-87-0

015-179-00-0

UVCB condensation product of:
tetrakis-hydroxymethylphospho-
nium chloride, urea and distilled
hydrogenated C 16-18 tallow
alkylamine

422-720-8

166242-53-1

016-001-00-4

hydrogen sulphide

231-977-3

7783-06-4

016-008-00-2

ammonium polysulphides

232-989-1

9080-17-5

016-012-00-4

disulphur dichloride
sulfur monochloride

233-036-2

10025-67-9

Xn; R20/21
C; R34
R52-53

Carc. Cat. 3; R40
Xn; R22-48/22
C; R34
R43
N; R50-53

F+; R12
T+; R26
N; R50

R31
C; R34
N; R50

R14
T; R25
Xn; R20
R29
C; R35
N; R50

C
R: 20/21-34-52/53
S: (1/2-)26-28-36/37/39-
45-61

C; N
R: 22-34-40-43-48/22-
50/53
S: (1/2-)26-36/37/39-45-
60-61

F+; T+; N
R: 12-26-50
S: (1/2-)9-16-36-38-45-
61

C; N
R: 31-34-50
S: (1/2-)26-45-61

C ≥ 25 %: C, N ; R31-34-50
5 % ≤ C < 25 %: C; R31-34
1 % ≤ C < 5 %: Xi; R31-36/38

T; C; N
R: 14-20-25-29-35-50
S: (1/2-)26-36/37/39-45-
61

C ≥ 25 %: T, C, N; R20-25-35-50
10 % ≤ C < 25 %: C; R22-35
5 % ≤ C < 10 %: C; R22-34
3 % ≤ C < 5 %: Xn; R22-36/37/38
1 % ≤ C < 3 %: Xi; R36/37/38

.

.

1
6
6
2
0
0
4

E
N

O

f
f
i
c
i
a
l

J
o
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r
n
a
l

o
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e

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/
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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

016-013-00-X

sulphur dichloride

234-129-0

10545-99-0

016-014-00-5

sulphur tetrachloride

—

13451-08-6

016-021-00-3 methanethiol

methyl mercaptan

200-822-1

74-93-1

016-023-00-4

dimethyl sulphate

E

201-058-1

77-78-1

016-059-00-0

N,N,N′,N′-tetramethyldithiobi-
s(ethylene)diamine dihydrochlor-
ide

405-300-9

17339-60-5

017-003-00-8

barium chlorate

236-760-7

13477-00-4

017-004-00-3

potassium chlorate

223-289-7

3811-04-9

017-005-00-9

sodium chlorate

231-887-4

7775-09-9

017-011-00-1

sodium hypochlorite, solution
… % Cl active

B

231-668-3

7681-52-9

R14
C; R34
Xi; R37
N; R50

R14
C; R34
N; R50

F+; R12
T; R23
N; R50-53

Carc. Cat. 2; R45
Muta. Cat. 3; R68
T+; R26
T; R25
C; R34
R43

Xn; R22
Xi; R36
R43
N; R50-53

O; R9
Xn; R20/22
N; R51-53

O; R9
Xn; R20/22
N; R51-53

O; R9
Xn; R22
N; R51-53

C; R34
R31
N; R50

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

C ≥ 25 %: C, N ; R34-50
10 % ≤ C < 25 %: C; R34
5 % ≤ C < 10 %: Xi; R36/37/38

C ≥ 25 %: C, N; R34-50
10 ≤ C < 25 %: C; R34
5 ≤ C < 10 %: Xi; R36/37/38

C ≥ 25 %: T+; R45-R25-R26-R34-R43-
R68
10 % ≤ C < 25 %: T+; R45-R22-R26-R34-
R43-R68
7 % ≤ C < 10 %: T+; R45-R22-R26-R36/
37/38-R43-R68
5 % ≤ C < 7 %: T; R45-R22-R23-R36/37/
38-R43-R68
3 % ≤ C < 5 %: T; R45-R22-R23-R43-
R68
1 % ≤ C < 3 %: T; R45-R23-R43-R68
0,1 % ≤ C < 1 %: T; R45-R20-R68
0,01 % ≤ C < 0,1 %: T; R45-R68

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

F+; T; N
R: 12-23-50/53
S: (2-)16-25-60-61

T+
R: 45-25-26-34-43-68
S: 53-45

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

O; Xn; N
R: 9-20/22-51/53
S: (2-)13-27-61

O; Xn; N
R: 9-20/22-51/53
S: (2-)13-16-27-61

O; Xn; N
R: 9-22-51/53
S: (2-)13-17-46-61

C; N
R: 31-34-50
S: (1/2-)28-45-50-61

C ≥ 25 %: C, N; R31-34-50
10 % ≤ C < 25 %: C; R31-34
5 % ≤ C < 10 %: Xi; R31-36/38

L

2
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J
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n
a
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t
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e

E
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U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

017-012-00-7

calcium hypochlorite

231-908-7

7778-54-3

024-001-00-0

chromium (VI) trioxide

E

215-607-8

1333-82-0

O; R8
Xn; R22
R31
C; R34
N; R50

O; R9
Carc. Cat. 1; R45
Muta. Cat. 2; R46
Repr. Cat. 3; R62
T+; R26
T; R24/25-48/23
C; R35
R42/43
N; R50-53

O; C; N
R: 8-22-31-34-50
S: (1/2-)26-36/37/39-45-
61

C ≥ 25 %: C, N; R22-34-50
10 % ≤ C < 25 %: C; R34
3 % ≤ C < 10 %: Xi; R37/38-41
0,5 % ≤ C < 3 %: Xi; R36

O; T+; N
R: 45-46-9-24/25-26-
35-42/43-48/23-62-50/
53
S: 53-45-60-61

024-002-00-6

potassium dichromate

E

231-906-6

7778-50-9

T+; N; O
R: 45-46-60-61-8-21-
25-26-34-42/43-48/23-
50/53
S: 53-45-60-61

O; R8
Carc. Cat. 2: R45
Muta. Cat. 2; R46
Repr. Cat. 2; R60-61
T+; R26
T; R25-48/23
Xn; R21
C; R34
R42/43
N; 50-53

.

.

1
6
6
2
0
0
4

E
N

O

f
f
i
c
i
a
l

J
o
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n
a
l

o
f

t
h
e

E
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o
p
e
a
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U
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o
n

i

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2
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6
/
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7

C ≥ 25 %: T+, N; R24/25-26-35-42/43-
45-46-48/23-50/53-62
10 % ≤ C < 25 %: T+, N; R21/22-26-35-
42/43-45-46-48/23-51/53-62
7 % ≤ C < 10 %: T+, N; R21/22-26-34-
42/43-45-46-48/20-51/53-62
5 % ≤ C < 7 %: T, N; R21/22-23-34-42/
43-45-46-48/20-51/53-62
3 % ≤ C < 5 %: T, N; R21/22-23-36/37/
38-42/43-45-46-48/20-51/53
2,5 % ≤ C < 3 %: T, N; R23-36/37/38-42/
43-45-46-48/20-51/53
1 % ≤ C < 2,5 %: T; R23-36/37/38-42/43-
45-46-48/20-52/53
0,25 % ≤ C < 1 %: T; R20-45-46-52/53
0,1 % ≤ C < 0,25 %: T; R20-45-46

3

C ≥ 25 %: T+, N; R45-46-60-61-21-25-
26-34-42/43-48/23-50/53
10 % ≤ C < 25 %: T+, N; R45-46-60-61-
22-26-34-42/43-48/23-51/53
7 % ≤ C < 10 %: T+, N; R45-46-60-61-
22-26-36/37/38-42/43-48/20-51/53
5 % ≤ C < 7 %: T, N; R45-46-60-61-22-
23-36/37/38-42/43-48/20-51/53
3 % ≤ C < 5 %: T, N; R45-46-60-61-22-
23-42/43-48/20-51/53
2,5 % ≤ C < 3 %: T, N; R45-46-60-61-23-
42/43-48/20-51/53
1 % ≤ C < 2,5 %: T; R45-46-60-61-23-42/
43-48/20-52/53
0,5 % ≤ C < 1 %: T; R45-46-60-61-20-42/
43-52/53
0,25 % ≤ C < 0,5 %: T; R45-46-20-42/43-
52/53
0,2 % ≤ C < 0,25 %: T; R45-46-20-42/43
0,1 % ≤ C < 0,2 %: T; R45-46-20

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

024-003-00-1

ammonium dichromate

E

232-143-1

7789-09-5

024-004-00-7

sodium dichromate anhydrate

E

234-190-3

10588-01-9

E; T+; N
R: 45-46-60-61-2-8-21-
25-26-34-42/43-48/23-
50/53
S: 53-45-60-61

E; R2
O; R8
Carc. Cat. 2; R45
Muta. Cat. 2; R46
Repr. Cat. 2; R60-61
T+; R26
T; R25-48/23
Xn; R21
C; R34
R42/43
N; R50-53

T+; N; O
R: 45-46-60-61-8-21-
25-26-34-42/43-48/23-
50/53
S: 53-45-60-61

O; R8
Carc. Cat. 2; R45
Muta. Cat. 2; R46
Repr. Cat. 2; R60-61
T+; R26
T; R25-48/23
Xn; R21
C; R34
R42/43
N; 50-53

3

3

C ≥ 25 %: T+, N; R45-46-60-61-21-25-
26-34-42/43-48/23-50/53
10 % ≤ C < 25 %: T+, N; R45-46-60-61-
22-26-34-42/43-48/23-50/53
7 % ≤ C < 10 %: T+, N; R45-46-60-61-
22-26-36/37/38-42/43-48/20-50/53
5 % ≤ C < 7 %: T, N; R45-46-60-61-22-
23-36/37/38-42/43-48/20-51/53
3 % ≤ C < 5 %: T, N; R45-46-60-61-22-
23-42/43-48/20-51/53
2,5 % ≤ C < 3 %: T, N; R45-46-60-61-23-
42/43-48/20-51/53
1 % ≤ C < 2,5 %: T; R45-46-60-61-23-42/
43-48/20-52/53
0,5 % ≤ C < 1 %: T; R45-46-60-61-20-42/
43-52/53
0,25 % ≤ C < 0,5 %: T; R45-46-20-42/43-
52/53
0,2 % ≤ C < 0,25 %: T; R45-46-20-42/43
0,1 % ≤ C < 0,2 %: T; R45-46-20

C ≥ 25 %: T+, N; R45-46-60-61-21-25-
26-34-42/43-48/23-50/53
10 % ≤ C < 25 %: T+, N; R45-46-60-61-
22-26-34-42/43-48/23-51/53
7 % ≤ C < 10 %: T+, N; R45-46-60-61-
22-26-36/37/38-42/43-48/20-51/53
5 % ≤ C < 7 %: T, N; R45-46-60-61-22-
23-36/37/38-42/43-48/20-51/53
3 % ≤ C < 5 %: T, N; R45-46-60-61-22-
23-42/43-48/20-51/53
2,5 % ≤ C < 3 %: T, N; R45-46-60-61-23-
42/43-48/20-51/53
1 % ≤ C < 2,5 %: T ; R45-46-60-61-23-
42/43-48/20-52/53
0,5 % ≤ C < 1 %: T; R45-46-60-61-20-42/
43-52/53
0,25 % ≤ C < 0,5 %: T; R45-46-20-42/43-
52/53
0,2 % ≤ C < 0,25 %: T; R45-46-20-42/43
0,1 % ≤ C < 0,2 %: T; R45-46-20

L

2
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O

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i
a
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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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p
e
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U
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o
n

i

.

.

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6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

024-004-01-4

sodium dichromate, dihydrate

E

234-190-3

7789-12-0

T+; N; O
R: 45-46-60-61-8-21-
25-26-34-42/43-48/23-
50/53
S: 53-45-60-61

O; R8
Carc. Cat.2; R45
Muta. Cat. 2; R46
Repr. Cat. 2; R60-61
T+; R26
T; R25-48/23
Xn; R21
C; R34
R42/43
N; R50-53

C ≥ 25 %: T+, N; R45-46-60-61-21-25-
26-34-42/43-48/23-50/53
10 % ≤ C < 25 %: T+, N; R45-46-60-61-
22-26-34-42/43-48/23-51/53
7 % ≤ C < 10 %: T+, N; R45-46-60-61-
22-26-36/37/38-42/43-48/20-51/53
5 % ≤ C < 7 %: T, N; R45-46-60-61-22-
23-36/37/38-42/43-48/20-51/53
3 % ≤ C < 5 %: T, N; R45-46-60-61-22-
23-42/43-48/20-51/53
2,5 % ≤ C < 3 %: T, N; R45-46-60-61-23-
42/43-48/20-51/53
1 % ≤ C < 2,5 %: T; R45-46-60-61-23-42/
43-48/20-52/53
0,5 % ≤ C < 1 %: T; R45-46-60-61-20-42/
43-52/53
0,25 % ≤ C < 0,5 %: T; R45-46-20-42/43-
52/53
0,2 % ≤ C < 0,25 %: T; R45-46-20-42/43
0,1 % ≤ C < 0,2 %: T; R45-46-20

Notes related
to preparations

3

024-011-00-5

ammonium bis(1-(3,5-dinitro-2-
oxidophenylazo)-3-(N-phenylcar-
bamoyl)-2-naphtholato)chro-
mate(1-)

400-110-2

—

F; R11
N; R50-53

F; N
R: 11-50/53
S: (2-)33-60-61

024-018-00-3

sodium chromate

E

231-889-5

7775-11-3

T+; N
R: 45-46-60-61-21-25-
26-34-42/43-48/23-50/
53
S: 53-45-60-61

Carc. Cat. 2; R45
Muta. Cat. 2; R46
Repr. Cat.2; R60-61
T+; R26
T; R25-48/23
Xn; R21
C; R34
R42/43
N; R50-53

3

C ≥ 25 %: T+, N; R45-46-60-61-21-25-
26-34-42/43-48/23-50/53
10 % ≤ C < 25 %: T+, N; R45-46-60-61-
22-26-34-42/43-48/23-51/53
7 % ≤ C < 10 %: T+, N; R45-46-60-61-
22-26-36/37/38-42/43-48/20-51/53
5 % ≤ C < 7 %: T, N; R45-46-60-61-22-
23-36/37/38-42/43-48/20-51/53
3 % ≤ C < 5 %: T, N; R45-46-60-61-22-
23-42/43-48/20-51/53
2,5 % ≤ C < 3 %: T, N; R45-46-60-61-23-
42/43-48/20-51/53
1 % ≤ C < 2,5 %: T; R45-46-60-61-23-42/
43-48/20-52/53
0,5 % ≤ C < 1 %: T; R45-46-60-61-20-42/
43-52/53
0,25 % ≤ C < 0,5 %: T; R45-46-20-42/43-
52/53
0,2 % ≤ C < 0,25 %: T; R45-46-20-42/43
0,1 % ≤ C < 0,2 %: T; R45-46-20

.

.

1
6
6
2
0
0
4

E
N

O

f
f
i
c
i
a
l

J
o
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r
n
a
l

o
f

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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

027-004-00-5

cobalt dichloride

027-005-00-0

cobalt sulphate

E

E

231-589-4

7646-79-9

Carc. Cat. 2; R49
Xn; R22
R42/43
N; R50-53

T; N
R: 49-22-42/43-50/53
S: (2-)22-53-45-60-61

233-334-2

10124-43-3

Carc. Cat. 2; R49
Xn; R22
R42/43
N; R50-53

T; N
R: 49-22-42/43-50/53
S: (2-)22-53-45-60-61

C ≥ 25 %: T, N; R49-22-42/43-50/53
2,5 % ≤ C < 25 %: T, N; R49-22-42/43-
51/53
1 % ≤ C < 2,5 %: T; R49-42/43-52/53
0,25 % ≤ C < 1 %: T; R49-52/53
0,01 % ≤ C < 0,25 %: T; R49

C ≥ 25 %: T, N; R49-22-42/43-50/53
2,5 % ≤ C < 25 %: T, N; R49-42/43-51/
53
1 % ≤ C < 2,5 %: T; R49-42/43-52/53
0,25 % ≤ C < 1 %: T; R49-52/53
0,01 % ≤ C < 0,25 %: T; R49

1

1

029-002-00-X

dicopper oxide
copper (I) oxide

215-270-7

1317-39-1

030-001-00-1

zinc powder - zinc dust
(pyrophoric)

231-175-3

7440-66-6

Xn; R22
N; 50-53

F; R15-17
N; R50-53

030-002-00-7

zinc powder - zinc dust
(stabilized)

231-175-3

7440-66-6

N; R50-53

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

F; N
R: 15-17-50/53
S: (2-)43-46-60-61

N
R: 50/53
S: 60-61

030-003-00-2

zinc chloride

231-592-0

7646-85-7

Xn; R22
C; R34
N; R50-53

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

C ≥ 25 %: C, N; R22-34-50/53
10 % ≤ C < 25 %: C, N; R34-51/53
5 % ≤ C < 10 %: Xn, N; R36/37/38-51/53
2.5 % ≤ C < 5 %: N; R51/53
0.25 % ≤ C < 2.5 %: R52/53

030-006-00-9

zinc sulphate (hydrous) (mono-,
hexa- and hepta hydrate)
[1]
zinc sulphate (anhydrous)
[2]

231-793-3 [1]
231-793-3 [2]

7446-19-7 [1]
7733-02-0 [2]

Xn; R22
R41
N; R50-53

Xn; N
R: 22-41-50/53
S: (2-)22-26-39-46-60-
61

033-001-00-X

arsenic

231-148-6

7440-38-2

T; R23/25
N; R50-53

T; N
R: 23/25-50/53
S: (1/2-)20/21-28-45-60-
61

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i
a
l

J
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o
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t
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e

E
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U
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o
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i

.

.

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6
2
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0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

033-002-00-5

arsenic compounds, with the
exception of those specified
elsewhere in this Annex

A

—

—

T; R23/25
N; R50-53

T; N
R: 23/25-50/53
S: (1/2-)20/21-28-45-60-
61

C ≥ 25 %: T, N; R23/25-50/53
2,5 % ≤ C < 25 %: T, N; R23/25-51/53
0,25 % ≤ C < 2,5 %: T; R23/25-52/53
0,2 % ≤ C < 0,25 %: T; R23/25
0,1 % ≤ C < 0,2 %: Xn; R20/22

042-002-00-4

tetrakis(dimethylditetradecylam-
monium) hexa-μ-oxotetra-μ3-
oxodi-μ5-oxotetradecaoxoocta-
molybdate(4-)

048-001-00-5

cadmium compounds, with the
exception of cadmium
sulphoselenide (xCdS.yCdSe),
mixture of cadmium sulphide
with zinc sulphide (xCdS.yZnS),
mixture of cadmium sulphide
with mercury sulphide
(xCdS.yHgS), and those specified
elsewhere in this Annex

404-760-8

117342-25-3

T; R23
Xi; R41
R53

T
R: 23-41-53
S: (1/2-)26-37/39-45-61

A

—

—

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

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

048-003-00-6

cadmium diformate
cadmiumformate

224-729-0

4464-23-7

T; R23/25
R33
Xn; R68
N; R50-53

T; N
R: 23/25-33-68-50/53
S: (1/2-)22-45-60-61

048-004-00-1

cadmium cyanide

208-829-1

542-83-6

T+; R26/27/28
R32
R33
Xn; R68
N; R50-53

T+; N
R: 26/27/28-32-33-68-
50/53
S: (1/2-)7-28-29-45-60-
61

C ≥ 25 %: Xn, N; R20/21/22-50/53
2,5 % ≤ C < 25 %: Xn, N; R20/21/22-51/
53
0,25 % ≤ C < 2,5 %: Xn; R20/21/22-52/
53
0,1 % ≤ C < 0,25 %: Xn; R20/21/22

C ≥ 25 %: T, N; R23/25-33-50/53-68
10 % ≤ C < 25 %: T, N; R23/25-33-51/
53-68
2,5 % ≤ C < 10 %: Xn, N; R20/22-33-51/
53-68
1 % ≤ C < 2,5 %: Xn; R20/22-33-52/53-
68
0,1 % ≤ C < 1 %: Xn; R20/22-33-52/53
0,25 % ≤ C < 0,1 %: Xn; R20/22-33-52/
53

C ≥ 25 %: T+, N; R26/27/28-32-33-50/
53-68
7 % ≤ C < 25 %: T+, N; R26/27/28-32-
33-51/53-68
2,5 % ≤ C < 7 %: T, N; R23/24/25-32-33-
51/53-68
1 % ≤ C < 2,5 %: T; R23/24/25-32-33-52/
53-68
0,25 % ≤ C < 1 %: Xn; R20/21/22-33-52/
53
0,1 % ≤ C < 0,25 %: Xn; R20/21/22-33

1

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

048-005-00-7

cadmiumhexafluorosilicate(2-)
cadmium fluorosilica

241-084-0

17010-21-8

T; R23/25
R33
Xn; R68
N; R50-53

T; N
R: 23/25-33-68-50/53
S: (1/2-)22-45-60-61

048-006-00-2

cadmium fluoride

E

232-222-0

7790-79-6

Carc. Cat. 2; R45
Muta. Cat. 2; R46
Repr. Cat. 2; R60-61
T+; R26
T; R25-48/23/25
N; R50-53

T+; N
R: 45-46-60-61-25-26-
48/23/25-50/53
S: 53-45-60-61

048-007-00-8

cadmium iodide

232-223-6

7790-80-9

T; R23/25
R33
Xn; R68
N; R50-53

T; N
R: 23/25-33-68-50/53
S: (1/2-)22-45-60-61

C ≥ 25 %: T, N; R23/25-33-50/53-68
10 % ≤ C < 25 %: T, N; R23/25-33-51/
53-68
2,5 % ≤ C < 10 %: Xn, N; R20/22-33-51/
53-68
1 % ≤ C < 2,5 %: Xn; R20/22-33-52/53-
68
0,25 % ≤ C < 1 %: Xn; R20/22-33-52/53
0,1 % ≤ C < 0,25 %: Xn; R20/22-33

C ≥ 25 %:: T+, N; R45-46-60-61-25-26-
48/23/25-50/53
10 % ≤ C < 25 %: T+, N; R45-46-60-61-
25-26-48/23/25-51/53
7 % ≤ C < 10 %: T+, N; R45-46-60-61-
22-26-48/23/25-51/53
2,5 % ≤ C < 7 %: T, N; R45-46-60-61-22-
23-48/20/22-51/53
1 % ≤ C < 2,5 %: T; R45-46-60-61-22-
23-48/20/22-52/53
0,5 % ≤ C < 1 %: T; R45-46-60-61-20/22-
48/20/22-52/53
0,25 % ≤ C < 0,5 %: T; R45-46-20/22-48/
20/22-52/53
0,1 % ≤ C < 0,25 %: T; R45-46-20/22-48/
20/22
0,01 % ≤ C < 0,1 %: T; R45

C ≥ 25 %: T, N; R23/25-33-50/53-68
10 % ≤ C < 25 %: T, N; R23/25-33-51/
53-68
2,5 % ≤ C < 10 %: Xn, N; R20/22-33-51/
53-68
1 % ≤ C < 2,5 %: Xn; R20/22-33-52/53-
68
0,25 % ≤ C < 1 %: Xn; R20/22-33-52/53
0,1 % ≤ C < 0,25 %: Xn; R20/22-33

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

048-008-00-3

cadmium chloride

E

233-296-7

10108-64-2

Carc. Cat. 2; R45
Muta. Cat. 2; R46
Repr. Cat. 2; R60-61
T+; R26
T; R25-48/23/25
N; R50-53

T+; N
R: 45-46-60-61-25-26-
48/23/25-50/53
S: 53-45-60-61

048-009-00-9

cadmium sulphate

E

233-331-6

10124-36-4

Carc. Cat. 2; R45
Muta. Cat. 2; R46
Repr. Cat. 2; R60-61
T; R48/23/25
T+; R26
T; R25
N; R50-53

T+; N
R: 45-46-60-61-25-26-
48/23/25-50/53
S: 53-45-60-61

C ≥ 25 %: T+, N; R45-46-60-61-25-26-
48/23/25-50/53
10 % ≤ C < 25 %: T+, N; R45-46-60-61-
25-26-48/23/25-51/53
7 % ≤ C < 10 %: T+, N; R45-46-60-61-
22-26-48/23/25-51/53
2,5 % ≤ C < 7 %: T, N; R45-46-60-61-22-
23-48/20/22-51/53
1 % ≤ C < 2,5 %: T; R45-46-60-61-22-
23-48/20/22-52/53
0,5 % ≤ C < 1 %: T; R45-46-60-61-20/22-
48/20/22-52/53
0,25 % ≤ C < 0,5 %: T; R45-46-20/22-48/
20/22-52/53
0,1 % ≤ C < 0,25 %: T; R45-46-20/22-48/
20/22
0,01 % ≤ C < 0,1 %: T; R45

C ≥ 25 %: T+, N; R45-46-60-61-25-26-
48/23/25-50/53
10 % ≤ C < 25 %: T+, N; R45-46-60-61-
25-26-48/23/25-51/53
7 % ≤ C < 10 %: T+, N; R45-46-60-61-
22-26-48/23/25-51/53
2,5 % ≤ C < 7 %: T, N; R45-46-60-61-22-
23-48/20/22-51/53
1 % ≤ C < 2,5 %: T; R45-46-60-61-22-
23-48/20/22-52/53
0,5 % ≤ C < 1 %: T; R45-46-60-61-20/22-
48/20/22-52/53
0,25 % ≤ C < 0,5 %: T; R45-46-20/22-48/
20/22-52/53
0,1 % ≤ C < 0,25 %: T; R45-46-20/22-48/
20/22
0,01 % ≤ C < 0,1 %: T; R45

048-010-00-4

cadmium sulphide

E

215-147-8

1306-23-6

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Repr. Cat. 3; R62-63
T; R48/23/25
Xn; R22
R53

T; N
R: 45-22-48/23/25-62-
63-68-53
S: 53-45-61

1

C ≥ 25 %: T; R45-22-48/23/25-62-63-68-
53
10 % ≤ C < 25 %: T; R45-22-48/23/25-
62-63-68
5 % ≤ C < 10 %: T; R45-48/20/22-62-63-
68
1 % ≤ C < 5 %: T; R45-48/20/22-68
0,1 % ≤ C < 1 %: T; R45-48/20/22

.

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

050-001-00-5

tin tetrachloride
stannic chloride

231-588-9

7646-78-8

C; R34
R52-53

C
R: 34-52/53
S: (1/2-)7/8-26-45-61

C ≥ 25 %: C; R34-52/53
10 % ≤ C < 25 %: C; R34
5 % ≤ C < 10 %: Xi; R36/37/38

050-005-00-7

trimethyltin compounds, with
the exception of those specified
elsewhere in this Annex

A

—

—

T+; R26/27/28
N; R50-53

050-006-00-2

triethyltin compounds, with the
exception of those specified
elsewhere in this Annex

A

—

—

T+; R26/27/28
N; R50-53

050-007-00-8

tripropyltin compounds, with
the exception of those specified
elsewhere in this Annex

A

—

—

T; R23/24/25
N; R50-53

T+; N
R: 26/27/28-50/53
S: (1/2-)26-27-28-45-60-
61

T+; N
R: 26/27/28-50/53
S: (1/2-)26-27-28-45-60-
61

T; N
R: 23/24/25-50/53
S: (1/2-)26-27-28-45-60-
61

050-008-00-3

tributyltin compounds, with the
exception of those specified
elsewhere in this Annex

A

—

—

T; R25-48/23/25
Xn; R21
Xi; R36/38
N; R50-53

T; N
R: 21-25-36/38-48/23/
25-50/53
S: (1/2-)35-36/37/39-45-
60-61

050-009-00-9

fluorotripentylstannane
[1]
hexapentyldistannoxane
[2]

243-546-7 [1]
247-143-7 [2]

20153-49-5 [1]
25637-27-8
[2]

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

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

050-010-00-4

fluorotrihexylstannane

243-547-2

20153-50-8

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

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

C ≥ 25 %: T+, N; R26/27/28-50/53
2,5 % ≤ C < 25 %: T+, N; R26/27/28-51/
53
0,5 % ≤ C < 2,5 %: T+; R26/27/28-52/53
0,25 % ≤ C < 0,5 %: T; R23/24/25-52/53
0,1 % ≤ C < 0,25 %: T; R23/24/25
0,05 % ≤ C < 0,1 %: Xn; R20/21/22

C ≥ 25 %: T+, N; R26/27/28-50/53
2,5 % ≤ C < 25 %: T+, N; R26/27/28-51/
53
0,5 % ≤ C < 2,5 %: T+; R26/27/28-52/53
0,25 % ≤ C < 0,5 %: T; R23/24/25-52/53
0,1 % ≤ C < 0,25 %: T; R23/24/25
0,05 % ≤ C < 0,1 %: Xn; R20/21/22

C ≥ 25 %: T, N; R23/24/25-50/53
2,5 % ≤ C < 25 %: T, N; R23/24/25-51/53
0,5 % ≤ C < 2,5 %: T; R23/24/25-52/53
0,25 % ≤ C < 0,5 %: Xn; R20/21/22-52/
53
0,1 % ≤ C < 0,25 %: Xn; R20/21/22

C ≥ 25 %: T, N; R21-25-36/38-48/23/25-
50/53
2,5 % ≤ C < 25 %: T, N; R21-25-36/38-
48/23/25-51/53
1 % ≤ C < 2,5 %: T; R21-25-36/38-48/23/
25-52/53
0,25 % ≤ C < 1 %: Xn; R22-48/20/22-52/
53

C ≥ 25 %: Xn, N; R20/21/22-50/53
2,5 % ≤ C < 25 %: Xn, N; R20/21/22-51/
53
1 % ≤ C < 2,5 %: Xn; R20/21/22-52/53
0,25 % ≤ C < 1 %: R52/53

C ≥ 25 %: Xn, N; R20/21/22-50/53
2,5 % ≤ C < 25 %: Xn, N; R20/21/22-51/
53
1 % ≤ C < 2,5 %: Xn; R20/21/22-52/53
0,25 % ≤ C < 1 %: R52/53

1

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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

050-011-00-X

triphenyltin compounds, with
the exception of those specified
elsewhere in this Annex

050-012-00-5

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

A

A

—

—

T; R23/24/25
N; R50-53

T; N
R: 23/24/25-50/53
S: (1/2-)26-27-28-45-60-
61

C ≥ 25 %: T, N; R23/24/25-50/53
2,5 % ≤ C < 25 %: T, N; R23/24/25-51/53
1 % ≤ C < 2,5 %: T; R23/24/25-52/53
0,25 % ≤ C < 1 %: Xn; R20/21/22-52/53

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 ≥ 25 %: Xn, N; R20/21/22-50/53
2,5 % ≤ C < 25 %: Xn, N; R20/21/22-51/
53
1 % ≤ C < 2,5 %: Xn; R20/21/22-52/53
0,25 % ≤ C < 1 %: R52/53

050-013-00-0

trioctyltin compounds, with the
exception of those specified
elsewhere in this Annex

A

—

—

Xi; R36/37/38
R53

Xi
R: 36/37/38-53
S: (2-)61

C ≥ 25 %: Xi; R36/37/38-53
1 % ≤ C < 25 %: Xi; R36/37/38

051-002-00-3

antimony pentachloride

231-601-8

7647-18-9

C; R34
N; R51-53

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

C ≥ 25 %: C, N; R34-51/53
10 % ≤ C < 25 %: C; R34-52/53
5 % ≤ C < 10 %: Xi; R36/37/38-52/53
2,5 % ≤ C < 5 %: R52/53

051-003-00-9

antimony compounds, with the
exception of the tetroxide
(Sb2O4), pentoxide (Sb2O5),
trisulphide (Sb2S3), pentasulphide
(Sb2S5) and those specified
elsewhere in this Annex

080-002-00-6

inorganic compounds of
mercury with the exception of
mercuric sulphide and those
specified elsewhere in this
Annex

A

A

—

—

Xn; R20/22
N; R51-53

Xn; N
R: 20/22-51/53
S: (2-)61

C ≥ 25 %: Xn, N; R20/22-51/53
2,5 % ≤ C < 25 %: Xn; R20/22-52/53
0,25 % ≤ C < 2,5 %: Xn; R20/22

—

—

T+; R26/27/28
R33
N; R50-53

T+; N
R: 26/27/28-33-50/53
S: (1/2-)13-28-45-60-61

C ≥ 25 %: T+, N; R26/27/28-33-50/53
2,5 % ≤ C < 25 %: T+, N; R26/27/28-33-
51/53
2 % ≤ C < 2,5 %: T+; R26/27/28-33-52/
53
0,5 % ≤ C < 2 %: T; R23/24/25-33-52/53
0,25 % ≤ C < 0,5 %: Xn; R20/21/22-33-
52/53
0,1 % ≤ C < 0,25 %: Xn; R20/21/22-33

1

1

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1

1

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6
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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

080-004-00-7

organic compounds of mercury
with the exception of those
specified elsewhere in this
Annex

A

—

—

T+; R26/27/28
R33
N; R50-53

T+; N
R: 26/27/28-33-50/53
S: (1/2-)13-28-36-45-60-
61

080-007-00-3

dimethylmercury
[1]
diethylmercury
[2]

209-805-3 [1]
211-000-7 [2]

593-74-8 [1]
627-44-1 [2]

T+; R26/27/28
R33
N; R50-53

T+; N
R: 26/27/28-33-50/53
S: (1/2-)13-28-36-45-60-
61

082-001-00-6

lead compounds with the
exception of those specified
elsewhere in this Annex

AE

—

—

Repr. Cat. 1; R61
Repr. Cat. 3; R62
Xn; R20/22
R33
N; R50-53

T; N
R: 61-20/22-33-62-50/
53
S: 53-45-60-61

082-002-00-1

lead alkyls

AE

—

—

Repr. Cat. 1; R61
Repr. Cat. 3; R62
T+; R26/27/28
R33
N; R50-53

T+; N
R: 61-26/27/28-33-62-
50/53
S: 53-45-60-61

1

1

1

1

C ≥ 25 %: T+, N; R26/27/28-33-50/53
2,5 % ≤ C < 25 %: T+, N; R26/27/28-33-
51/53
1 % ≤ C < 2,5 %: T+; R26/27/28-33-52/
53
0,5 % ≤ C < 1 %: T; R23/24/25-33-52/53
0,25 % ≤ C < 0,5 %: Xn; R20/21/22-33-
52/53
0,05 % ≤ C < 0,25 %: Xn; R20/21/22-33

C ≥ 25 %: T+, N; R26/27/28-33-50/53
2,5 % ≤ C < 25 %: T+, N; R26/27/28-33-
51/53
0,5 % ≤ C < 2,5 %: T+; R26/27/28-33-52/
53
0,25 % ≤ C < 0,5 %: T; R23/24/25-33-52/
53
0,1 % ≤ C < 0,25 %: T; R23/24/25-33
0,05 % ≤ C < 0,1 %: Xn; R20/21/22-33

C ≥ 25 %: T, N; R61-20/22-33-62-50/53
5 % ≤ C < 25 %: T, N; R61-20/22-33-62-
51/53
2,5 % ≤ C < 5 %: T, N; R61-20/22-33-62-
51/53
1 % ≤ C < 2,5 %: T; R61-20/22-33-52/53
0,5 % ≤ C < 1 %: T; R61-33-52/53
0,25 % ≤ C < 0,5 %: R52/53

C ≥ 25 %: T+, N; R61-26/27/28-33-62-
50/53
5 % ≤ C < 25 %: T+, N; R61-26/27/28-
33-62-51/53
2,5 % ≤ C < 5 %: T+, N; R61-26/27/28-
33-51/53
0,5 % ≤ C < 2,5 %: T+; R61-26/27/28-33-
52/53
0,25 % ≤ C < 0,5 %: T; R61-26/27/28-33-
52/53
0,1 % ≤ C < 0,25 %: T; R61-23/24/25-33
0,05 % ≤ C < 0,1 %: Xn; R20/21/22-33

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

601-010-00-3

ethylene

200-815-3

74-85-1

601-014-00-5

isoprene (stabilized)
2-methyl-1,3-butadiene

D

201-143-3

78-79-5

601-017-00-1

cyclohexane

203-806-2

110-82-7

601-020-00-8

benzene

E

200-753-7

71-43-2

601-021-00-3

toluene

203-625-9

108-88-3

601-025-00-5 mesitylene

1,3,5-trimethylbenzene

203-604-4

108-67-8

601-027-00-6

2-phenylpropene
α-methylstryene

202-705-0

98-83-9

601-028-00-1

2-methylstyrene
2-vinyltoluene

210-256-7

611-15-4

F+; R12
R67

F+; R12
Carc. Cat. 2; R45
Muta. Cat. 3; R68
R52-53

F; R11
Xn; R65
Xi; R38
R67
N; R50-53

F; R11
Carc. Cat. 1; R45
Muta. Cat. 2; R46
T; R48/23/24/25
Xn; R65
Xi; R36/38

F; R11
Repr.Cat.3; R63
Xn; R48/20-65
Xi; R38
R67

R10
Xi; R37
N; R51-53

R10
Xi; R36/37
N; R51-53

Xn; R20
N; R51-53

F+
R: 12-67
S: (2-)9-16-33-46

F+; T
R: 45-12-68-52/53
S: 53-45-61

F; Xn; N
R: 11-38-65-67-50/53
S: (2-)9-16-25-33-60-61-
62

F; T
R: 45-46-11-36/38-48/
23/24/25-65
S: 53-45

F; Xn
R: 11-38-48/20-63-65-
67
S: (2-)36/37-62-46

Xi; N
R: 10-37-51/53
S: (2-)61

Xi; N
R: 10-36/37-51/53
S: (2-)61

Xn; N
R: 20-51/53
S: (2-)24-61

C ≥ 25 %: Xi, N; R37-51/53
2,5 % ≤ C < 25 %: R52/53

C ≥ 25 %: Xi, N; R36/37-51/53
2,5 % ≤ C < 25 %: R52/53

C ≥ 25 %: Xn, N; R20-51/53
2,5 % ≤ C < 25 %: R52/53

4 6

4, 6

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
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e
a
n
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i

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6
/
2
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

601-032-00-3

benzo[a]pyrene
benzo[def]chrysene

200-028-5

50-32-8

601-037-00-0

n-hexane

203-777-6

110-54-3

601-041-00-2

dibenz[a,h]anthracene

200-181-8

53-70-3

Carc. Cat. 2; R45
Muta. Cat. 2; R46
Repr. Cat. 2; R60-61
R43
N; R50-53

T; N
R: 45-46-60-61-43-50/
53
S: 53-45-60-61

C ≥ 25 %: T, N; R43-45-46-50-53-60-61
2,5 % ≤ C < 25 %: T, N; R43-45-46-51-
53-60-61
1 % ≤ C < 2,5 %: T; R43-45-46-52-53-
60-61
0,5 % ≤ C < 1 %: T; R45-46-52-53-60-61
0,25 % ≤ C < 0,5 %: T; R45-46-52-53
0,1 % ≤ C < 0,25 %: T; R45-46
0,01 % ≤ C < 0,1 %: T; R45

F; R11
Repr. Cat. 3; R62
Xn; R65-48/20
Xi; R38
R67
N; R51-53

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

F; Xn; N
R: 11-38-48/20-62-65-
67-51/53
S: (2-)9-16-29-33-36/37-
61-62

C ≥ 25 %: Xn, N; R38-48/20-62-51/53
20 % ≤ C < 25 %: Xn; R38-48/20-62-52/
53
5 % ≤ C < 20 %: Xn; R48/20-62-52/53
2,5 % ≤ C < 5 %: R52/53

4 6

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

C ≥ 25 %: T, N; R45-50/53
2,5 % ≤ C < 25 %: T, N; R45-51/53
0,25 % ≤ C < 2,5 %: T; R45-52/53
0,01 % ≤ C < 0,25 %: T; R45

601-048-00-0

chrysene

205-923-4

218-01-9

Carc. Cat. 2; R45
Muta. Cat. 3; R68
N; R50-53

T; N
R: 45-68-50/53
S: 53-45-60-61

601-052-00-2

naphthalene

202-049-5

91-20-3

601-053-00-8

nonylphenol
[1]
4-nonylphenol, branched
[2]

246-672-0 [1]
284-325-5 [2]

25154-52-3 [1]
84852-15-3
[2]

602-003-00-8

dibromomethane

200-824-2

74-95-3

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

Repr.Cat.3; R62
Repr.Cat.3; R63
Xn; R22
C; R34
N; R50-53

Xn; R20
R52-53

Xn; N
R: 22-40-50/53
S: (2-)36/37-46-60-61

C; N
R: 22-34-62-63-50/53
S: (1/2-)26-36/37/39-45-
46-60-61

Xn
R: 20-52/53
S: (2-)24-61

C ≥ 25 %: Xn; R20-52/53
12,5 % ≤ C < 25 %: Xn; R20

602-008-00-5

carbon tetrachloride
tetrachloromethane

200-262-8

56-23-5

Carc. Cat. 3; R40
T; R23/24/25-48/23
R52-53
N; R59

T; N
R: 23/24/25-40-48/23-
59-52/53
S: (1/2-)23-36/37-45-59-
61

C ≥ 25 %: T, N; R23/24/25-40-48/23-52/
53-59
1 % ≤ C < 25 %: T, N; R23/24/25-40-48/
23-59
0,2 % ≤ C < 1 %: Xn, N; R20/21/22-48/
20-59
0,1 % ≤ C < 0,2 %: N; R59

L

2
1
6
/
2
8

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
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r
o
p
e
a
n
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

602-010-00-6

1,2-dibromoethane

E

203-444-5

106-93-4

602-011-00-1

1,1-dichloroethane

200-863-5

75-34-3

602-014-00-8

1,1,2-trichloroethane

201-166-9

79-00-5

Carc. Cat. 2; R45
T; R23/24/25
Xi; R36/37/38
N; R51-53

T; N
R: 45-23/24/25-36/37/
38-51/53
S: 53-45-61

C ≥ 25 %: T, N; R45-23/24/25-36/37/38-
51/53
20 % ≤ C < 25 %: T, N; R45-23/24/25-
36/37/38-52/53
2,5 % ≤ C < 20 %: T, N; R45-23/24/25-
52/53
1 % ≤ C < 2,5 %: T; R45-23/24/25
0,1 % ≤ C < 1 %: T; R45-20/21/22

F; R11
Xn; R22
Xi; R36/37
R52-53

F; Xn
R: 11-22-36/37-52/53
S: (2-)16-23-61

C ≥ 25 %: Xn; R22-36/37-52/53
20 % ≤ C < 25 %: Xn; R22-36/37
12,5 % ≤ C < 20 %: Xn; R22

Carc.Cat.3; R40
Xn; R20/21/22
R66

Xn
R: 20/21/22-40-66
S: (2-)9-36/37-46

C ≥ 5 %: Xn; R20/21/22

602-015-00-3

1,1,2,2-tetrachloroethane

201-197-8

79-34-5

T+; R26/27
N; R51-53

T+; N
R: 26/27-51/53
S: (1/2-)38-45-61

602-016-00-9

1,1,2,2-tetrabromoethane

201-191-5

79-27-6

T+; R26
Xi; R36
R52-53

T+
R: 26-36-52/53
S: (1/2-)24-27-45-61

C ≥ 25 %: T+, N; R26/27-51/53
7 % ≤ C < 25 %: T+; R26/27-52/53
2,5 % ≤ C < 7 %: T; R23/24-52/53
1 % ≤ C < 2,5 %: T; R23/24
0,1 % ≤ C < 1 %: Xn; R20/21

C ≥ 25 %: T+; R26-36-52/53
20 % ≤ C < 25 %: T+; R26-36
7 % ≤ C < 20 %: T+; R26
1 % ≤ C < 7 %: T; R23
0,1 % ≤ C < 1 %: Xn; R20

602-017-00-4

pentachloroethane

200-925-1

76-01-7

Carc. Cat. 3; R40
T; R48/23
N; R51-53

T; N
R: 40-48/23-51/53
S: (1/2-)23-36/37-45-61

C ≥ 25 %: T, N; R40-48/23-51/53
2,5 % ≤ C < 25 %: T; R40-48/23-52/53
1 % ≤ C < 2,5 %: T; R40-48/23
0,2 % ≤ C < 1 %: Xn; R48/20

602-019-00-5

1-bromopropane
n-propyl bromide

203-445-0

106-94-5

F; R11
Rep. Cat. 2; R60
Rep. Cat. 3; R63
Xn; R48/20
Xi; R36/37/38
R67

T; F
R: 60-11-36/37/38-48/
20-63-67
S: 53-45

.

.

1
6
6
2
0
0
4

E
N

O

f
f
i
c
i
a
l

J
o
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r
n
a
l

o
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e

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2
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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

602-025-00-8

1,1-dichloroethylene
vinylidene chloride

602-026-00-3

1,2-dichloroethylene
[1]
cis-dichloroethylene
[2]
trans-dichloroethylene
[3]

D

C

200-864-0

75-35-4

F; R12
Carc.Cat.3; R40
Xn; R20

F+; Xn
R: 12-20-40
S: (2-)7-16-29-36/37-46

C ≥ 12,5 %: Xn; R20-40
1 % ≤ C < 12,5 %: Xn; R40

208-750-2 [1]
205-859-7 [2]
205-860-2 [3]

540-59-0 [1]
156-59-2 [2]
156-60-5 [3]

F; R11
Xn; R20
R52-53

F; Xn
R: 11-20-52/53
S: (2-)7-16-29-61

C ≥ 25 %: Xn; R20-52/53
12,5 % ≤ C < 25 %: Xn; R20

602-029-00-X

3-chloropropene
allyl chloride

D

203-457-6

107-05-1

F; R11
Carc.Cat.3; R40
Muta.Cat.3; R68
Xn; R20/21/22-48/20
Xi; R36/37/38
N; R50

F; Xn; N
R: 11-20/21/22-36/37/
38-40-48/20-68-50
S: (2-)16-25-26-36/37-
46-61

602-033-00-1

chlorobenzene

203-628-5

108-90-7

R10
Xn; R20
N; R51-53

Xn; N
R: 10-20-51/53
S: (2-)24/25-61

C ≥ 25 %: Xn, N; R20-51/53
5 % ≤ C < 25 %: Xn, N; R20-52/53
2,5 % ≤ C < 5 %: R52/53

602-034-00-7

1,2-dichlorobenzene
o-dichlorobenzene

202-425-9

95-50-1

Xn; R22
Xi; R36/37/38
N; R50-53

Xn; N
R: 22-36/37/38-50/53
S: (2-)23-60-61

C ≥ 25 %: Xn, N; R22-36/37/38-50/53
20 % ≤ C < 25 %: Xn, N; R22-36/37/38-
51/53
5 % ≤ C < 20 %: Xn, N; R22-51/53
2,5 % ≤ C < 5 %: N; R51/53
0,25 % ≤ C < 2,5 %: R52/53

602-035-00-2

1,4-dichlorobenzene
p-dichlorobenzene

203-400-5

106-46-7

Xi; R36
Carc. Cat. 3; R40
N; R50-53

Xn; N
R: 36-40-50/53
S: (2-)36/37-46-60-61

602-036-00-8

chloroprene (stabilized)
2-chlorobuta-1,3-diene

D E

204-818-0

126-99-8

F; R11
Carc. Cat. 2; R45
Xn; R20/22-48/20
Xi; R36/37/38

F; T
R: 45-11-20/22-36/37/
38-48/20
S: 53-45

602-039-00-4

polychlorobiphenyls
PCB

C

215-648-1

1336-36-3

R33
N; R50-53

Xn; N
R: 33-50/53
S: (2-)35-60-61

C ≥ 25 %: Xn, N; R33-50/53
2,5 % ≤ C < 25 %: Xn, N; R33-51/53
0,25 % ≤ C < 2,5 %: Xn, N; R33-52/53
0,005 % ≤ C < 0,25 %: Xn; R33

L

2
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6
/
3
0

E
N

O

f
f
i
c
i
a
l

J
o
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r
n
a
l

o
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t
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e

E
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n

i

.

.

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6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

602-043-00-6

γ-HCH or γ-BHC
γ-1,2,3,4,5,6-hexachlorocyclo-
hexane
lindane

200-401-2

58-89-9

T; R25
Xn; R20/21-48/22
R64
N; R50-53

T; N
R: 20/21-25-48/22-64-
50/53
S: (1/2-)36/37-45-60-61

602-062-00-X

1,2,3-trichloropropane

602-073-00-X

1,4-dichlorobut-2-ene

D

E

202-486-1

96-18-4

Carc. Cat. 2; R45
Repr. Cat. 2; R60
Xn; R20/21/22

T
R: 45-60-20/21/22
S: 53-45

212-121-8

764-41-0

Carc. Cat. 2; R45
T+; R26
T; R24/25
C; R34
N; R50-53

T+; N
R: 45-24/25-26-34-50/
53
S: 53-45-60-61

C ≥ 25 %: T, N; R20/21-25-48/22-64-50-
53
10 % ≤ C < 25 %: Xn, N; R22-48/22-64-
50-53
3 % ≤ C < 10 %: Xn, N; R22-64-50-53
2,5 % ≤ C < 3 %: N; R64-50-53
1 % ≤ C < 2,5 %: N; R64-51-53
0,25 % ≤ C < 1 %: N; R51-53
0,025 % ≤ C < 0,25 %: R52-53

C ≥ 25 %: T+, N; R45-24/25-26-34-50/53
10 % ≤ C < 25 %: T+, N; R45-21/22-26-
34-51/53
7 % ≤ C < 10 %: T+, N; R45-21/22-26-
36/37/38-51/53
5 % ≤ C < 7 %: T, N; R45-21/22-23-36/
37/38-51/53
3 % ≤ C < 5 %: T, N; R45-21/22-23-51/
53
2,5 % ≤ C < 3 %: T, N; R45-23-51/53
1 % ≤ C < 2,5 %: T; R45-23-52/53
0,25 % ≤ C < 1 %: T; R45-20-52/53
0,1 % ≤ C < 0,25 %: T; R45-20
0,01 % ≤ C < 0,1 %: T; R45

603-006-00-7

pentanol isomers, with the
exception fo those specified
elsewhere in this Annex

C

250-378-8

30899-19-5

603-007-00-2

2-methylbutan-2-ol
tert-pentanol

200-908-9

75-85-4

603-029-00-2

bis(2-chloroethyl) ether

203-870-1

111-44-4

R10
Xn; R20
Xi; R37
R66

F; R11
Xn; R20
Xi; R37/38

R10
Carc.Cat.3; R40
T+; R26/27/28

603-030-00-8

2-aminoethanol
ethanolamine

205-483-3

141-43-5

Xn; R20/21/22
C; R34

Xn
R: 10-20-37-66
S: (2-)46

F; Xn
R: 11-20-37/38
S: (2-)46

T+
R: 10-26/27/28-40
S: (1/2-)7/9-27-28-36/
37-45

C ≥ 7 %: T+; R26/27/28-40
1 % ≤ C < 7 %: T; R23/24/25-40
0,1 % ≤ C < 1 %: Xn; R20/21/22

C
R: 20/21/22-34
S: (1/2-)26-36/37/39-45

C ≥ 25 %: C; R20/21/22-34
10 % ≤ C < 25 %: C; R34
5 % ≤ C < 10 %: Xi; R36/37/38

.

.

1
6
6
2
0
0
4

E
N

O

f
f
i
c
i
a
l

J
o
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r
n
a
l

o
f

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h
e

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/
3
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

603-031-00-3

1,2-dimethoxyethane
ethylene glycol dimethyl ether
EGDME

203-794-9

110-71-4

603-054-00-9

di-n-butyl ether
dibutyl ether

205-575-3

142-96-1

603-063-00-8

2,3-epoxypropan-1-ol
glycidol
oxiranemethanol

E

209-128-3

556-52-5

Repr.Cat.2; R60
Repr.Cat.2; R61
F; R11
R19
Xn; R20

R10
Xi; R36/37/38
R52-53

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Repr. Cat. 2; R60
T; R23
Xn; R21/22
Xi; R36/37/38

603-066-00-4

1,2-epoxy-4-epoxyethylcyclo-
hexane
vinylcyclohexane diepoxide

603-067-00-X

phenyl glycidyl ether
2,3-epoxypropyl phenyl ether
1,2-epoxy-3-phenoxypropane

203-437-7

106-87-6

T; R23/24/25
Xn; R68

E

204-557-2

122-60-1

603-070-00-6

2-amino-2-methylpropanol

204-709-8

124-68-5

603-074-00-8

reaction product: bisphenol-A-
(epichlorhydrin)
epoxy resin (number average
molecular weight ≤ 700)

500-033-5

25068-38-6

603-076-00-9

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

D

203-788-6

110-65-6

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Xn; R20
Xi; R37/38
R43
R52-53

Xi; R36/38
R52-53

Xi; R36/38
R43
N; R51-53

C; R34
T; R23/25
Xn; R21-48/22
R43

F; T
R: 60-61-11-19-20
S: 53-45

Xi
R: 10-36/37/38-52/53
S: (2-)61

T
R: 45-60-21/22-23-36/
37/38-68
S: 53-45

T
R: 23/24/25-68
S: (1/2-)23-24-45

T
R: 45-20-37/38-43-68-
52/53
S: 53-45-61

C ≥ 10 %: Xi; R36/37/38

C ≥ 1 %: T; R23/24/25-68
0,1 % ≤ C < 1 %: Xn; R20/21/22

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

Xi; N
R: 36/38-43-51/53
S: (2-)28-37/39-61

C ≥ 25 %: Xi; R36/38-52/53
10 % ≤ C < 25 %: Xi; R36/38

C ≥ 25 %: Xi, N; R36/38-43-51/53
5 % ≤ C < 25 %: Xi; R36/38-43-52/53
2,5 % ≤ C < 5 %: Xi; R43-52/53
1 % ≤ C < 2,5 %: Xi; R43

C; T
R: 21-23/25-34-43-48/
22
S: (1/2-)25-26-36/37/39-
45-46

C ≥ 50 %: T, C; R21-23/25-34-48/22-43
25 % ≤ C < 50 %: T; R21-23/25-36/38-
48/22-43
10 % ≤ C < 25 %: Xn; R20/22-48/22-43
3 % ≤ C < 10 %: Xn; R20/22-43
1 % ≤ C < 3 %: Xi; R43

L

2
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6
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3
2

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N

O

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f
i
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i
a
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J
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t
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e

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i

.

.

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6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

603-095-00-2

2-(propyloxy)ethanol
EGPE

220-548-6

2807-30-9

Xn; R21
Xi; R36

603-105-00-5

furan

E

203-727-3

110-00-9

604-001-00-2

phenol
carbolic acid
monohydroxybenzene
phenylalcohol

203-632-7

108-95-2

F+; R12
R19
Carc. Cat. 2; R45
Muta. Cat. 3; R68
Xn; R20/22-48/22
Xi; R38
R52-53

Muta.Cat.3; R68
T; R23/24/25
Xn; R48/20/21/22
C; R34

Xn
R: 21-36
S: (2-)26-36/37-46

F+; T
R: 45-12-19-20/22-38-
48/22-68-52/53
S: 53-45-61

T; C
R: 23/24/25-34-48/20/
21/22-68
S: (1/2-)24/25-26-28-36/
37/39-45

C ≥ 10 %: T; R23/24/25-48/20/21/22-34-
68
3 % ≤ C < 10 %: C ; Xn ; R20/21/22-34-
68
1 % ≤ C < 3 %: Xn ; R36/38-68

604-009-00-6

pyrogallol
1,2,3-trihydroxybenzene

201-762-9

87-66-1

Muta. Cat. 3; R68
Xn; R20/21/22
R52-53

Xn
R: 20/21/22-68-52/53
S: (2-)36/37-61

C ≥ 25 %: Xn; R20/21/22-68-52/53
10 % ≤ C < 25 %: Xn; R20/21/22-68
1 % ≤ C < 10 %: Xn; R68

604-010-00-1

resorcinol
1,3-benzenediol

203-585-2

108-46-3

604-012-00-2

4-chloro-o-cresol
4-chloro-2-methyl phenol

216-381-3

1570-64-5

604-013-00-8

2,3,4,6-tetrachlorophenol

200-402-8

58-90-2

Xn; R22
Xi; R36/38
N; R50

T; R23
C; R35
N; R50

T; R25
Xi; R36/38
N; R50-53

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

C ≥ 25 %: Xn, N; R22-36/38-50
20 % ≤ C < 25 %: Xn; R22-36/38
10 % ≤ C < 20 %: Xn; R22

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

C ≥ 25 %: T, C, N; R23-35-50
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

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

C ≥ 25 %: T, N; R25-36/38-50/53
20 % ≤ C < 25 %: T, N; R25-51/53
5 % ≤ C < 20 %: T, N; R25-36/38-51/53
2,5 % ≤ C < 5 %: Xn, N; R22-51/53
0,5 % ≤ C < 2,5 %: Xn; R22-52/53
0,25 % ≤ C < 0,5 %: R52/53

604-014-00-3

chlorocresol
4-chloro-m-cresol
4-chloro-3-methylphenol

200-431-6

59-50-7

Xn; R21/22
Xi; R41
R43
N; R50

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

C ≥ 25 %: Xn, N; R21/22-41-43-50
10 % ≤ C < 25 %: Xn; R21/22-41-43
5 % ≤ C < 10 %: Xn; R21/22-36-43
1 % ≤ C < 5 %: Xi; R43

.

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6
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4

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

604-015-00-9

2,2′-methylenebis-(3,4,6-tri-
chlorophenol)
hexachlorophene

200-733-8

70-30-4

T; R24/25
N; R50-53

T; N
R: 24/25-50/53
S: (1/2-)20-37-45-60-61

604-017-00-X

2,4,5-trichlorophenol

202-467-8

95-95-4

Xn; R22
Xi; R36/38
N; R50-53

Xn; N
R: 22-36/38-50/53
S: (2-)26-28-60-61

604-030-00-0

bisphenol A
4,4′-isopropylidenediphenol

201-245-8

80-05-7

Repr. Cat. 3; R62
Xi; R37-41
R43

Xn
R: 37-41-43-62
S: (2-)26-36/37-39-46

605-002-00-0

1,3,5-trioxan
trioxymethylene

605-016-00-7

glyoxal…%
ethandial…%

605-020-00-9

safrole
5-allyl-1,3-benzodioxole

605-022-00-X

glutaral
glutaraldehyde
1,5-pentanedial

B

E

203-812-5

110-88-3

203-474-9

107-22-2

202-345-4

94-59-7

203-856-5

111-30-8

F; R11
Repr.Cat.3; R63
Xi; R37

Muta. Cat. 3; R68
Xn; R20
Xi; R36/38
R43

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Xn; R22

T; R23/25
C; R34
R42/43
N; R50

F; Xn
R: 11-37-63
S: (2-)36/37-46

Xn
R: 20-36/38-43-68
S: (2-)36/37

T
R: 45-22-68
S: 53-45

T; N
R: 23/25-34-42/43-50
S: (1/2-)26-36/37/39-45-
61

605-025-00-6

chloroacetaldehyde

203-472-8

107-20-0

Carc. Cat. 3; R40
T+; R26
T; R24/25
C; R34
N; R50

T+; N
R: 24/25-26-34-40-50
S: (1/2-)26-28-36/37/39-
45-61

C ≥ 25 %: T, N; R24/25-50/53
2,5 % ≤ C < 25 %: T, N; R24/25-51/53
2 % ≤ C < 2,5 %: T; R24/25-52/53
0,25 % ≤ C < 2 %: Xn; R21/22-52/53
0,2 % ≤ C < 0,25 %: Xn; R21/22

C ≥ 25 %: Xn, N; R22-36/38-50/53
20 % ≤ C < 25 %: Xn, N; R22-36/38-51/
53
5 % ≤ C < 20 %: Xn, N; R36/38-51/53
2,5 % ≤ C < 5 %: N; R51/53
0,25 % ≤ C < 2,5 %: R52/53

C ≥ 10 %: Xn; R20-36/38-43-68
1 % ≤ C < 10 %: Xn; R43-68

C ≥ 50 %: T, N; R23/25-34-42/43-50
25 % ≤ C < 50 %: T; R22-23-34-42/43
10 % ≤ C < 25 %: C; R20/22-34-42/43
2 % ≤ C < 10 %: Xn; R20/22-37/38-41-
42/43
1 % ≤ C < 2 %: Xn; R36/37/38-42/43
0,5 % ≤ C < 1 %: Xi; R36/37/38-43

C ≥ 25 %: T+, N; R24/25-26-34-40-50
10 % ≤ C < 25 %: T+; R21/22-26-34-40
7 % ≤ C < 10 %: T+; R21/22-26-36/37/
38-40
5 % ≤ C < 7 %: T; R21/22-23-36/37/38-
40
3 % ≤ C < 5 %: T; R21/22-23-40
1 % ≤ C < 3 %: T; R23-40
0,1 % ≤ C < 1 %: Xn; R20

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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

606-037-00-4

triadimefon (ISO)
1-(4-chlorophenoxy)-3,3-
dimethyl-1-(1,2,4-triazol-1-yl)bu-
tanone

256-103-8

43121-43-3

Xn; R22
R43
N; R51-53

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

606-048-00-4

2′-anilino-3′-methyl-6′-dipenty-
laminospiro(isobenzofuran-
1(1H),9′-xanthen)-3-one

406-480-1

—

R53

R: 53
S: 61

607-004-00-7

trichloroacetic acid

200-927-2

76-03-9

607-019-00-9 methyl chloroformate

201-187-3

79-22-1

607-049-00-2 mecoprop (ISO) [1] and its salts
2-(4-chloro-o-tolyloxy) propionic
acid
(RS)-2-(4-chloro-o-tolyloxy)pro-
pionic acid
[1]
2-(4-chloro-2-methylphenoxy)-
propionic acid
[2]

230-386-8 [1]
202-264-4 [2]

7085-19-0 [1]
93-65-2 [2]

C; R35
N; R50-53

F; R11
T+; R26
Xn; R21/22
C; R34

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

C; N
R: 35-50/53
S: (1/2-)26-36/37/39-45-
60-61

C ≥ 25 %: C, N; R35-50/53
10 % ≤ C < 25 %: C, N; R35-51/53
5 % ≤ C < 10 %: C, N; R34-51/53
2,5 % ≤ C < 5 %: Xi, N; R36/37/38-51/53
1 % ≤ C < 2,5 %: Xi; R36/37/38-52/53
0,25 % ≤ C < 1 %: R52/53

F; T+
R: 11-21/22-26-34
S: (1/2-)26-14-28-36/37-
39-36/37/39-45-46-63

Xn; N
R: 22-38-41-50/53
S: (2-)13-26-37/39-60-
61

C ≥ 25 %: Xn, N; R22-38-41-50-53
20 % ≤ C < 25 %: Xi, N; R38-41-50-53
10 % ≤ C < 20 %: Xi, N; R41-50-53
5 % ≤ C < 10 %: Xi, N; R36-50-53
0,25 % ≤ C < 5 %: N; R50-53
0,025 % ≤ C < 0,25 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

607-053-00-4 MCPB (ISO)

202-365-3

94-81-5

N; R50-53

4-(4-chloro-o-tolyloxy) butyric
acid

N
R: 50/53
S: 60-61

607-061-00-8

acrylic acid
prop-2-enoic acid

D

201-177-9

79-10-7

R10
Xn; R20/21/22
C; R35
N; R50

C; N
R: 10-20/21/22-35-50
S: (1/2-)26-36/37/39-45-
61

C ≥ 25 %: C, N; R20/21/22-35-50
10 % ≤ C < 25 %: C; R35
5 % ≤ C < 10 %: C; R34
1 …% ≤ C < 5 %: Xi; R36/37/38

.

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-064-00-4

benzyl chloroformate

207-925-0

501-53-1

607-072-00-8

2-hydroxyethyl acrylate

D

212-454-9

818-61-1

607-086-00-4

diallyl phthalate

205-016-3

131-17-9

607-091-00-1

trifluoroacetic acid … %

B

200-929-3

76-05-1

607-094-00-8

peracetic acid … %

201-186-8

79-21-0

C; R34
N; R50-53

C; N
R: 34-50/53
S: (1/2-)26-45-60-61

C ≥ 25 %: C, N; R34-50/53
10 % ≤ C < 25 %: C, N; R34-51/53
5 % ≤ C < 10 %: Xi, N; R36/37/38-51/53
2,5 % ≤ C < 5 %: N; R51/53
0,25 % ≤ C < 2,5 %: R52/53

T; R24
C; R34
R43
N; R50

Xn; R22
N; R50-53

Xn; R20
C; R35
R52-53

R10
O; R7
Xn; R20/21/22
C; R35
N; R50

T; N
R: 24-34-43-50
S: (1/2-)26-36/39-45-61

C ≥ 25 %: T; R24-34-43-50
10 % ≤ C < 25 %: T; R24-34-43
5 % ≤ C < 10 %: T; R24-36/38-43
2 % ≤ C < 5 %: T; R24-43
0,2 % ≤ C < 2 %: Xn; R21-43

Xn; N
R: 22-50/53
S: (2-)24/25-60-61

C ≥ 25 %: Xn, N; R22-50/53
2,5 % ≤ C < 25 %: N; R51/53
0,25 % ≤ C < 2,5 %: R52/53

C
R: 20-35-52/53
S: (1/2-)9-26-27-28-45-
61

C ≥ 25 %: C; R20-35-52/53
10 % ≤ C < 25 %: C; R20-35
5 % ≤ C < 10 %: C; R34
1 % ≤ C < 5 %: Xi; R36/38

O; C; N
R: 7-10-20/21/22-35-50
S: (1/2-)3/7-14-36/37/
39-45-61

C ≥ 25 %: C, N; R20/21/22-35-50
10 % ≤ C < 25 %: C; R20/21/22-35
5 % ≤ C < 10 %: C; R34
1 % ≤ C < 5 %: Xi, R36/37/38

607-107-00-7

2-ethylhexyl acrylate

607-113-00-X

isobutyl methacrylate

607-116-00-6

cyclohexyl acrylate

D

D

D

203-080-7

103-11-7

Xi; R37/38
R43

Xi
R: 37/38-43
S: (2-)36/37-46

202-613-0

97-86-9

R10
Xi; R36/37/38
R43
N; R50

Xi; N
R: 10-36/37/38-43-50
S: (2-)24-37-61

C ≥ 25 %: Xi, N; R36/37/38-43-50
20 % ≤ C < 25 %: Xi; R36/37/38-43
1 % ≤ C < 20 %: Xi; R43

221-319-3

3066-71-5

Xi; R37/38
N; R51-53

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

C ≥ 25 %: Xi, N; R37/38-51/53
10 % ≤ C < 25 %: Xi; R37/38-52/53
2,5 % ≤ C < 10 %: R52/53

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-133-00-9 monoalkyl or monoaryl or

A

—

—

monoalkylaryl esters of acrylic
acid with the exception of those
specified elsewhere in this
Annex

Xi; R36/37/38
N; R51-53

Xi; N
R: 36/37/38-51/53
S: (2-)26-28-61

C ≥ 25 %: Xi, N; R36/37/38-51/53
10 % ≤ C < 25 %: Xi; R36/37/38-52/53
2,5 % ≤ C < 10 %: R52/53

607-151-00-7

propargite (ISO)
2-(4-tert-butylphenoxy) cyclo-
hexyl prop-2-ynyl sulphite

219-006-1

2312-35-8

Carc.Cat.3; R40
T; R23
Xi; R38-41
N; R50-53

T; N
R: 23-38-40-41-50/53
S: (1/2-)26-36/37/39-45-
60-61

C ≥ 25 %: T, N; R23-38-40-41-50-53
20 % ≤ C < 25 %: Xn, N; R20-38-40-41-
50-53
10 % ≤ C < 20 %: Xn, N; R20-40-41-50-
53
5 % ≤ C < 10 %: Xn, N; R20-40-36-50-53
3 % ≤ C < 5 %: Xn, N; R20-40-50-53
2,5 % ≤ C < 3 %: Xn, N; R40-50-53
1 % ≤ C < 2,5 %: Xn, N; R40-51-53
0,25 % ≤ C < 1 %: N; R51-53
0,025 % ≤ C < 0,25 %: R52-53

607-189-00-4

trimethylenediaminetetraacetic
acid

400-400-9

1939-36-2

Xn; R22
Xi; R41
N; R50-53

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

607-244-00-2

isooctyl acrylate

249-707-8

29590-42-9

607-245-00-8

tert-butyl acrylate

D

216-768-7

1663-39-4

607-247-00-9

dodecyl methacrylate

205-570-6

142-90-5

Xi; R36/37/38
N; R50-53

Xi; N
R: 36/37/38-50/53
S: (2-)26-28-60-61

C ≥ 25 %: Xi, N; R36/37/38-50/53
10 % ≤ C < 25 %: Xi, N; R36/37/38-51/
53
2,5 % ≤ C < 10 %: N; R51/53
0,25 % ≤ C < 2,5 %: R52/53

F; R11
Xn; R20/21/22
Xi; R37/38
R43
N; R52-53

Xi; 36/37/38
N; R50-53

F; Xn
R: 11-20/21/22-37/38-
43-52/53
S: (2-)16-25-37-61

C ≥ 25 %: Xn; R20/21/22-37/38-43-52-
53
20 % ≤ C < 25 %: Xi; R37/38-43
1 % ≤ C < 20 %: Xi; R43

Xi; N
R: 36/37/38-50/53
S: (2-)26-28-60-61

C ≥ 25 %: Xi, N; R36/37/38-50/53
10 % ≤ C < 25 %: Xi, N; R36/37/38-51/
53
2,5 % ≤ C < 10 %: N; R51/53
0,25 % ≤ C < 2,50 %: R52/53

.

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6
6
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4

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O

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J
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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-ethanediyl)] diacry-
late

256-032-2

42978-66-5

Xi; R36/37/38
R43
N; R51-53

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

608-003-00-4

acrylonitrile

D E

203-466-5

107-13-1

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

F; T; N
R: 45-11-23/24/25-37/
38-41-43-51/53
S: 9-16-53-45-61

608-006-00-0

bromoxynil (ISO) and its salts
3,5-dibromo-4-hydroxybenzoni-
trile
bromoxynil phenol

216-882-7

1689-84-5

Repr. Cat. 3; R63
T+; R26
T; R25
R43
N; R50-53

T+; N
R: 25-26-43-63-50/53
S: (1/2-)27/28-36/37-45-
63-60-61

608-007-00-6

ioxynil (ISO) and its salts
4-hydroxy-3,5-diiodobenzoni-
trile

216-881-1

1689-83-4

Repr. Cat. 3; R63
T; R23/25
Xn; R21-48/22
Xi; R36
N; R50-53

T; N
R: 21-23/25-36-48/22-
63-50/53
S: (1/2-)36/37-45-60-61-
63

C ≥ 25 %: Xi, N; R36/37/38-43-51/53
10 % ≤ C < 25 %: Xi; R36/37/38-43-52/
53
2,5 % ≤ C < 10 %: Xi; R43-52/53
1 % ≤ C < 2,5 %: Xi; R43

C ≥ 25 %: T, N; R45-23/24/25-37/38-41-
43-51/53
20 % ≤ C < 25 %: T; R45-23/24/25-37/
38-41-43-52/53
10 % ≤ C < 20 %: T; R45-23/24/25-41-
43-52/53
5 % ≤ C < 10 %: T; R45-23/24/25-36-43-
52/53
2,5 % ≤ C < 5 %: T; R45-23/24/25-43-52/
53
1 % ≤ C < 2,5 %: T; R45-23/24/25-43
0,2 % ≤ C < 1 %: T; R45-20/21/22
0,1 % ≤ C < 0,2 %: T; R45

C ≥ 25 %: T+, N; R25-26-43-63-50-53
7 % ≤ C < 25 %: T+, N; R22-26-43-63-
50-53
5 % ≤ C < 7 %: T, N; R22-23-43-63-50-
53
3 % ≤ C < 5 %: T, N; R22-23-43-50-53
2,5 % ≤ C < 3 %: T, N; R23-43-50-53
1 % ≤ C < 2,5 %: T, N; R23-43-51-53
0,25 % ≤ C < 1 %: Xn, N; R20-51-53
0,1 % ≤ C < 0,25 %: Xn; R20-52-53
0,025 % ≤ C < 0,1 %: R52-53

C ≥ 25 %: T, N; R21-23/25-36-48/22-63-
50-53
20 % ≤ C < 25 %: Xn, N; R20/22-36-48/
22-63-50-53
10 % ≤ C < 20 %: Xn, N; R20/22-48/22-
63-50-53
5 % ≤ C < 10 %: Xn, N; R20/22-63-50-53
3 % ≤ C < 5 %: Xn, N; R20/22-50-53
2,5 % ≤ C < 3 %: N; R50-53
0,25 % ≤ C < 2,5 %: N; R51-53
0,025 % ≤ C < 0,25 %: R52-53

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

608-010-00-2 methacrylonitrile

D

204-817-5

126-98-7

2-methyl-2-propene nitrile

F; R11
T; R23/24/25
R43

F; T
R: 11-23/24/25-43
S: (1/2-)9-16-18-29-45

C ≥ 1 %: T; R23/24/25-43
0,2 % ≤ C < 1 %: Xn; R20/21/22-43

608-014-00-4

chlorothalonil (ISO)
tetrachloroisophthalonitrile

217-588-1

1897-45-6

608-017-00-0

bromoxynil octanoate (ISO)
2,6-dibromo-4-cyanophenyl
octanoate

216-885-3

1689-99-2

Carc. Cat. 3; R40
T+; R26
Xi; R41
Xi; R37
R43
N; R50-53

T+; N
R: 26-37-40-41-43-50/
53
S: (2-)28-36/37/39-45-
60-61

C ≥ 20 %: T+, N; R26-37-40-41-43-50-53
10 % ≤ C < 20 %: T+, N; R26-40-41-43-
50-53
7 % ≤ C < 10 %: T+, N; R26-40-36-43-
50-53
5 % ≤ C < 7 %: T, N; R23-40-36-43-50-
53
2,5 % ≤ C < 5 %: T, N; R23-40-43-50-53
1 % ≤ C < 2,5 %: T, N; R23-40-43-51-53
0,25 % ≤ C < 1: Xn, N; R20-51-53
0,1 % ≤ C < 0,25 %: Xn; R20-52-53
0,025 % ≤ C < 0,1 %: R52-53

Repr. Cat. 3; R63
T; R23
Xn; R22
R43
N; R50-53

T; N
R: 22-23-43-63-50/53
S: (1/2-)36/37-45-63-60-
61

C ≥ 25 %: T, N; R22-23-43-63-50-53
5 % ≤ C < 25 %: Xn, N; R20-43-63-50-53
3 % ≤ C < 5 %: Xn, N; R20-43-50-53
2,5 % ≤ C < 3 %: Xi, N; R43-50-53
1 % ≤ C < 2,5 %: Xi, N; R43-51-53
0,25 % ≤ C < 1 %: N; R51-53
0,025 % ≤ C < 0,25 %: R52-53

608-018-00-6

ioxynil octanoate (ISO)
4-cyano-2,6-diiodophenyl
octanoate

223-375-4

3861-47-0

Repr. Cat. 3; R63
T; R25
Xi; R36
R43
N; R50-53

T; N
R: 25-36-43-63-50/53
S: (1/2-)26-36/37-45-60-
61

C ≥ 25 %: T, N; R25-36-43-63-50-53
20 % ≤ C < 25 %: Xn, N; R22-36-43-63-
50-53
5 % ≤ C < 20 %: Xn, N; R22-43-63-50-53
3 % ≤ C < 5 %: Xn, N; R22-43-50-53
2,5 % ≤ C < 3 %: N; R43-50-53
1 % ≤ C < 2,5 %: N; R43-51-53
0,25 % ≤ C < 1 %: N; R51-53
0,025 % ≤ C < 0,25 %: R52-53

608-021-00-2

3-(2-(diaminomethyleneamino)
thiazol-4-ylmethylthio)propioni-
trile

403-710-2

76823-93-3

Xn; R22
R43

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

.

.

1
6
6
2
0
0
4

E
N

O

f
f
i
c
i
a
l

J
o
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r
n
a
l

o
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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

609-007-00-9

2,4-dinitrotoluene
dinitrotoluene, technical grade
[1]
dinitrotoluene
[2]

609-023-00-6

dinocap (ISO)

609-043-00-5

quintozene (ISO)
pentachloronitrobenzene

609-049-00-8

2,6-dinitrotoluene

609-050-00-3

2,3-dinitrotoluene

609-051-00-9

3,4-dinitrotoluene

E

E

E

E

E

204-450-0 [1]
246-836-1 [2]

121-14-2 [1]
25321-14-6 [2]

254-408-0

39300-45-3

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Repr. Cat. 3; R62
T; R23/24/25
Xn; R48/22
N; R51-53

Repr. Cat. 2; R61
Xn; R20-48/22
Xi; R38
R43
N; R50-53

201-435-0

82-68-8

R43
N; R50-53

210-106-0

606-20-2

210-013-5

602-01-7

210-222-1

610-39-9

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Repr. Cat. 3; R62
T; R23/24/25
Xn; R48/22
R52-53

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Repr. Cat. 3; R62
T; R23/24/25
Xn; R48/22
N; R50-53

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Repr. Cat. 3; R62
T; R23/24/25
Xn; R48/22
N; R51-53

T; N
R: 45-23/24/25-48/22-
62-68-51/53
S: 53-45-61

T; N
R: 61-20-22-38-43-48/
22-50/53
S: 53-45-60-61

Xi; N
R: 43-50/53
S: (2-)13-24-37-60-61

T
R: 45-23/24/25-48/22-
62-68-52/53
S: 53-45-61

T; N
R: 45-23/24/25-48/22-
62-68-50/53
S: 53-45-60-61

T; N
R: 45-23/24/25-48/22-
62-68-51/53
S: 53-45-61

L

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4
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J
o
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a
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e

E
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i

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.

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4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

609-052-00-4

3,5-dinitrotoluene

609-055-00-0

2,5-dinitrotoluene

E

E

210-566-2

618-85-9

210-581-4

619-15-8

609-056-00-6

2,2-dibromo-2-nitroethanol

412-380-9

69094-18-4

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Repr. Cat. 3; R62
T; R23/24/25
Xn; R48/22
R52-53

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Repr. Cat. 3; R62
T; R23/24/25
Xn; R48/22
N; R51-53

E; R2
Carc. Cat. 3; R40
Xn; R22-48/22
C; R35
R43
N; R50-53

T
R: 45-23/24/25-48/22-
62-68-52/53
S: 53-45-61

T; N
R: 45-23/24/25-48/22-
62-68-51/53
S: 53-45-61

E; C; N
R: 2-22-35-40-43-48/
22-50/53
S: (1/2-)23-26-35-36/37/
39-45-60-61

C ≥ 25 %: C, N; R22-35-40-43-48/22-50/
53
10 % ≤ C < 25 %: C, N; R22-35-40-43-
48/22-51/53
5 % ≤ C < 10 %: C, N; R34-40-43-51/53
2,5 % ≤ C < 5 %: Xn, N; R36/37/38-40-
43-51/53
1 % ≤ C < 2,5 %: Xn; R36/37/38-40-43-
52/53
0,25 % ≤ C < 1 %: R52/53

610-005-00-5

1-chloro-4-nitrobenzene

202-809-6

100-00-5

611-001-00-6

azobenzene

E

203-102-5

103-33-3

Carc.Cat.3;R40
Mut.Cat.3;R68
T; R23/24/25
Xn; R48/20/21/22
N; R51-53

T; N
R: 23/24/25-40-48/20/
21/22-68-51/53
S: (1/2-)28-36/37-45-61

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Xn; R20/22-48/22
N; R50-53

T; N
R: 45-20/22-48/22-68-
50/53
S: 53-45-60-61

.

.

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6
2
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4

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O

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J
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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

413-180-4

—

Xi; R41

Xi
R: 41
S: (2-)22-26-39

611-060-00-8

A mixture of: sodium 5-[8-[4-
[4-[4-[7-(3,5-dicarboxylatophe-
nylazo)-8-hydroxy-3,6-disulfona-
tonaphthalen-1-ylamino]-6-
hydroxy-1,3,5-triazin-2-yl]-2,5-
dimethylpiperazin-1-yl]-6-
hydroxy-1,3,5-triazin-2-yla-
mino]-1-hydroxy-3,6-disulfona-
tonaphthalen-2-ylazo]-isophtha-
late; ammonium 5-[8-[4-[4-[4-
[7-(3,5-dicarboxylatophenylazo)-
8-hydroxy-3,6-disulfonato-
naphthalen-1-ylamino]-6-
hydroxy-1,3,5-triazin-2-yl]-2,5-
dimethylpiperazin-1-yl]-6-
hydroxy-1,3,5-triazin-2-yla-
mino]-1-hydroxy-3,6-disulfona-
tonaphthalen-2-ylazo]-isophtha-
late; 5-[8-[4-[4-[4-[7-(3,5-
dicarboxylatophenylazo)-8-
hydroxy-3,6-disulfonatonaphtha-
len-1-ylamino]-6-hydroxy-1,3,5-
triazin-2-yl]-2,5-dimethylpipera-
zin-1-yl]-6-hydroxy-1,3,5-tria-
zin-2-ylamino]-1-hydroxy-3,6-
disulfonaphthalen-2-ylazo]-
isophthalic acid

611-063-00-4

trisodium [4′-(8-acetylamino-
3,6-disulfonato-2-naphthylazo)-
4″-(6-benzoylamino-3-sulfonato-
2-naphthylazo)-biphenyl-
1,3′,3″,1‴-tetraolato-
O,O′,O″,O‴]copper(II)

413-590-3

164058-22-4

Carc. Cat. 2; R45

T
R: 45
S: 53-45

612-008-00-7

aniline

200-539-3

62-53-3

Carc. Cat. 3; R40
Muta.Cat.3; R68
T; R23/24/25-48/23/24/
25
Xi; R41
R43
N; R50

T; N
R: 23/24/25-40-41-43-
48/23/24/25-68-50
S: (1/2-)26-27-36/37/39-
45-46-61-63

C ≥ 25 %: T, N; R23/24/25-40-41-43-48/
23/24/25-50-68
10 % ≤ C < 25 %: T; R20/21/22-40-41-
43-48/23/24/25-68
1 % ≤ C < 10 %: T; R20/21/22-40-43-48/
23/24/25-68
0,2 % ≤ C < 1 %: Xn; R48/20/21/22

L

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J
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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

612-009-00-2

salts of aniline

A

—

—

Carc. Cat. 3; R40
Muta. Cat. 3; R68
T; R23/24/25
Xi; R41
R43
N; R50

T; N
R: 23/24/25-40-41-43-
48/23/24/25-68-50
S: (1/2-)26-27-36/37/39-
45-61-63

C ≥ 25 %: T, N; R23/24/25-40-41-43-48/
23/24/25-50-68
10 % ≤ C < 25 %: T; R20/21/22-40-41-
43-48/23/24/25-68
1 % ≤ C < 10 %: T; R20/21/22-40-43-48/
23/24/25-68
0,2 % ≤ C < 1 %: Xn; R48/20/21/22

612-010-00-8

chloroanilines (with exception of
those specified elsewhere in this
Annex)

C

—

—

T; R23/24/25
R33
N; R50-53

T; N
R: 23/24/25-33-50/53
S: (1/2-)28-36/37-45-60-
61

612-022-00-3

2-naphthylamine

612-023-00-9

phenylhydrazine
[1]
phenylhydrazinium chloride
[2]
phenylhydrazine hydrochloride
[3]
phenylhydrazinium sulphate
(2:1)
[4]

612-025-00-X

nitrotoluidines, with the
exception of those specified
elsewhere in this Annex

612-035-00-4

2-methoxyaniline
o-anisidine

612-042-00-2

benzidine
1,1′-biphenyl-4,4′-diamine
4,4′-diaminobiphenyl
biphenyl-4,4′-ylenediamine

E

E

C

E

E

202-080-4

91-59-8

Carc. Cat. 1; R45
Xn; R22
N; R51-53

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

C ≥ 25 %: T, N; R45-22-51/53
2,5 % ≤ C < 25 %: T; R45-52/53
0,01 % ≤ C < 2,5 %: T; R45

202-873-5 [1]
200-444-7 [2]
248-259-0 [3]
257-622-2 [4]

100-63-0 [1]
59-88-1 [2]
27140-08-5 [3]
52033-74-6 [4]

Carc. Cat. 2; R45
Muta. Cat. 3; R68
T; R23/24/25-48/23/24/
25
Xi; R36/3
R438
N; R50

T; N
R: 45-23/24/25-36/38-
43-48/23/24/25-68-50
S: 53-45-61

—

—

T; R23/24/25
R33
N; R51-53

T; N
R: 23/24/25-33-51/53
S: (1/2-)28-36/37-45-61

201-963-1

90-04-0

Carc. Cat. 2; R45
Muta Cat. 3; R68
T; R23/24/25

T
R: 45-23/24/25-68
S: 53-45

202-199-1

92-87-5

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

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

C ≥ 25 %: T, N; R45-22-50/53
2,5 % ≤ C < 25 %: T, N; R45-51/53
0,01 % ≤ C < 2,5 %: T; R45

.

.

1
6
6
2
0
0
4

E
N

O

f
f
i
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i
a
l

J
o
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r
n
a
l

o
f

t
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e

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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

612-051-00-1

4,4′-diaminodiphenylmethane
4,4′-methylenedianiline

E

202-974-4

101-77-9

612-054-00-8

N,N-diethylaniline

202-088-8

91-66-7

Carc. Cat. 2; R45
Muta. Cat. 3; R68
T; R39/23/24/25
Xn; R48/20/21/22
R43
N; R51-53

T; N
R: 45-39/23/24/25-43-
48/20/21/22-68-51/53
S: 53-45-61

T; R23/24/25
R33
N; R51-53

T; N
R: 23/24/25-33-51/53
S: (1/2-)28-37-45-61

C ≥ 25 %: T, N; R23/24/25-33-51/53
5 % ≤ C < 25 %: T; R23/24/25-33-52/53
2,5 % ≤ C < 5 %: Xn; R20/21/22-33-52/
53
1 % ≤ C < 2,5 %: Xn; R20/21/22-33

612-056-00-9

N,N-dimethyl-p-toluidine
[1]
N,N-dimethyl-m-toluidine
[2]
N,N-dimethyl-o-toluidine
[3]

C

202-805-4 [1]
204-495-6 [2]
210-199-8 [3]

99-97-8 [1]
121-72-2 [2]
609-72-3 [3]

T; R23/24/25
R33
R52-53

T
R: 23/24/25-33-52/53
S: (1/2-)28-36/37-45-61

C ≥ 25 %: T; R23/24/25-33-52-53
5 % ≤ C < 25 %: T; R23/24/25-33
1 % ≤ C < 5 %: Xn; R20/21/22-33

612-059-00-5

3,6-diazaoctanethylenediamin
triethylenetetramine

203-950-6

112-24-3

612-060-00-0

3,6,9-triazaundecamethylenedia-
mine
tetraethylenepentamine

203-986-2

112-57-2

612-064-00-2

3,6,9,12-tetra-azatetradeca-
methylenediamine
pentacthylenehexamine

223-775-9

4067-16-7

612-065-00-8

polyethlyenepolyamines with the
exception of those specified
elsewhere in this Annex

—

—

Xn; R21
C; R34
R43
R52-53

Xn; R21/22
C; R34
R43
N; R51-53

C; R34
R43
N; R50-53

Xn; R21/22
C; R34
R43
N; R50-53

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

C ≥ 25 %: C; R21-34-43-52/53
10 % ≤ C < 25 %: C; R34-43
5 % ≤ C < 10 %: Xi; R36/38-43
1 % ≤ C < 5 %: Xi; R43

C; N
R: 21/22-34-43-51/53
S: (1/2-)26-36/37/39-45-
61

C ≥ 25 %: C, N; R21/22-34-43-51/53
10 % ≤ C < 25 %: C; R34-43-52/53
5 % ≤ C < 10 %: Xi; R36/38-43-52/53
2,5 % ≤ C < 5 %: Xi; R43-52/53
1 % ≤ C < 2,5 %: Xi; R43

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

C ≥ 25 %: C, N; R34-43-50/53
10 % ≤ C < 25 %: C, N; R34-43-51/53
5 % ≤ C < 10 %: Xi, N; R36/38-43-51/53
2,5 % ≤ C < 5 %: Xi, N; R43-51/53
1 % ≤ C < 2,5 %: Xi; R43-52/53
0,25 % ≤ C < 1 %: R52/53

C; N
R: 21/22-34-43-50/53
S: (1/2-)26-36/37/39-45-
60-61

C ≥ 25 %: C, N; R21/22-34-43-50/53
10 % ≤ C < 25 %: C, N; R34-43-51/53
5 % ≤ C < 10 %: Xi, N; R36/38-43-51/53
1 % ≤ C < 2,5 %: Xi; R43-52/53
0,25 % ≤ C < 1 %: R52/53

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O

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f
i
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i
a
l

J
o
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a
l

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t
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e

E
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U
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i

.

.

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6
2
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4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

612-066-00-3

dicyclohexylamine

202-980-7

101-83-7

612-067-00-9

3-aminomethyl-3,5,5-trimethyl-
cyclohexylamine

220-666-8

2855-13-2

Xn; R22
C; R34
N; R50-53

Xn; R21/22
C; R34
R43
R52-53

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

C ≥ 25 %: C, N; R22-34-50/53
10 % ≤ C < 25 %: C, N; R34-51/53
2,5 % ≤ C < 10 %: Xi, N; R36/38-51/53
2 % ≤ C < 2,5 %: Xi; R36/38-52/53
0,25 % ≤ C < 2 %: R52/53

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

C ≥ 25 %: C; R21/22-34-43-52/53
10 % ≤ C < 25 %: C; R34-43
5 % ≤ C < 10 %: Xi; R36/38-43
1 % ≤ C < 5 %: Xi; R43

612-077-00-3

dimethylnitrosoamine
N-nitrosodimethylamine

E

200-549-8

62-75-9

Carc. Cat. 2; R45
T+; R26
T; R25-48/25
N; R51-53

T+; N
R: 45-25-26-48/25-51/
53
S: 53-45-61

612-086-00-2

amitraz (ISO)
N,N-bis(2,4-xylyliminomethyl)
methylamine

251-375-4

33089-61-1

Xn; R22-48/22
R43
N; R50-53

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

612-087-00-8

guazatine

236-855-3

13516-27-3

612-094-00-6

4-(2-chloro-4-trifluoromethyl)-
phenoxy-2-fluoroaniline hydro-
chloride

402-190-4

—

T+; R26
Xn; R21/22
Xi; R37/38-41
N; R50-53

T; R48/25
Xn; R22-48/20
Xi; R41
R43
N; R50-53

T+; N
R: 21/22-26-37/38-41-
50/53
S: (1/2-)26-28-36/37/39-
38-45-46-60-61-63

T; N
R: 22-41-43-48/20-48/
25-50/53
S: (1/2-)26-36/37/39-45-
60-61

C ≥ 25 %: T+, N; R45-25-26-48/25-51/53
10 % ≤ C < 25 %: T+; R45-22-26-48/25-
52/53
7 % ≤ C < 10 %: T+; R45-22-26-48/22-
52/53
3 % ≤ C < 7 %: T; R45-22-23-48/22-52/
53
2,5 % ≤ C < 3 %: T; R45-23-48/22-52/53
1 % ≤ C < 2,5 %: T; R45-23-48/22
0,1 % ≤ C < 1 %: T; R45-20
0,001 % ≤ C < 0,1 %: T; R45

C ≥ 25 %: Xn, N; R22-43-48/22-50-53
10 % ≤ C < 25 %: Xn, N; R43-48/22-50-
53
2,5 % ≤ C < 10 %: N; R43-50-53
1 % ≤ C < 2,5 %: N; R43-51-53
0,25 % ≤ C < 1 %: N; R51-53
0,025 % ≤ C < 0,25 %: R52-53

.

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6
6
2
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4

E
N

O

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a
l

J
o
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n
a
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4
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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

612-121-00-1

amines, polyethylenepoly-
HEPA

268-626-9

68131-73-7

612-136-00-3

N-isopropyl-N′-phenyl-p-pheny-
lenediamine

202-969-7

101-72-4

612-151-00-5

E

diaminotoluene, technical
product - mixture of [2] and [3]
methyl-phenylenediamine
[1]
4-methyl-m-phenylene diamine
[2]
2-methyl-m-phenylene diamine
[3]

246-910-3 [1]
202-453-1 [2]
212-513-9 [3]

25376-45-8 [1]
95-80-7 [2]
823-40-5 [3]

613-009-00-5

2,4,6-trichloro-1,3,5-triazine
cyanuric chloride

203-614-9

108-77-0

613-011-00-6

amitrole (ISO)
1,2,4-triazol-3-ylamine

200-521-5

61-82-5

613-033-00-6

2-methylaziridine
propyleneimine

E

200-878-7

75-55-8

Xn; R21/22
C; R34
R43
N; R50-53

Xn; R22
R43
N; R50-53

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

T+; R26
Xn; R22
C; R34
R43
R14

Repr.Cat.3; R63
Xn; R48/22
N; R51-53

F; R11
Carc. Cat. 2; R45
T+; R26/27/28
Xi; R41
N; R51-53

C; N
R: 21/22-34-43-50/53
S: (1/2-)26-36/37/39-45-
60-61

C ≥ 25 %: C, N; R21/22-34-43-50/53
10 % ≤ C < 25 %: C, N; R34-43-51/53
5 % ≤ C < 10 %: Xi, N; R36/38-43-51/53
2,5 % ≤ C < 5 %: Xi, N; R43-51/53
1 % ≤ C < 2,5 %: Xi; R43-52/53
0,25 % ≤ C < 1 %: R52/53

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

C ≥ 25 %: Xn, N; R22-43-50/53
2,5 % ≤ C < 25 %: Xi, N; R43-51/53
0,25 % ≤ C < 2,5 %: Xi; R43-52/53
0,1 % ≤ C < 0,25 %: Xi; R43

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

T+; C
R: 14-22-26-34-43
S: (1/2-)26-28-36/37/39-
45-46-63

C ≥ 25 %: T+; R22-26-34-43
10 % ≤ C < 25 %: T+; R26-34-43
7 % ≤ C < 10 %: T+; R26-36/37/38-43
5 % ≤ C < 7 %: T; R23-36/37/38-43
1 % ≤ C < 5 %: T; R23-43
0,1 % ≤ C < 1 %: Xn; R20

Xn; N
R: 48/22-63-51/53
S: (2-)13-36/37-61

F; T+; N
R: 45-11-26/27/28-41-
51/53
S: 53-45-61

C ≥ 25 %: T+, N; R45-26/27/28-41-51/53
10 % ≤ C < 25 %: T+; R45-26/27/28-41-
52/53
7 % ≤ C < 10 %: T+; R45-26/27/28-36-
52/53
5 % ≤ C < 7 %: T; R45-23/24/25-36-52/
53
2,5 % ≤ C < 5 %: T; R45-23/24/25-52/53
1 % ≤ C < 2,5 %: T; R45-23/24/25
0,1 % ≤ C < 1 %: T; R45-20/21/22
0,01 % ≤ C < 0,1 %: T; R45

613-040-00-4

azaconazole (ISO)
1-{[2-(2,4-dichlorophenyl)-1,3-
dioxolan-2-yl]methyl}-1H-1,2.4-
triazole

262-102-3

60207-31-0

Xn; R22

Xn
R: 22
S: (2-)46

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0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

613-043-00-0

imazalil sulphate (ISO) powder
1-[2-(allyloxy)ethyl-2-(2,4-
dichlorophenyl)]-1H-imidazo-
lium hydrogen sulphate
[1]
(±)-1-[2-(allyloxy)ethyl-2-(2,4-
dichlorophenyl)]-1H-imidazo-
lium hydrogen sulphate
[2]

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

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

Xn; R22
R43
N; R50-53

Xn; N
R: 22-43-50/53
S: (2-)24/25-37-46-60-
61

613-048-00-8

carbendazim (ISO)
methyl benzimidazol-2-
ylcarbamate

234-232-0

10605-21-7

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

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

613-049-00-3

benomyl (ISO)
methyl
1-(butylcarbamoyl)benzimidazol-
2-ylcarbamate

241-775-7

17804-35-2

Muta. Cat. 2; R46
Repr.Cat.2; R60-61
Xi; R37/38
R43
N; R50-53

T; N
R: 46-60-61-37/38-43-
50/53
S: 53-45-60-61

613-051-00-4 molinate (ISO)

218-661-0

2212-67-1

S-ethyl
1-perhydroazepinecarbothioate
S-ethyl perhydroazepine-1-
carbothioate

Carc.Cat3; R40
Repr.Cat3; R62
Xn; R20/22
Xn; R48/22
R43
N; R50-53

T; N
R: 20/22-40-43-48/22-
63-50/53
S: (2-)36/37-46-60-61

C ≥ 20 %: T, N; R46-60-61-37/38-43-50-
53
2,5 % ≤ C < 20 %: T, N; R46-60-61-43-
50-53
1 % ≤ C < 2,5 %: T, N; R46-60-61-43-51-
53
0,5 % ≤ C < 1 %: T, N; R46-60-61-51-53
0,25 % ≤ C < 0,5 %: T, N; R46-51-53
0,1 % ≤ C < 0,25 %: T; R46-52-53
0,025 % ≤ C < 0,1 %: R52-53

C ≥ 25 %: Xn, N; R20/22-40-43-48/22-
62-50-53
10 % ≤ C < 25 %: Xn, N; R40-43-48/22-
62-50-53
5 % ≤ C < 10 %: Xn, N; R40-43-62-50-53
1 % ≤ C < 5 %: Xn, N; R40-43-50-53
0,25 % ≤ C < 1 %: N; R50-53
0,025 % ≤ C < 0,25 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

613-058-00-2

permethrin (ISO)
m-phenoxybenzyl 3-(2,2-
dichlorovinyl)-2,2-dimethylcyclo-
propanecarboxylate

258-067-9

52645-53-1

Xn; R20/22
R43
N; R50-53

Xn; N
R: 20/22-43-50/53
S: (2-)13-24-36/37/39-
60-61

C ≥ 25 %: Xn, N; R20/22-43-50-53
1 % ≤ C < 25 %: N; R43-50-53
0,025 % ≤ C < 1 %: N; R50-53
0,0025 % ≤ C < 0,025 %: N; R51-53
0,00025 % ≤ C < 0,0025 %: R52-53

.

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6
6
2
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4

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O

f
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a
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J
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chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

613-075-00-5

1,3-dichloro-5-ethyl-5-methyli-
midazolidine-2,4-dione

401-570-7

89415-87-2

613-088-00-6

1,2-benzisothiazol-3(2H)-one
1,2-benzisothiazolin-3-one

220-120-9

2634-33-5

613-112-00-5

2-octyl-2H-isothiazol-3-one

247-761-7

26530-20-1

O; R8
T; R23
C; R34
Xn; R22
R43
N; R50

Xn; R22
Xi; R38-41
R43
N; R50

T; R23/24
Xn; R22
C; R34
R43
N; R50-53

O; T; N
R: 8-22-23-34-43-50
S: (1/2-)8-26-36/37/39-
45-61

Xn; N
R: 22-38-41-43-50
S: (2-)24-26-37/39-61

C ≥ 25 %: Xn, N; R22-38-41-43-50
20 % ≤ C < 25 %: Xi; R38-41-43
10 % ≤ C < 20 %: Xi; R41-43
5 % ≤ C < 10 %: Xi; R36-43
0,05 % ≤ C < 5 %: Xi; R43

T; N
R: 22-23/24-34-43-50/
53
S: (1/2-)26-36/37/39-45-
60-61

C ≥ 25 %: T, N; R22-23/24-34-43-50/53
10 % ≤ C < 25 %: C, N; R20/21-34-43-
51/53
5 % ≤ C < 10 %: Xn, N; R20/21-36/38-
43-51/53
3 % ≤ C < 5 %: Xn, N; R20/21-43-51/53
2,5 % ≤ C < 3 %: Xi, N; R43-51/53
0,25 % ≤ C < 2,5 %: Xi; R43-52/53
0,05 % ≤ C < 0,25 %: Xi; R43

613-124-00-0

fenpropimorph
cis-4-[3-(p-tert-butylphenyl)-2-
methylpropyl]-2,6-dimethylmor-
pholine

266-719-9

67564-91-4

Repr. Cat. 3; R63
Xn; R22
Xi; R38
N; R51-53

Xn; N
R: 22-38-63-51/53
S: (2-)36/37-46-61

613-129-00-8 metamitron

255-349-3

41394-05-2

613-167-00-5

4-amino-3-methyl-6-phenyl-
1,2,4-triazin-5-one

mixture of: 5-chloro-2-methyl-4-
isothiazolin-3-one [EC no. 247-
500-7]
and 2-methyl-2Hisothiazol-3-
one [EC no. 220-239-6] (3:1)
mixture of: 5-chloro-2-methyl-4-
isothiazolin-3-one [EC no. 247-
500-7]
and 2-methyl-4-isothiazolin-3-
one [EC no. 220-239-6] (3:1)

—

55965-84-9

Xn; R22
N; R50

T; R23/24/25
C; R34
R43
N; R50-53

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

T; N
R: 23/24/25-34-43-50/
53
S: (2-)26-28-36/37/39-
45-60-61

C ≥ 25 %: T, N; R23/24/25-34-43-50/53
3 % ≤ C < 25 %: C, N; R20/21/22-34-43-
51/53
2,5 % ≤ C < 3 %: C, N; R34-43-51/53
0,6 % ≤ C < 2,5 %: Xi; R34-43-52/53
0,25 % ≤ C < 0,6 %: Xi; R33/38-43-52/53
0,06 % ≤ C < 0,25 %: Xi; R36/38-43
0,0015 % ≤ C < 0,06 %: Xi; R43

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4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

613-175-00-9

Epoxiconazole
(2RS,3SR)-3-(2-chlorophenyl)-2-
(4-fluorophenyl)-[(1H-1,2,4-tria-
zol-1-yl)methyl]oxirane

406-850-2

133855-98-8

Carc. Cat. 3; R40
Repr. Cat. 3; R62
Repr. Cat. 3; R63
N; R51-53

Xn; N
R: 40-62-63-51/53
S: (2-)36/37-46-61

615-001-00-7 methyl isocyanate

210-866-3

624-83-9

615-004-00-3

salts of thiocyanic acid

A

—

—

F+; R12
Repr.Cat.3; R63
T+; R26
T; R24/25
R42/43
Xi; R37/38-41

F+; T+
R: 12-24/25-26-37/38-
41-42/43-63
S: (1/2-)26-27/28-36/37/
39-45-63

Xn; R20/21/22
R32
R52-53

Xn
R: 20/21/22-32-52/53
S: (2-)13-61

615-006-00-4

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

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

C ≥ 25 %: T+; R26-36/37/38-40-42/43-
52/53
20 % ≤ C < 25 %: 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

615-008-00-5

3-isocyanatomethyl-3,5,5-tri-
methylcyclohexyl isocyanate
isophorone di-isocyanate

223-861-6

4098-71-9

T; R23
Xi; R36/37/38
R42/43
N; R51-53

T; N
R: 23-36/37/38-42/43-
51/53
S: (1/2-)26-28-38-45-61

2

C ≥ 25 %: T, N; R23-36/37/38-42/43-51/
53
20 % ≤ C < 25 %: T; R23-36/37/38-42/
43-52/53
2,5 % ≤ C < 20 %: T; R23-42/43-52/53
2 % ≤ C < 2,5 %: T; R23-42/43
0,5 % ≤ C < 2 %: Xn; R20-42/43

615-015-00-3

1,7,7-trimethylbicyclo(2,2,1)-
hept-2-yl thiocyanatoacetate
isobornyl thiocyanoacetate

204-081-5

115-31-1

Xn; R22
N; R50-53

Xn; N
R: 22-50/53
S: (2-)24/25-60-61

.

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6
6
2
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4

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O

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J
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a
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4
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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

616-015-00-6

alachlor (ISO)
2-chloro-2′,6′-diethyl-N-(meth-
oxymethyl)acetanilide

240-110-8

15972-60-8

Carc. Cat. 3; R40
Xn; R22
R43
N; R50-53

Xn; N
R: 22-40-43-50/53
S: (2-)36/37-46-60-61

C ≥ 25 %: Xn, N; R22-40-43-50-53
1 % ≤ C < 25 %: Xn, N; R40-43-50-53
0,25 % ≤ C < 1 %: N; R50-53
0,025 % ≤ C < 0,25 %: N; R51-53
0,0025 % ≤ C < 0,025 %: R52-53

616-024-00-5

2-(4,4-dimethyl-2,5-dioxooxazo-
lidin-1-yl)-2-chloro-5-(2-(2,4-di-
tert-pentylphenoxy)butyramido)-
4,4-dimethyl-3-oxovaleranilide

402-260-4

—

R53

R: 53
S: 61

617-002-00-8

α,α-dimethylbenzyl
hydroperoxide
cumene hydroperoxide

201-254-7

80-15-9

O; R7
T; R23
Xn; R21/22-48/20/22
C; R34
N; R51-53

O; T; N
R: 7-21/22-23-34-48/
20/22-51/53
S: (1/2-)3/7-14-36/37/
39-45-50-61

C ≥ 25 %: T, N; R21/22-23-34-48/20/22-
51/53
10 % ≤ C < 25 %: C; R20-34-48/20/22-
52/53
3 % ≤ C < 10 %: Xn; R20-37/38-41-52/53
2,5 % ≤ C < 3 %: Xi; R36/37-52/53
1 % ≤ C < 2,5 %: Xi; R36/37

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

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

C ≥ 25 %: C, N; R22-34-50/53
10 % ≤ C < 25 %: C, N; R34-51/53
5 % ≤ C < 10 %: Xi, N; R36/37/38-51/53
2,5 % ≤ C < 5 %: N; R51/53
0,25 % ≤ C < 2,5 %: R52/53

L

2
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6
/
5
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
U
n
o
n

i

H

292-606-9

90640-85-0

Carc. Cat. 2; R45

T
R: 45
S: 53-45

648-043-00-X

Creosote oil, acenaphthene
fraction, acenaphthene-free
Wash Oil Redistillate
[The oil remaining after removal
by a crystallization process of
acenaphthene from
acenaphthene oil from coal tar.
Composed primarily of
naphthalene and
alkylnaphthalenes.]

.

.

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6
2
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Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

H

295-506-3

92061-93-3

Carc. Cat. 2; R45

H

292-605-3

90640-84-9

Carc. Cat. 2; R45

H

263-047-8

61789-28-4

Carc. Cat. 2; R45

T
R: 45
S: 53-45

T
R: 45
S: 53-45

T
R: 45
S: 53-45

648-080-00-1

Residues (coal tar), creosote oil
distn.
Wash Oil Redistillate
[The residue from the fractional
distillation of wash oil boiling in
the approximate range of 270 °C
to 330 °C (518 °F to 626 °F). It
consists predominantly of
dinuclear aromatic and
heterocyclic hydrocarbons.]

648-098-00-X

Creosote oil, acenaphthene
fraction
Wash Oil
[A complex combination of
hydrocarbons produced by the
distillation of coal tar and
boiling in the range of
approximately 240 °C to 280 °C
(464 °F to 536 °F). Composed
primarily of acenaphthene,
naphthalene and alkyl
naphthalene.]

648-099-00-5

Creosote oil
[A complex combination of
hydrocarbons obtained by the
distillation of coal tar. It consists
primarily of aromatic
hydrocarbons and may contain
appreciable quantities of tar
acids and tar bases. It distills at
the approximate range of 200 °C
to 325 °C (392 °F to 617 °F).]

E
N

O

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J
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a
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o
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E
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e
a
n
U
n
o
n

i

L

2
1
6
/
5
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H

274-565-9

70321-79-8

Carc. Cat. 2; R45

H

232-287-5

8001-58-9

Carc. Cat. 2; R45

H

310-189-4

122384-77-4

Carc. Cat. 2; R45

T
R: 45
S: 53-45

T
R: 45
S: 53-45

T
R: 45
S: 53-45

648-100-00-9

Creosote oil, high-boiling
distillate
Wash Oil
[The high-boiling distillation
fraction obtained from the high
temperature carbonization of
bituminous coal which is further
refined to remove excess
crystalline salts. It consists
primarily of creosote oil with
some of the normal polynuclear
aromatic salts, which are
components of coal tar
distillates, removed. It is crystal
free at approximately 5 °C
(41 °F).]

648-101-00-4

Creosote
[The distillate of coal tar
produced by the high
temperature carbonization of
bituminous coal. It consists
primarily of aromatic
hydrocarbons, tar acids and tar
bases.]

648-102-00-X

Extract residues (coal), creosote
oil acid
Wash Oil Extract Residue
[A complex combination of
hydrocarbons from the base-
freed fraction from the
distillation of coal tar, boiling in
the range of approximately
250 °C to 280 °C (482 °F to
536 °F). It consists
predominantly of biphenyl and
isomeric diphenylnaphthalenes.]

L

2
1
6
/
5
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H

274-566-4

70321-80-1

Carc. Cat. 2; R45

648-138-00-6

Creosote oil, low-boiling
distillate
Wash Oil
[The low-boiling distillation
fraction obtained from the high
temperature carbonization of
bituminous coal, which is
further refined to remove excess
crystalline salts. It consists
primarily of creosote oil with
some of the normal polynuclear
aromatic salts, which are
components of coal tar distillate,
removed. It is crystal free at
approximately 38 °C (100 °F).]

649-001-00-3

Extracts (petroleum), light
naphthenic distillate solvent

649-002-00-9

Extracts (petroleum), heavy
paraffinic distillate solvent

649-003-00-4

Extracts (petroleum), light
paraffinic distillate solvent

649-004-00-X

Extracts (petroleum), heavy
naphthenic distillate solvent

649-005-00-5

Extracts (petroleum), light
vacuum gas oil solvent

H

H

H

H

H

265-102-1

64742-03-6

Carc. Cat. 2; R45

265-103-7

64742-04-7

Carc. Cat. 2; R45

265-104-2

64742-05-8

Carc. Cat. 2; R45

265-111-0

64742-11-6

Carc. Cat. 2; R45

295-341-7

91995-78-7

Carc. Cat. 2; R45

649-006-00-0

hydrocarbons C26-55, arom-rich

H

307-753-7

97722-04-8

Carc. Cat. 2; R45

T
R: 45
S: 53-45

T
R: 45
S: 53-45

T
R: 45
S: 53-45

T
R: 45
S: 53-45

T
R: 45
S: 53-45

T
R: 45
S: 53-45

T
R: 45
S: 53-45

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
5
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

649-062-00-6 Gases (petroleum), catalytic

H K

270-755-0

68477-73-6

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

cracked naphtha depropanizer
overhead, C3-rich acid-free
Petroleum gas
[A complex combination of
hydrocarbons obtained from
fractionation of catalytic cracked
hydrocarbons and treated to
remove acidic impurities. It
consists of hydrocarbons having
carbon numbers in the range of
C2 through C4, predominantly
C3.]

649-063-00-1 Gases (petroleum), catalytic
cracker
Petroleum gas
[A complex combination of
hydrocarbons produced by the
distillation of the products from
a catalytic cracking process. It
consists predominantly of
aliphatic hydrocarbons having
carbon numbers predominantly
in the range of C1 through C6.]

H K

270-756-6

68477-74-7

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-064-00-7 Gases (petroleum), catalytic

H K

270-757-1

68477-75-8

cracker, C1-5-rich
Petroleum gas
[A complex combination of
hydrocarbons produced by the
distillation of products from a
catalytic cracking process. It
consists of aliphatic
hydrocarbons having carbon
numbers in the range of C1
through C6, predominantly C1
through C5.]

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

L

2
1
6
/
5
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

270-758-7

68477-76-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-065-00-2 Gases (petroleum), catalytic
polymd. naphtha stabilizer
overhead, C2-4-rich
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
fractionation stabilization of
catalytic polymerized naphtha. It
consists of aliphatic
hydrocarbons having carbon
numbers in the range of C2
through C6, predominantly C2
through C4.]

649-066-00-8 Gases (petroleum), catalytic

H K

270-760-8

68477-79-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

reformer, C1-4-rich
Petroleum gas
[A complex combination of
hydrocarbons produced by
distillation of products from a
catalytic reforming process. It
consists of hydrocarbons having
carbon numbers in the range of
C1 through C6, predominantly C1
through C4.]

Gases (petroleum), C3-5 olefinic-
paraffinic alkylation feed
Petroleum gas
[A complex combination of
olefinic and paraffinic
hydrocarbons having carbon
numbers in the range of C3
through C5 which are used as
alkylation feed. Ambient
temperatures normally exceed
the critical temperature of these
combinations.]

649-067-00-3

H K

270-765-5

68477-83-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
5
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

649-068-00-9 Gases (petroleum), C4-rich

H K

270-767-6

68477-85-0

Petroleum gas
[A complex combination of
hydrocarbons produced by
distillation of products from a
catalytic fractionation process. It
consists of aliphatic
hydrocarbons having carbon
numbers in the range of C3
through C5, predominantly C4.]

649-069-00-4 Gases (petroleum), deethanizer

H K

270-768-1

68477-86-1

overheads
Petroleum gas
[A complex combination of
hydrocarbons produced from
distillation of the gas and
gasoline fractions from the
catalytic cracking process. It
contains predominantly ethane
and ethylene.]

649-070-00-X Gases (petroleum),

H K

270-769-7

68477-87-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-772-3

68477-90-7

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

deisobutanizer tower overheads
Petroleum gas
[A complex combination of
hydrocarbons produced by the
atmospheric distillation of a
butane-butylene stream. It
consists of aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C3 through C4.]

Gases (petroleum), depropanizer
dry, propene-rich
Petroleum gas
[A complex combination of
hydrocarbons produced by the
distillation of products from the
gas and gasoline fractions of a
catalytic cracking process. It
consists predominantly of
propylene with some ethane and
propane.]

649-071-00-5

L

2
1
6
/
5
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

H K

270-773-9

68477-91-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-777-0

68477-94-1

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-778-6

68477-95-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-072-00-0

649-073-00-6

649-074-00-1

Gases (petroleum), depropanizer
overheads
Petroleum gas
[A complex combination of
hydrocarbons produced by
distillation of products from the
gas and gasoline fractions of a
catalytic cracking process. It
consists of aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C2 through C4.]

Gases (petroleum), gas recovery
plant depropanizer overheads
Petroleum gas
[A complex combination of
hydrocarbons obtained by
fractionation of miscellaneous
hydrocarbon streams. It consists
predominantly of hydrocarbons
having carbon numbers in the
range of C1 through C4,
predominantly propane.]

Gases (petroleum), Girbatol unit
feed
Petroleum gas
[A complex combination of
hydrocarbons that is used as the
feed into the Girbatol unit to
remove hydrogen sulfide. It
consists of aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C2 through C4.]

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
U
n
o
n

i

L

2
1
6
/
5
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

649-075-00-7

Gases (petroleum), isomerized
naphtha fractionator, C4-rich,
hydrogen sulfide-free
Petroleum gas

H K

270-782-8

68477-99-6

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-802-5

68478-21-7

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-803-0

68478-22-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-076-00-2

Tail gas (petroleum), catalytic
cracked clarified oil and thermal
cracked vacuum residue
fractionation reflux drum
Petroleum gas
[A complex combination of
hydrocarbons obtained from
fractionation of catalytic cracked
clarified oil and thermal cracked
vacuum residue. It consists
predominantly of hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C6.]

649-077-00-8

Tail gas (petroleum), catalytic
cracked naphtha stabilization
absorber
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
stabilization of catalytic cracked
naphtha. It consists
predominantly of hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C6.]

L

2
1
6
/
5
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

270-804-6

68478-24-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-806-7

68478-26-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-078-00-3

Tail gas (petroleum), catalytic
cracker, catalytic reformer and
hydrodesulfurizer combined
fractionater
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
fractionation of products from
catalytic cracking, catalytic
reforming and
hydrodesulfurizing processes
treated to remove acidic
impurities. It consists
predominantly of hydrocarbons
having cabon numbers
predominantly in the range of
C1 through C5.]

649-079-00-9

Tail gas (petroleum), catalytic
reformed naphtha fractionation
stabilizer
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
fractionation stabilization of
catalytic reformed naphtha. It
consists predominantly of
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C4.]

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
5
9

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

270-813-5

68478-32-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-814-0

68478-33-1

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-815-6

68478-34-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-080-00-4

649-081-00-X

649-082-00-5

Tail gas (petroleum), saturate gas
plant mixed stream, C4-rich
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
fractionation stabilization of
straight-run naphtha, distillation
tail gas and catalytic reformed
naphtha stabilizer tail gas. It
consists of hydrocarbons having
carbon numbers in the range of
C3 through C6, predominantly
butane and isobutane.]

Tail gas (petroleum), saturate gas
recovery plant, C1-2-rich
Petroleum gas
[A complex combination of
hydrocarbons obtained from
fractionation of distillate tail gas,
straight-run naphtha, catalytic
reformed naphtha stabilizer tail
gas. It consists predominantly of
hydrocarbons having carbon
numbers in the range of C1
through C5, predominantly
methane and ethane.]

Tail gas (petroleum), vacuum
residues thermal cracker
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
thermal cracking of vacuum
residues. It consists of
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C5.]

L

2
1
6
/
6
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

649-083-00-0 Hydrocarbons, C3-4-rich,

H K

270-990-9

68512-91-4

petroleum distillate
Petroleum gas
[A complex combination of
hydrocarbons produced by
distillation and condensation of
crude oil. It consists of
hydrocarbons having carbon
numbers in the range of C3
through C5, predominantly C3
through C4.]

649-084-00-6 Gases (petroleum), full-range

H K

271-000-8

68513-15-5

straight-run naphtha
dehexanizer off
petroleum gas
[A complex combination of
hydrocarbons obtained by the
fractionation of the full-range
straight-run naphtha. It consists
of hydrocarbons having carbon
numbers predominantly in the
range of C2 through C6.]

649-085-00-1 Gases (petroleum),

H K

271-001-3

68513-16-6

hydrocracking depropanizer off,
hydrocarbon-rich
Petroleum gas
[A complex combination of
hydrocarbon produced by the
distillation of products from a
hydrocracking process. It
consists predominantly of
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C4. It may
also contain small amounts of
hydrogen and hydrogen sulfide.]

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

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
U
n
o
n

i

L

2
1
6
/
6
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

649-086-00-7 Gases (petroleum), light straight-

H K

271-002-9

68513-17-7

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

run naphtha stabilizer off
Petroleum gas
[A complex combination of
hydrocarbons obtained by the
stabilization of light straight-run
naphtha. It consists of saturated
aliphatic hydrocarbons having
carbon numbers predominantly
in the range of C2 through C6.]

Residues (petroleum), alkylation
splitter, C4-rich
Petroleum gas
[A complex residuum from the
distillation of streams various
refinery operations. It consists of
hydrocarbons having carbon
numbers in the range of C4
through C5, predominantly
butane and boiling in the range
of approximately -11.7 °C to
27.8 °C (11 °F to 82 °F).]

649-087-00-2

H K

271-010-2

68513-66-6

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-088-00-8 Hydrocarbons, C1-4

H K

271-032-2

68514-31-8

Petroleum gas
[A complex combination of
hydrocarbons provided by
thermal cracking and absorber
operations and by distillation of
crude oil. It consists of
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C4 and
boiling in the range of
approximately minus 164 °C to
minus 0.5 °C (-263 °F to 31 °F).]

Carc. Cat 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

L

2
1
6
/
6
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

271-038-5

68514-36-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-089-00-3 Hydrocarbons, C1-4, sweetened
Petroleum gas
[A complex combination of
hydrocarbons obtained by
subjecting hydrocarbon gases to
a sweetening process to convert
mercaptans or to remove acidic
impurities. It consists of
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C4 and
boiling in the range of
approximately -164 °C to -0.5 °C
(-263 °F to 31 °F).]

649-090-00-9 Hydrocarbons, C1-3

H K

271-259-7

68527-16-2

Petroleum gas
[A complex combination of
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C3 and
boiling in the range of
approximately minus 164 °C to
minus 42 °C (-263 °F to -44 °F).]

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-091-00-4

Hydrocarbons, C1-4, debutanizer
fraction
Petroleum gas

H K

271-261-8

68527-19-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

649-092-00-X Gases (petroleum), C1-5, wet

H K

271-624-0

68602-83-5

Petroleum gas
[A complex combination of
hydrocarbons produced by the
distillation of crude oil and/or
the cracking of tower gas oil. It
consists of hydrocarbons having
carbon numbers predominantly
in the range of C1 through C5.]

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

T
R: 45-46
S: 53-45

649-093-00-5 Hydrocarbons, C2-4

H K

271-734-9

68606-25-7

Petroleum gas

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
6
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

649-094-00-0 Hydrocarbons, C3

H K

271-735-4

68606-26-8

Petroleum gas

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-095-00-6 Gases (petroleum), alkylation

H K

271-737-5

68606-27-9

feed
Petroleum gas
[A complex combination of
hydrocarbons produced by the
catalytic cracking of gas oil. It
consists of hydrocarbons having
carbon numbers predominantly
in the range of C3 through C4.]

649-096-00-1 Gases (petroleum), depropanizer

H K

271-742-2

68606-34-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-183-7

68783-07-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

bottoms fractionation off
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
fractionation of depropanizer
bottoms. It consists
predominantly of butane,
isobutane and butadiene.]

Gases (petroleum), refinery
blend
Petroleum gas
[A complex combination
obtained from various processes.
It consists of hydrogen,
hydrogen sulfide and
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C5.]

649-097-00-7

L

2
1
6
/
6
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

272-203-4

68783-64-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-098-00-2 Gases (petroleum), catalytic
cracking
Petroleum gas
[A complex combination of
hydrocarbons produced by the
distillation of the products from
a catalytic cracking process. It
consists predominantly of
hydrocarbons having carbon
numbers predominantly in the
range of C3 through C5.]

649-099-00-8 Gases (petroleum), C2-4,

H K

272-205-5

68783-65-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

sweetened
Petroleum gas
[A complex combination of
hydrocarbons obtained by
subjecting a petroleum distillate
to a sweetening process to
convert mercaptans or to
remove acidic impurities. It
consists predominantly of
saturated and unsaturated
hydrocarbons having carbon
numbers predominantly in the
range of C2 through C4 and
boiling in the range of
approximately -51 °C to -34 °C
(-60 °F to -30 °F).]

Gases (petroleum), crude oil
fractionation off
Petroleum gas
[A complex combination of
hydrocarbons produced by the
fractionation of crude oil. It
consists of saturated aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C5.]

649-100-00-1

H K

272-871-7

68918-99-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
6
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

272-872-2

68919-00-6

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-878-5

68919-05-1

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-879-0

68919-06-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-101-00-7

649-102-00-2

649-103-00-8

Gases (petroleum), dehexanizer
off
Petroleum gas
[A complex combination of
hydrocarbons obtained by the
fractionation of combined
naphtha streams. It consists of
saturated aliphatic hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C5.]

Gases (petroleum), light straight
run gasoline fractionation
stabilizer off
Petroleum gas
[A complex combination of
hydrocarbons obtained by the
fractionation of light straight-
run gasoline. It consists of
saturated aliphatic hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C5.]

Gases (petroleum), naphtha
unifiner desulfurization stripper
off
Petroleum gas
[A complex combination of
hydrocarbons produced by a
naphtha unifiner desulfurization
process and stripped from the
naphtha product. It consists of
saturated aliphatic hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C4.]

L

2
1
6
/
6
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

H K

272-882-7

68919-09-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-893-7

68919-20-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-883-2

68919-10-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45
S: 53-45

649-104-00-3

Gases (petroleum), straight-run
naphtha catalytic reforming off
Petroleum gas
[A complex combination of
hydrocarbons obtained by the
catalytic reforming of straight-
run naphtha and fractionation of
the total effluent. It consists of
methane, ethane, and propane.]

649-105-00-9

Gases (petroleum), fluidized
catalytic cracker splitter
overheads
Petroleum gas
[A complex combination of
hydrocarbons produced by the
fractionation of the charge to
the C3-C4 splitter. It consists
predominantly of C3
hydrocarbons.]

649-106-00-4

Gases (petroleum), straight-run
stabilizer off
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
fractionation of the liquid from
the first tower used in the
distillation of crude oil. It
consists of saturated aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C4.]

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
U
n
o
n

i

L

2
1
6
/
6
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

273-169-3

68952-76-1

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

273-170-9

68952-77-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

273-175-6

68952-81-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-107-00-X

Gases (petroleum), catalytic
cracked naphtha debutanizer
Petroleum gas
[A complex combination of
hydrocarbons obtained from
fractionation of catalytic cracked
naphtha. It consists of
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C4.]

649-108-00-5

649-109-00-0

Tail gas (petroleum), catalytic
cracked distillate and naphtha
stabilizer
Petroleum gas
[A complex combination of
hydrocarbons obtained by the
fractionation of catalytic cracked
naphtha and distillate. It consists
predominantly of hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C4.]

Tail gas (petroleum), thermal-
cracked distillate, gas oil and
naphtha absorber
petroleum gas
[A complex combination of
hydrocarbons obtained from the
separation of thermal-cracked
distillates, naphtha and gas oil. It
consists pedrominantly of
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C6.]

L

2
1
6
/
6
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

273-176-1

68952-82-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

273-265-5

68955-28-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

273-270-2

68955-34-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-110-00-6

649-111-00-1

649-112-00-7

Tail gas (petroleum), thermal
cracked hydrocarbon
fractionation stabilizer,
petroleum coking
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
fractionation stabilization of
thermal cracked hydrocarbons
from petroleum coking process.
It consists of hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C6.]

Gases (petroleum, light steam-
cracked, butadiene conc.
Petroleum gas
[A complex combination of
hydrocarbons produced by the
distillation of products from a
thermal cracking process, It
consists of hydrocarbons having
a carbon number predominantly
of C4.]

Gases (petroleum), straight-run
naphtha catalytic reformer
stabilizer overhead
Petroleum gas
[A complex combination of
hydrocarbons obtained by the
catalytic reforming of straight-
run naphtha and the
fractionation of the total
effluent. It consists of saturated
aliphatic hydrocarbons having
carbon numbers predominantly
in the range of C2 through C4.]

649-113-00-2

Hydrocarbons, C4
Petroleum gas

H K

289-339-5

87741-01-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
6
9

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

649-114-00-8

Alkanes, C1-4, C3-rich
Petroleum gas

H K

292-456-4

90622-55-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

H K

295-404-9

92045-22-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

T
R: 45-46
S: 53-45

649-115-00-3

649-116-00-9

649-117-00-4

Gases (petroleum), steam-cracker
C3-rich
Petroleum gas
[A complex combination of
hydrocarbons produced by the
distillation of products from a
steam cracking process. It
consists predominantly of
propylene with some propane
and boils in the range of
approximately -70 °C to 0 °C
(-94 °F to 32 °F).]

Hydrocarbons, C4, steam-cracker
distillate
Petroleum gas
[A complex combination of
hydrocarbons produced by the
distillation of the products of a
steam cracking process. It
consists predominantly of
hydrocarbons having a carbon
number of C4, predominantly 1-
butene and 2-butene, containing
also butane and isobutene and
boiling in the range of
approximately minus 12 °C to
5 °C (10.4 °F to 41 °F).]

Petroleum gases, liquefied,
sweetened, C4 fraction
Petroleum gas
[A complex combination of
hydrocarbons obtained by
subjecting a liquified petroleum
gas mix to a sweetening process
to oxidize mercaptans or to
remove acidic impurities. It
consists predominantly of C4
saturated and unsaturated
hydrocarbons.]

H K

295-405-4

92045-23-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

HKS

295-463-0

92045-80-2

F+; R12
Carc. Cat. 1; R45
Muta. Cat. 2; R46

F+; T
R: 12-45-46
S: 53-45

L

2
1
6
/
7
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

649-119-00-5

Raffinates (petroleum), steam-
cracked C4 fraction cuprous
ammonium acetate extn., C3-5
and C3-5 unsatd., butadiene-free
Petroleum gas

H K

307-769-4

97722-19-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-746-1

68477-65-6

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-747-7

68477-66-7

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-120-00-0

Gases (petroleum), amine system
feed
Refinery gas
[The feed gas to the amine
system for removal of hydrogen
sulfide. It consists of hydrogen.
Carbon monoxide, carbon
dioxide, hydrogen sulfide and
aliphatic hydrocarbons having
carbon numbers predominantly
in the range of C1 through C5
may also be present.]

649-121-00-6

Gases (petroleum), benzene unit
hydrodesulfurizer off
Refinery gas
[Off gases produced by the
benzene unit. It consists
primarily of hydrogen. Carbon
monoxide and hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C6, including
benzene, may also be present.]

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
7
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

270-748-2

68477-67-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-749-8

68477-68-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-759-2

68477-77-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-122-00-1

649-123-00-7

649-124-00-2

Gases (petroleum), benzene unit
recycle, hydrogen-rich
Refinery gas
[A complex combination of
hydrocarbons obtained by
recycling the gases of the
benzene unit. It consists
primarily of hydrogen with
various small amounts of carbon
monoxide and hydrocarbons
having carbon numbers in the
range of C1 through C6.]

Gases (petroleum), blend oil,
hydrogen-nitrogen-rich
Refinery gas
[A complex combination of
hydrocarbons obtained by
distillation of a blend oil. It
consists primarily of hydrogen
and nitrogen with various small
amounts of carbon monoxide,
carbon dioxide, and aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C5.]

Gases (petroleum), catalytic
reformed naphtha stripper
overheads
Refinery gas
[A complex combination of
hydrocarbons obtained from
stabilization of catalytic
reformed naphtha. Its consists of
hydrogen and saturated
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C4.]

L

2
1
6
/
7
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

H K

270-761-3

68477-80-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-762-9

68477-81-6

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-125-00-8

Gases (petroleum), C6-8 catalytic
reformer recycle
Refinery gas
[A complex combination of
hydrocarbons produced by
distillation of products from
catalytic reforming of C6-C8 feed
and recycled to conserve
hydrogen. It consists primarily
of hydrogen. It may also contain
various small amounts of carbon
monoxide, carbon dioxide,
nitrogen, and hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C6.]

649-126-00-3

Gases (petroleum), C6-8 catalytic
reformer
Refinery gas
[A complex combination of
hydrocarbons produced by
distillation of products from
catalytic reforming of C6-C8 feed.
It consists of hydrocarbons
having carbon numbers in the
range of C1 through C5 and
hydrogen.]

649-127-00-9

Gases (petroleum), C6-8 catalytic
reformer recycle, hydrogen-rich
Refinery gas

H K

270-763-4

68477-82-7

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

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
U
n
o
n

i

L

2
1
6
/
7
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

270-766-0

68477-84-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-774-4

68477-92-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-776-5

68477-93-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-128-00-4

Gases (petroleum), C2-return
stream
Refinery gas
[A complex combination of
hydrocarbons obtained by the
extraction of hydrogen from a
gas stream which consists
primarily of hydrogen with
small amounts of nitrogen,
carbon monoxide, methane,
ethane, and ethylene. It contains
predominantly hydrocarbons
such as methane, ethane, and
ethylene with small amounts of
hydrogen, nitrogen and carbon
monoxide.]

649-129-00-X Gases (petroleum), dry sour, gas-
concn.-unit-off
Refinery gas
[The complex combination of
dry gases from a gas
concentration unit. It consists of
hydrogen, hydrogen sulfide and
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C3.]

649-130-00-5

Gases (petroleum), gas concn.
reabsorber distn.
Refinery gas
[A complex combination of
hydrocarbons produced by
distillation of products from
combined gas streams in a gas
concentration reabsorber. It
consists predominantly of
hydrogen, carbon monoxide,
carbon dioxide, nitrogen,
hydrogen sulfide and
hydrocarbons having carbon
numbers in the range of C1
through C3.]

L

2
1
6
/
7
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

H K

270-779-1

68477-96-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-780-7

68477-97-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-781-2

68477-98-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-131-00-0

Gases (petroleum), hydrogen
absorber off
Refinery gas
[A complex combination
obtained by absorbing hydrogen
from a hydrogen rich stream. It
consists of hydrogen, carbon
monoxide, nitrogen, and
methane with small amounts of
C2 hydrocarbons.]

649-132-00-6

Gases (petroleum), hydrogen-
rich
Refinery gas
[A complex combination
separated as a gas from
hydrocarbon gases by chilling. It
consists primarily of hydrogen
with various small amounts of
carbon monoxide, nitrogen,
methane, and C2 hydrocarbons.]

649-133-00-1

Gases (petroleum), hydrotreater
blend oil recycle, hydrogen-
nitrogen-rich
Refinery gas
[A complex combination
obtained from recycled
hydrotreated blend oil. It
consists primarily of hydrogen
and nitrogen with various small
amounts of carbon monoxide,
carbon dioxide and
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C5.]

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
U
n
o
n

i

L

2
1
6
/
7
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

270-783-3

68478-00-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-784-9

68478-01-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-785-4

68478-02-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-134-00-7

649-135-00-2

649-136-00-8

Gases (petroleum), recycle,
hydrogen-rich
Refinery gas
[A complex combination
obtained from recycled reactor
gases. It consists primarily of
hydrogen with various small
amounts of carbon monoxide,
carbon dioxide, nitrogen,
hydrogen sulfide, and saturated
aliphatic hydrocarbons having
carbon numbers in the range of
C1 through C5.]

Gases (petroleum), reformer
make-up, hydrogen-rich
Refinery gas
[A complex combination
obtained from the reformers. It
consists primarily of hydrogen
with various small amounts of
carbon monoxide and aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C5.]

Gases (petroleum), reforming
hydrotreater
Refinery gas
[A complex combination
obtained from the reforming
hydrotreating process. It consists
primarily of hydrogen, methane,
and ethane with various small
amounts of hydrogen sulfide
and aliphatic hydrocarbons
having carbon numbers
predominantly in the range of
C3 thorugh C5.]

L

2
1
6
/
7
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

270-787-5

68478-03-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-788-0

68478-04-6

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-789-6

68478-05-7

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-137-00-3

649-138-00-9

649-139-00-4

Gases (petroleum), reforming
hydrotreater, hydrogen-methane-
rich
Refinery gas
[A complex combination
obtained from the reforming
hydrotreating process. It consists
primarily of hydrogen and
methane with various small
amounts of carbon monoxide,
carbon dioxide, nitrogen and
saturated aliphatic hydrocarbons
having carbon numbers
predominantly in the range of
C2 through C5.]

Gases (petroleum), reforming
hydrotreater make-up, hydrogen-
rich
Refinery gas
[A complex combination
obtained from the reforming
hydrotreating process. It consists
primarily of hydrogen with
various small amounts of carbon
monoxide and aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C5.]

Gases (petroleum), thermal
cracking distn.
Refinery gas
[A complex combination
produced by distillation of
products from a thermal
cracking process. It consists of
hydrogen, hydrogen sulfide,
carbon monoxide, carbon
dioxide and hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C6.]

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
7
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

270-805-1

68478-25-1

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-807-2

68478-27-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-808-8

68478-28-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-140-00-X

Tail gas (petroleum), catalytic
cracker refractionation absorber
Refinery gas
[A complex combination of
hydrocarbons obtained from
refractionation of products from
a catalytic cracking process. It
consists of hydrogen and
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C3.]

649-141-00-5

Tail gas (petroleum), catalytic
reformed naphtha separator
Refinery gas
[A complex combination of
hydrocarbons obtained from the
catalytic reforming of straight
run naphtha. It consists of
hydrogen and hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C6.]

649-142-00-0

Tail gas (petroleum), catalytic
reformed naphtha stabilizer
Refinery gas
[A complex combination of
hydrocarbons obtained from the
stabilization of catalytic
reformed naphtha. It consists of
hydrogen and hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C6.]

L

2
1
6
/
7
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

H K

270-809-3

68478-29-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-810-9

68478-30-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-999-8

68513-14-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-143-00-6

649-144-00-1

649-145-00-7

Tail gas (petroleum), cracked
distillate hydrotreater separator
Refinery gas
[A complex combination of
hydrocarbons obtained by
treating cracked distillates with
hydrogen in the presence of a
catalyst. It consists of hydrogen
and saturated aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C5.]

Tail gas (petroleum),
hydrodesulfurized straight-run
naphtha separator
Refinery gas
[A complex combination of
hydrocarbons obtained from
hydrodesulfurization of straight-
run naphtha. It consists of
hydrogen and saturated aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C6.]

Gases (petroleum), catalytic
reformed straight-run naphtha
stabilizer overheads
Refinery gas
[A complex combination of
hydrocarbons obtained from the
catalytic reforming of straight-
run naphtha followed by
fractionation of the total
effluent. It consists of hydrogen,
methane, ethane and propane.]

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
U
n
o
n

i

L

2
1
6
/
7
9

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

271-003-4

68513-18-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

271-005-5

68513-19-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

271-258-1

68527-15-1

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-146-00-2

649-147-00-8

649-148-00-3

Gases (petroleum), reformer
effluent high-pressure flash
drum off
Refinery gas
[A complex combination
produced by the high-pressure
flashing of the effluent from the
reforming reactor. It consists
primarily of hydrogen with
various small amounts of
methane, ethane, and propane.]

Gases (petroleum), reformer
effluent low-pressure flash drum
off
Refinery gas
[A complex combination
produced by low-pressure
flashing of the effluent from the
reforming reactor. It consists
primarily of hydrogen with
various small amounts of
methane, ethane, and propane.]

Gases (petroleum), oil refinery
gas distn. off
Refinery gas
[A complex combination
separated by distillation of a gas
stream containing hydrogen,
carbon monoxide, carbon
dioxide and hydrocarbons
having carbon numbers in the
range of C1 through C6 or
obtained by cracking ethane and
propane. It consists of
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C2,
hydrogen, nitrogen, and carbon
monoxide.]

L

2
1
6
/
8
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

H K

271-623-5

68602-82-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

271-625-6

68602-84-6

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-149-00-9

Gases (petroleum), benzene unit
hydrotreater depentanizer
overheads
Refinery gas
[A complex combination
produced by treating the feed
from the benzene unit with
hydrogen in the presence of a
catalyst followed by
depentanizing. It consists
primarily of hydrogen, ethane
and propane with various small
amounts of nitrogen, carbon
monoxide, carbon dioxide and
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C6. It may
contain trace amounts of
benzene.]

649-150-00-4

Gases (petroleum), secondary
absorber off, fluidized catalytic
cracker overheads fractionator
Refinery gas
[A complex combination
produced by the fractionation of
the overhead products from the
catalytic cracking process in the
fluidized catalytic cracker. It
consists of hydrogen, nitrogen,
and hydrocarbons having carbon
numbers predominantly in the
range of C1 through C3.]

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
U
n
o
n

i

L

2
1
6
/
8
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

271-750-6

68607-11-4

Car. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-182-1

68783-06-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-338-9

68814-67-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-343-6

68814-90-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-151-00-X

649-152-00-5

649-153-00-0

649-154-00-6

Petroleum products, refinery
gases
Refinery gas
[A complex combination which
consists primarily of hydrogen
with various small amounts of
methane, ethane, and propane.]

Gases (petroleum),
hydrocracking low-pressure
separator
Refinery gas
[A complex combination
obtained by the liquid-vapor
separation of the hydrocracking
process reactor effluent. It
consists predominantly of
hydrogen and saturated
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C3.]

Gases (petroleum), refinery
Refinery gas
[A complex combination
obtained from various
petroleum refining operations. It
consists of hydrogen and
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C3.]

Gases (petroleum), platformer
products separator off
Refinery gas
[A complex combination
obtained from the chemical
reforming of naphthenes to
aromatics. It consists of
hydrogen and saturated aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C2 through C4.]

L

2
1
6
/
8
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

272-775-5

68911-58-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-776-0

68911-59-1

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-873-8

68919-01-7

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-155-00-1

649-156-00-7

649-157-00-2

Gases (petroleum), hydrotreated
sour kerosine depentanizer
stabilizer off
Refinery gas
[The complex combination
obtained from the depentanizer
stabilization of hydrotreated
kerosine. It consists primarily of
hydrogen, methane, ethane, and
propane with various small
amounts of nitrogen, hydrogen
sulfide, carbon monoxide and
hydrocarbons having carbon
numbers predominantly in the
range of C4 through C5.]

Gases (petroleum), hydrotreated
sour kerosine flash drum
Refinery gas
[A complex combination
obtained from the flash drum of
the unit treating sour kerosine
with hydrogen in the presence
of a catalyst. It consists
primarily of hydrogen and
methane with various small
amounts of nitrogen, carbon
monoxide, and hydro-carbons
having carbon numbers
predominantly in the range of
C2 through C5.]

Gases (petroleum), distillate
unifiner desulfurization stripper
off
Refinery gas
[A complex combination
stripped from the liquid product
of the unifiner desulfurization
process. It consists of hydrogen
sulfide, methane, ethane, and
propane.]

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
8
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

272-874-3

68919-02-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-875-9

68919-03-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-876-4

68919-04-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-158-00-8

Gases (petroleum), fluidized
catalytic cracker fractionation off
Refinery gas
[A complex combination
produced by the fractionation of
the overhead product of the
fluidized catalytic cracking
process. It consists of hydrogen,
hydrogen sulfide, nitrogen, and
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C5.]

649-159-00-3

Gases (petroleum), fluidized
catalytic cracker scrubbing
secondary absorber off
Refinery gas
[A complex combination
produced by scrubbing the
overhead gas from the fluidized
catalytic cracker. It consists of
hydrogen, nitrogen, methane,
ethane and propane.]

649-160-00-9

Gases (petroleum), heavy
distillate hydrotreater
desulfurization stripper off
Refinery gas
[A complex combination
stripped from the liquid product
of the heavy distillate
hydrotreater desulfurization
process. It consists of hydrogen,
hydrogen sulfide, and saturated
aliphatic hydrocarbons having
carbon numbers predominantly
in the range of C1 through C5.]

L

2
1
6
/
8
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

H K

272-880-6

68919-07-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-881-1

68919-08-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

272-884-8

68919-11-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-161-00-4

Gases (petroleum), platformer
stabilizer off, light ends
fractionation
Refinery gas
[A complex combination
obtained by the fractionation of
the light ends of the platinum
reactors of the plattformer unit.
It consists of hydrogen,
methane, ethane and propane.]

649-162-00-X

Gases (petroleum), preflash
tower off, crude distn.
Refinery gas
[A complex combination
produced from the first tower
used in the distillation of crude
oil. It consists of nitrogen and
saturated aliphatic hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C5.]

649-163-00-5

Gases (petroleum), tar stripper
off
Refinery gas
[A complex combination
obtained by the fractionation of
reduced crude oil. It consists of
hydrogen and hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C4.]

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
U
n
o
n

i

L

2
1
6
/
8
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

649-164-00-0

Gases (petroleum), unifiner
stripper off
Refinery gas
[A combination of hydrogen
and methane obtained by
fractionation of the products
from the unifiner unit.]

H K

272-885-3

68919-12-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

273-173-5

68952-79-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

273-174-0

68952-80-7

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-165-00-6

Tail gas (petroleum), catalytic
hydrodesulfurized naphtha
separator
Refinery gas
[A complex combination of
hydrocarbons obtained from the
hydrodesulfurization of naphtha.
It consists of hydrogen,
methane, ethane, and propane.]

649-166-00-1

Tail gas (petroleum), straight-run
naphtha hydrodesulfurizer
Refinery gas
[A complex combination
obtained from the
hydrodesulfurization of straight-
run naphtha. It consists of
hydrogen and hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C5.]

L

2
1
6
/
8
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

H K

273-269-7

68955-33-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

273-563-5

68989-88-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

295-397-2

92045-15-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-167-00-7

649-168-00-2

649-169-00-8

Gases (petroleum), sponge
absorber off, fluidized catalytic
cracker and gas oil desulfurizer
overhead fractionation
Refinery gas
[A complex combination
obtained by the fractionation of
products from the fluidized
catalytic cracker and gas oil
desulfurizer. It consists of
hydrogen and hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C4.]

Gases (petroleum), crude distn.
and catalytic cracking
Refinery gas
[A complex combination
produced by crude distillation
and catalytic cracking processes.
It consists of hydrogen,
hydrogen sulfide, nitrogen,
carbon monoxide and paraffinic
and olefinic hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C6.]

Gases (petroleum), gas oil
diethanolamine scrubber off
Refinery gas
[A complex combination
produced by desulfurization of
gas oils with diethanolamine. It
consists predominantly of
hydrogen sulfide, hydrogen and
aliphatic hydrocarbons having
carbon numbers in the range of
C1 through C5.]

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
U
n
o
n

i

L

2
1
6
/
8
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

295-398-8

92045-16-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

295-399-3

92045-17-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

295-400-7

92045-18-6

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-170-00-3

649-171-00-9

649-172-00-4

Gases (petroleum), gas oil
hydrodesulfurization effluent
Refinery gas
[A complex combination
obtained by separation of the
liquid phase from the effluent
from the hydrogenation
reaction. It consists
predominantly of hydrogen,
hydrogen sulfide and aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C3.]

Gases (petroleum), gas oil
hydrodesulfurization purge
Refinery gas
[A complex combination of
gases obtained from the
reformer and from the purges
from the hydrogenation reactor.
It consists predominantly of
hydrogen and aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C4.]

Gases (petroleum), hydrogenator
effluent flash drum off
Refinery gas
[A complex combination of
gases obtained from flash of the
effluents after the hydrogenation
reaction. It consists
predominantly of hydrogen and
aliphatic hydrocarbons having
carbon numbers predominantly
in the range of C1 through C6.]

L

2
1
6
/
8
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

295-401-2

92045-19-7

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

295-402-8

92045-20-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

268-629-5

68131-75-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-173-00-X

649-174-00-5

649-177-00-1

Gases (petroleum), naphtha
steam cracking high-pressure
residual
Refinery gas
[A complex combination
obtained as a mixture of the
non-condensable portions from
the product of a naphtha steam
cracking process as well as
residual gases obtained during
the preparation of subsequent
products. It consists
predominantly of hydrogen and
paraffinic and olefinic
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C5 with
which natural gas may also be
mixed.]

Gases (petroleum), residue
visbaking off
Refinery gas
[A complex combination
obtained from viscosity
reduction of residues in a
furnace. It consists
predominantly of hydrogen
sulfide and paraffinic and
olefinic hydrocarbons having
carbon numbers predominantly
in the range of C1 through C5.]

Gases (petroleum), C3-4
Petroleum gas
[A complex combination of
hydrocarbons produced by
distillation of products from the
cracking of crude oil. It consists
of hydrocarbons having carbon
numbers in the range of C3
through C4, predominantly of
propane and propylene, and
boiling in the range of
approximately -51 °C to -1 °C
(-60 °F to 30 °F.)]

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
8
9

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

269-617-2

68307-98-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

269-618-8

68307-99-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-178-00-7

Tail gas (petroleum), catalytic
cracked distillate and catalytic
cracked naphtha fractionation
absorber
Petroleum gas
[The complex combination of
hydrocarbons from the
distillation of the products from
catalytic cracked distillates and
catalytic cracked naphtha. It
consists predominantly of
hydrocarbons having carbon
numbers in the range of C1
through C4.]

649-179-00-2

Tail gas (petroleum), catalytic
polymn. naphtha fractionation
stabilizer
Petroleum gas
[A complex combination of
hydrocarbons from the
fractionation stabilization
products from polymerization of
naphtha. It consists
predominantly of hydrocarbons
having carbon numbers in the
range of C1 through C4.]

L

2
1
6
/
9
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

269-619-3

68308-00-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

269-620-9

68308-01-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-180-00-8

Tail gas (petroleum), catalytic
reformed naphtha fractionation
stabilizer, hydrogen sulfide-free
Petroleum gas
[A complex combination of
hydrocarbons obtained from
fractionation stabilization of
catalytic reformed naphtha and
from which hydrogen sulfide has
been removed by amine
treatment. It consists
predominantly of hydrocarbons
having carbon numbers
predominantly in the range of
C1 through C4.]

649-181-00-3

Tail gas (petroleum), cracked
distillate hydrotreater stripper
Petroleum gas
[A complex combination of
hydrocarbons obtained by
treating thermal cracked
distillates with hydrogen in the
presence of a catalyst. It consists
predominantly of saturated
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C6.]

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
9
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

269-630-3

68308-10-1

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

269-623-5

68308-03-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

269-624-0

68308-04-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-182-00-9

649-183-00-4

649-184-00-X

Tail gas (petroleum), straight-run
distillate hydrodesulfurizer,
hydrogen sulfide-free
Petroleum gas
[A complex combination of
hydrocarbons obtained from
catalytic hydrodesulfurization of
straight run distillates and from
which hydrogen sulfide has been
removed by amine treatment. It
consists predominantly of
hydrocarbons having carbon
numbers predominantly in the
range of C1 through C4.]

Tail gas (petroleum), gas oil
catalytic cracking absorber
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
distillation of products from the
catalytic cracking of gas oil. It
consists predominantly of
hydrocarbons having carbon
numbers predominantly in the
range of C1through C5.]

Tail gas (petroleum), gas
recovery plant
Petroleum gas
[A complex combination of
hydrocarbons from the
distillation of products from
miscellaneous hydrocarbon
streams. It consists
predominantly of hydrocarbons
having carbon numbers
predominantly in the range of
C1through C5.]

L

2
1
6
/
9
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

269-625-6

68308-05-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

269-626-1

68308-06-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

269-627-7

68308-07-6

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-185-00-5

649-186-00-0

649-187-00-6

Tail gas (petroleum), gas
recovery plant deethanizer
Petroleum gas
[A complex combination of
hydrocarbons from the
distillation of products from
miscellaneous hydrocarbon
streams. It consists of
hydrocarbon having carbon
numbers predominantly in the
range of C1through C4.]

Tail gas (petroleum),
hydrodesulfurized distillate and
hydrodesulfurized naphtha
fractionator, acid-free
Petroleum gas
[A complex combination of
hydrocarbons obtained from
fractionation of
hydrodesulfurized naphtha and
distillate hydrocarbon streams
and treated to remove acidic
impurities. It consists
predominantly of hydrocarbons
having carbon numbers
predominantly in the range of
C1through C5.]

Tail gas (petroleum),
hydrodesulfurized vacuum gas
oil stripper, hydrogen sulfide-
free
Petroleum gas
[A complex combination of
hydrocarbons obtained from
stripping stabilization of
catalytic hydrodesulfurized
vacuum gas oil and from which
hydrogen sulfide has been
removed by amine treatment. It
consists predominantly of
hydrocarbons having carbon
numbers predominantly in the
range of C1through C6.]

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
9
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

269-629-8

68308-09-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

269-631-9

68308-11-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-188-00-1

Tail gas (petroleum), light
straight-run naphtha stabilizer,
hydrogen sulfide-free
petroleum gas
[A complex combination of
hydrocarbons obtained from
fractionation stabilization of
light straight run naphtha and
from which hydrogen sulfide has
been removed by amine
treatment. It consists
predominantly of hydrocarbons
having carbon numbers
predominantly in the range of
C1through C5.]

649-189-00-7

Tail gas (petroleum), propane-
propylene alkylation feed prep
deethanizer
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
distillation of the reaction
products of propane with
propylene. It consists of
hydrocarbons having carbon
numbers predominantly in the
range of C1through C4.]

L

2
1
6
/
9
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H K

269-632-4

68308-12-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-071-2

68409-99-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-190-00-2

649-191-00-8

Tail gas (petroleum), vacuum gas
oil hydrodesulfurizer, hydrogen
sulfide-free
Petroleum gas
[A complex combination of
hydrocarbons obtained from
catalytic hydrodesulfurization of
vacuum gas oil and from which
hydrogen sulfide has been
removed by amine treatment. It
consists predominantly of
hydrocarbons having carbon
numbers predominantly in the
range of C1through C6.]

Gases (petroleum), catalytic
cracked overheads
Petroleum gas
[A complex combination of
hydrocarbons produced by the
distillation of products from the
catalytic cracking process. It
consists of hydrocarbons having
carbon numbers predominantly
in the range of C3through C5and
boiling in the range of
approximately -48 °C to 32 °C
(-54 °F to 90 °F).]

649-193-00-9

Alkanes, C1-2
Petroleum gas

H K

270-651-5

68475-57-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

649-194-00-4

Alkanes, C2-3
Petroleum gas

H K

270-652-0

68475-58-1

Carc. Cat. 1; R45
Muta. Cat. 2; R46

649-195-00-X

Alkanes, C3-4
petroleum gas

H K

270-653-6

68475-59-2

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

T
R: 45-46
S: 53-45

T
R: 45-46
S: 53-45

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
9
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

649-196-00-5

Alkanes, C4-5
Petroleum gas

H K

270-654-1

68475-60-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

H K

270-667-2

68476-26-6

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

T
R: 45-46
S: 53-45

H K

270-670-9

68476-29-9

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-197-00-0

Fuel gases
Petroleum gas
[A combination of light gases. It
consists predominantly of
hydrogen and/or low molecular
weight hydrocarbons.]

649-198-00-6

Fuel gases, crude oil of distillates
Petroleum gas
[A complex combination of
light gases produced by
distillation of crude oil and by
catalytic reforming of naphtha.
It consists of hydrogen and
hydrocarbons having carbon
numbers predominantly in the
range of C1through C4and
boiling in the range of
approximately -217 °C to -12 °C
(-423 °F to 10 °F).]

649-199-00-1

Hydrocarbons, C3-4
Petroleum gas

H K

270-681-9

68476-40-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

649-200-00-5 Hydrocarbons, C4-5

H K

270-682-4

68476-42-6

Petroleum gas

Carc. Cat. 1; R45
Muta. Cat. 2; R46

649-201-00-0

Hydrocarbons, C2-4, C3-rich
Petroleum gas

H K

270-689-2

68476-49-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

T
R: 45-46
S: 53-45

T
R: 45-46
S: 53-45

L

2
1
6
/
9
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

HKS

270-704-2

68476-85-7

F+; R12
Carc. Cat. 1; R45
Muta. Cat. 2; R46

F+; T
R: 12-45-46
S: 53-45

HKS

270-705-8

68476-86-8

F+; R12
Carc. Cat. 1; R45
Muta. Cat. 2; R46

F+; T
R: 12-45-46
S: 45-53

H K

270-724-1

68477-33-8

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-202-00-6

649-203-00-1

649-204-00-7

Petroleum gases, liquefied
Petroleum gas
[A complex combination of
hydrocarbons produced by the
distillation of crude oil. It
consists of hydrocarbons having
carbon numbers predominantly
in the range of C3through C7and
boiling in the range of
approximately -40 °C to 80 °C
(-40 °F to 176 °F).]

Petroleum gases, liquefied,
sweetened
Petroleum gas
[A complex combination of
hydrocarbons obtained by
subjecting liquefied petroleum
gas mix to a sweetening process
to convert mercaptans or to
remove acidic impurities. It
consists of hydrocarbons having
carbon numbers predominantly
in the range of C3through C7and
boiling in the range of
approximately -40 °C to 80 °C
(-40 °F to 176 °F).]

gases (petroleum), C3-4,
isobutane-rich
Petroleum gas
[A complex combination of
hydrocarbons from the
distillation of saturated and
unsaturated hydrocarbons
usually ranging in carbon
numbers from C3through C6,
predominantly butane and
isobutane. It consists of
saturated and unsaturated
hydrocarbons having carbon
numbers in the range of
C3through C4, predominantly
isobutane.]

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
9
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

649-205-00-2 Distillates (petroleum), C3-6,

H K

270-726-2

68477-35-0

piperylene-rich
Petroleum gas
[A complex combination of
hydrocarbons from the
distillation of saturated and
unsaturated aliphatic
hydrocarbons usually ranging in
the carbon numbers C3through
C6. It consists of saturated and
unsaturated hydrocarbons
having carbon numbers in the
range of C3through C6,
predominantly piperylenes.]

649-206-00-8 Gases (petroleum), butane

H K

270-750-3

68477-69-0

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

splitter overheads
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
distillation of the butane stream.
It consists of aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C3through C4.]

649-207-00-3

Gases (petroleum), C2-
Petroleum gas
[A complex combination of
hydrocarbons produced by the
distillation of products from a
catalytic fractionation process. It
contains predominantly ethane,
ethylene, propane, and
propylene.]

H K

270-751-9

68477-70-3

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

L

2
1
6
/
9
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

H K

270-752-4

68477-71-4

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

270-754-5

68477-72-5

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

H K

269-628-2

68308-08-7

Carc. Cat. 1; R45
Muta. Cat. 2; R46

T
R: 45-46
S: 53-45

649-208-00-9 Gases (petroleum), catalytic-
cracked gas oil depropanizer
bottoms, C4-rich acid-free
Petroleum gas
[A complex combination of
hydrocarbons obtained from
fractionation of catalytic cracked
gas oil hydrocarbon stream and
treated to remove hydrogen
sulfide and other acidic
components. It consists of
hydrocarbons having carbon
numbers in the range of
C3through C5, predominantly
C4.]

649-209-00-4 Gases (petroleum), catalytic-
cracked naphtha debutanizer
bottoms, C3-5-rich
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
stabilization of catalytic cracked
naphtha. It consists of aliphatic
hydrocarbons having carbon
numbers predominantly in the
range of C3through C5.]

649-210-00-X

Tail gas (petroleum), isomerized
naphtha fractionation stabilizer
Petroleum gas
[A complex combination of
hydrocarbons obtained from the
fractionation stabilization
products from isomerized
naphtha. It consists
predominantly of hydrocarbons
having carbon numbers
predominantly in the range of
C1through C4.]

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
U
n
o
n

i

L

2
1
6
/
9
9

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

H N

269-822-7

68334-30-5

Carc. Cat. 3; R40

Xn
R: 40
S: (2-)36/37’

649-224-00-6

Fuels, diesel
Gasoil - unspecified
[A complex combination of
hydrocarbons produced by the
distillation of crude oil. It
consists of hydrocarbons having
carbon numbers predominantly
in the range of C9through
C20and boiling in the range of
approximately 163 °C to 357 °C
(325 °F to 675 °F).]

L

2
1
6
/
1
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

ANNEX 1C

‘005-009-00-3

tetrabutylammonium butyltri-
phenylborate

418-080-4

120307-06-4

R 43
N; R50-53

Xi; N
R: 43-50/53
S: (2-)24-37-56-61

005-010-00-9

N,N-dimethylanilinium tetrakis
(pentafluorophenyl)borate

422-050-6

118612-00-3

Carc.Cat.3; R40
Xn; R22
Xi; R38-41

Xn
R: 22-38-40-41
S: (2-)22-26-36/37/39

005-012-00-X

diethyl{4-[1,5,5-tris(4-diethyla-
minophenyl)penta-2,4-dienylide-
ne]cyclohexa-2,5-dienylide-
ne}ammonium butyltriphenylbo-
rate

418-070-1

141714-54-7

R 43
N; R50-53

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

011-007-00-3

propoxycarbazone-sodium

—

013-009-00-X

sodium((n-butyl)x(ethyl)y-1,5-
dihydro)aluminate) x = 0.5
y = 1.5

014-026-00-5

dichloro-(3-(3-chloro-4-fluoro-
phenyl)propyl)methylsilane

014-027-00-0

chloro(3-(3-chloro-4-fluorophe-
nyl)propyl)dimethylsilane

014-028-00-6

α-[3-(1-oxoprop-2-eny)l-1-oxy-
propyl]dimethoxysilyloxy-ω-[3(1-
oxoprop-2-enyl)-1-oxypropyl]di-
methoxysilyl poly(dimethylsilox-
ane)

—

—

—

—

N; R50-53

F; R11
R14/15
R 17
Xn; R20
C; R35

C; R35

C; R35

418-720-2

407-180-3

410-270-5

415-290-8

—

R 43

N
R: 50/53
S: 60-61

C ≥ 2,5 %: N; R50/53
0,25 % ≤ C < 2,5 %: N; R51/53
0,025 ≤ C < 0,25 %: R52/53

F; C
R: 11-14/15-17-20-35
S: (1/2-)6-16-26-30-36/
37/39-43-45

C
R: 35
S: (1/2-)26-36/37/39-45

C
R: 35
S: (1/2-)8-26-28-36/37/
39-45

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

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
0
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

014-029-00-1 O,O′-(ethenylmethylsilyle-

421-870-1

—

ne)di[(4-methylpentan-2-one)
oxime]

Repr.Cat.3; R62
Xn; R22-48/22

Xn
R: 22-48/22-62
S: (2-)36/37

014-030-00-7

[(dimethylsilylene)bis
((1,2,3,3a,7a-η)-1H-inden-1-yli-
dene)dimethyl]hafnium

422-060-0

137390-08-0

T+; R28

014-031-00-2

bis(1-methylethyl)-dimethoxysi-
lane

421-540-7

18230-61-0

014-032-00-8

dicyclopentyldimethoxysilane

404-370-8

126990-35-0

015-180-00-6

[R-(R*,S*)]-[[2-methyl-1-(1-oxo-
propoxy)propoxy]-(4-phenylbu-
tyl)phosphinyl] acetic acid,
(-)-cinchonidine (1:1) salt

415-820-8

137590-32-0

015-181-00-1

phosphine

232-260-8

7803-51-2

R 10
Xi; R38
R43
R 52-53

Xi; R38-41
N; R50-53

Xi; R41
R 43
R 52-53

F+; R12
R17
T+; R26
C; R34
N; R50

T+
R: 28
S: (1/2-)6-22-28-36/37-
45

Xi
R: 10-38-43-52/53
S: (2-)24-37-61

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

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

F+; T+; N
R: 12-17-26-34-50
S: (1/2-)28-36/37-45-61-
63

015-184-00-8

Salts of glyphosate, with the
exception of those specified
elsewhere in this Annex

—

—

N; R51-53

N
R: 51/53
S: 61

015-186-00-9

chlorpyrifos-methyl

227-011-5

5598-13-0

R43
N; R50-53

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

C ≥ 1 %: N; R43-50-53
0,0025 % ≤C < 1 %: N; R50-53
0,00025 % ≤C < 0,0025 %: N; R51-53
0,000025 % ≤C < 0,00025 %: R52-53

L

2
1
6
/
1
0
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

015-187-00-4

A mixture of: tetrasodium(((2-
hydroxyethyl)imino)bis(methyle-
ne))bisphosphonate, N-oxide;
trisodium ((tetrahydro-2-
hydroxy-4H-1,4,2-oxazapho-
sphorin-4-yl)-methyl)phospho-
nate, N-oxide, P-oxide

417-540-1

—

Xi; R41
N; R51-53

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

015-189-00-5

phenyl bis(2,4,6-
trimethylbenzoyl)-phosphine
oxide

423-340-5

162881-26-7

R43
R53

402-590-9

109125-56-6

Xi; R41

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

Xi
R: 41
S: (2-)22-26-39

016-086-00-8

016-087-00-3

tetrasodium 10-amino-6,13-
dichloro-3-(3-(4-(2,5-disulfona-
toanilino)-6-fluoro-1,3,5-triazin-
2-ylamino)prop-3-ylamino)-
5,12-dioxa-7,14-diazapentacene-
4,11-disulfonate

A mixture of: thiobis(4,1-
phenylene)-S,S,S′,S′-tetraphenyl-
disulfonium bishexafluoropho-
sphate
diphenyl(4-phenylthiophenyl)sul-
fonium hexafluorophosphate
propylene carbonate

403-490-8

74227-35-3

Xi; R36
R 43
N; R50-53

Xi; N
R: 36-43-50/53
S: (2-)24-26-37-60-61

016-088-00-9

4-(bis(4-(diethylamino)phenyl)-
methyl)benzene-1,2-dimethane-
sulfonic acid

407-280-7

71297-11-5

R 52-53

R: 52/53
S: 61

016-089-00-4

A mixture of esters of
5,5′,6,6′,7,7′-hexahydroxy-
3,3,3′,3′-tetramethyl-1,1′-
spirobiindan and 2-diazo-1,2-
dihydro-1-oxo-5-
sulfonaphthalene

413-840-1

—

E; R2
F; R11
R 53

E
R: 2-11-53
S: (2-)33-35-40-61

016-090-00-X

4-methyl-N-(methylsulfonyl)ben-
zenesulfonamide

415-040-8

14653-91-9

Xn; R22
Xi; R37-41

Xn
R: 22-37-41
S: (2-)26-39

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
0
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

016-091-00-5

C12-14-tert-alkyl ammonium 1-
amino-9,10-dihydro-9,10-dioxo-
4-(2,4,6-trimethylanilino)-
anthracen-2-sulfonate

414-110-5

—

Xi; R41
N; R50-53

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

414-770-4

140698-96-0

F; R11
Carc.Cat.3; R40

F; Xn
R: 11-40
S: (2-)7-36/37

417-980-4

—

F; R11
Carc.Cat.3; R40

F; Xn
R: 11-40
S: (2-)7-36/37

016-093-00-6

016-095-00-7

A 2:1 mixture of: 4-(7-hydroxy-
2,4,4-trimethyl-2-chromanyl)re-
sorcinol-4-yl-tris(6-diazo-5,6-
dihydro-5-oxonaphthalen-1-sul-
fonate)
4-(7-hydroxy-2,4,4-trimethyl-2-
chromanyl)resorcinolbis(6-diazo-
5,6-dihydro-5-oxonaphthalen-1-
sulfonate)

A mixture of: reaction product
of 4,4′-methylenebis[2-(4-
hydroxybenzyl)-3,6-dimethyl-
phenol] and 6-diazo-5,6-
dihydro-5-oxo-naphthalenesulfo-
nate (1:2)
Reaction product of 4,4′-
methylenebis[2-(4-hydroxyben-
zyl)-3,6-dimethylphenol] and 6-
diazo-5,6-dihydro-5-oxo-
naphthalenesulfonate (1:3)

016-096-00-2

thifensulfuron-methyl

—

79277-27-3

N; R50-53

017-015-00-3

(2-(aminomethyl)phenyl)ace-
tylchloride hydrochloride

417-410-4

61807-67-8

017-016-00-9 methyltriphenylphosphonium

418-400-2

1031-15-8

chloride

Xn; R22
C; R35
R43

Xn; R21/22
Xi; R38-41
N; R51-53

N
R: 50/53
S: 60-61

C
R: 22-35-43
S: (1/2-)26-36/37/39-45

Xn; N
R: 21/22-38-41-51/53
S: (2-)22-26-36/37/39-
61

L

2
1
6
/
1
0
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

017-017-00-4

(Z)-13-docosenyl-N,N-bis(2-
hydroxyethyl)-N-methyl-ammo-
nium-chloride

426-210-6

120086-58-0

C; R34
N; R50-53

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

017-018-00-X

N,N,N-trimethyl-2,3-bis(stearoy-
loxy)propylammonium chloride

405-660-7

—

N;R51-53

017-019-00-5

(R)-1,2,3,4-tetrahydro-6,7-
dimethoxy-1-veratrylisoquinoline
hydrochloride

415-110-8

54417-53-7

Xn; R22
R52-53

017-020-00-0

ethyl propoxy aluminium
chloride

421-790-7

—

C; R35
F; R14/15

017-021-00-6

behenamidopropyl-dimethyl-
(dihydroxypropyl) ammonium
chloride

423-420-1

136920-10-0

Xi; R41
R43
N; R50-53

N
R: 51/53
S: 61

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

C; F
R: 14/15-35
S: (1/2-)16-23-26-30-36/
37/39-43-45

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

020-003-00-0 A mixture of: dicalcium (bis(2-

420-470-4

hydroxy-5-tetra-propenylphenyl-
methyl)methylamine)dihydroxide
tri-calcium (tris(2-hydroxy-5-
tetra-propenylphenylmethyl)-
methylamine)tri-hydroxide
poly[calcium ((2-hydroxy-5-
tetra-propenyl-phenylmethyl)-
methylamine)hydroxide]

—

Xi; R36/38
R43

Xi
R: 36/38-43
S: (2-)24-26-37

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
0
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

R 43
R52-53

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

024-019-00-9 Main component: acetoacetic

419-230-1

—

acid anilide / 3-amino-1-
hydroxybenzene (ATAN-MAP):
trisodium {6-[(2 or 3 or 4)-
amino-(4 or 5 or 6)-
hydroxyphenylazo]-5′-(phenyl-
sulfamoyl)-3-sulfonatonaphtha-
lene-2-azobenzene-1,2′-diolato}-
{6″-[1-(phenylcarbamoyl)ethy-
lazo]-5‴-(phenylsulfamoyl)-3″-
sulfonatonaphthalene-2″-azoben-
zene-1″,2‴-diolato}chromate (III)
by-product 1: acetoacetic acid
anilide / acetoacetic acid anilide
(ATAN-ATAN): trisodium bis{6-
[1-(phenylcarbamoyl)ethylazo]-
5′-(phenylsulfonyl)-3-sulfonato-
naphthalene-2-azobenzene-1,2′-
diolato}chromate (III)
by-product 2: 3-amino-1-
hydroxybenzene / 3-amino-1-
hydroxybenzene (MAP-MAP):
trisodium bis{6-[(2 or 3 or 4)-
amino-(4 or 5 or 6)-
hydroxyphenylazo]-5′-(phenyl-
sulfamoyl)-3-sulfonatonaphtha-
lene-2-azobenzene-1,2′-diolato}
chromate (III)

024-020-00-4

trisodium bis[(3′-nitro-5′-
sulfonato(6-amino-2-[4-(2-
hydroxy-1-naphtylazo)phenylsul-
fonylamino]pyrimidin-5-azo)-
benzene-2′,4-diolato)]chroma-
te(III)

418-220-4

—

R43
R52-53

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

L

2
1
6
/
1
0
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

025-005-00-5 A mixture of: tri-sodium [29H,

417-660-4

—

N; R50-53

31H-phthalocyanine-C,C,C-trisul-
fonato (6-)-N29,N30,N31,N32]
manganate (3-)
tetrasodium [29H,31H-
phthalocyanine-C,C,C,C-tetrasul-
fonato (6-)-N29,N30,N31,N32],
manganate (3-)
pentasodium [29H,31H-
phthalocyanine-C,C,C,C,C-penta-
sulfonato (6-)-
N29,N30,N31,N32] manganate
(3-)

029-012-00-4

sodium ((N-(3-
trimethylammoniopropyl)sulfa-
moyl)methylsulfonatophthalo-
cyaninato)copper(II)

029-013-00-X

trisodium(2-(α-(3-(4-chloro-6-(2-
(2-(vinylsulfonyl)ethoxy)ethyla-
mino)-1,3,5-triazin-2-ylamino)-
2-oxido-5-sulfonatophenylazo)-
benzylidenehydrazino)-4-sulfo-
natobenzoato)copper(II)

407-340-2

124719-24-0

Xi; R41

407-580-8

130201-51-3

Xi; R41
R52-53

030-011-00-6

trizinc bis(orthophosphate)

231-944-3

7779-90-0

N; R50-53

030-013-00-7

zinc oxide

215-222-5

1314-13-2

N; R50-53

N
R: 50/53
S: 60-61

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

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

N
R: 50/53
S: 60-61

N
R: 50/53
S: 60-61

034-003-00-3

sodium selenite

233-267-9

10102-18-8

T+; R28
T; R23
R31
R43
N; R51-53

T+; N
R: 23-28-31-43-51/53
S: (1/2-)28-36/37-45-61

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
0
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

053-005-00-5

(4-(1-methylethyl)phenyl)-(4-
methylphenyl)iodonium tetra-
kis(pentafluorophenyl)borate (1-)

422-960-3

178233-72-2

Xn; R21/22-48/22
N; R50-53

601-056-00-4

A mixture of isomers of:
methyldiphenylmethane
dimethyldiphenylmethane

405-470-4

—

601-057-00-X

N-dodecyl-[3-(4-dimethylamino)-
benzamido)-propyl]dimethylam-
monium tosylate

421-130-8

156679-41-3

601-058-00-5

di-L-para-menthene

417-870-6

—

601-059-00-0 methyl 2-benzylidene-3-

420-940-9

15768-07-7

oxobutyrate

Xi; R38
N; R50-53

Xi; R41
R43
N; R50-53

Xi; R38
R 43
N; R50-53

Xi; R36/38
N; R51-53

601-060-00-6

1,2-bis[4-fluoro-6-{4-sulfo-5-(2-
(4-sulfonaphtalene-3-ylazo)-1-
hydroxy-3,6-disulfo-8-amino-
naphthalene-7-ylazo)phenyla-
mino}-1,3,5-triazin-2ylami-
no]ethane;x-sodium, y-
potassium salts x = 7,755
y = 0,245

417-610-1

155522-09-1

R 43

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

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

Xi; N
R: 41-43-50/53
S: (2-)24-26-37/39-60-
61

Xi; N
R: 38-43-50/53
S: (2-)23-24-37-60-61

Xi; N
R: 36/38-51/53
S: (2-)26-37/39-61

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

601-061-00-1

(ethyl-1,2-ethanediyl)[-2-[[[(2-
hydroxyethyl)methylamino]ace-
tyl]-propyl]ω-(nonylphenoxy)po-
ly]oxy-(methyl-1,2-ethanediyl)

418-960-8

—

C; R34
R 43
N; R51-53

C; N
R: 34-43-51/53
S: (1/2-)26-28-36/37/39-
45-61

601-062-00-7

A mixture of: branched
triacontane
branched dotriacontane
branched tetratriacontane
branched hexatriacontane

417-030-9

151006-59-6

R 53

R: 53
S: 61

L

2
1
6
/
1
0
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

601-063-00-2

A mixture of isomers of
branched tetracosane

417-060-2

151006-61-0

Xn; R20
R53

601-064-00-8

branched hexatriacontane

417-070-7

151006-62-1

R53

Xn
R: 20-53
S: (2-)61

R: 53
S: 61

601-065-00-3

A mixture of: (1′-α,3′-α,6′-α-
2,2,3′,7′,7′-pentamethyl-
spiro(1,3-dioxane-5,2′-norcar-
ane)
(1′α,3′β,6′α)-2,2,3′,7′,7′-penta-
methylspiro(1,3-dioxane-5,2′-
norcarane)

416-930-9

—

Xn; R48/22
Xi; R41
N; R51-53

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

601-066-00-9

1-(4-(trans-4-heptylcyclohexyl)-
phenyl)ethane

426-820-2

78531-60-9

R43
R53

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

601-067-00-4

triethyl arsenate

427-700-2

15606-95-8

Carc.Cat.1; R45
T; R23/25
N; R50-53

T; N
R: 45-23/25-50/53
S: 53-45-60-61

601-068-00-X

1,2-diacetoxybut-3-ene

421-720-5

18085-02-4

Xn; R22

601-069-00-5

2-ethyl-1-(2-(1,3-dioxanyl)ethyl)-
pyridinium bromide

422-680-1

—

R52-53

601-071-00-6

1-dimethoxymethyl-2-nitro-ben-
zene

423-830-9

20627-73-0

R43
N; R51-53

601-073-00-7

1-bromo-3,5-difluorobenzene

416-710-2

461-96-1

R10
Xn; R22-48/22
Xi; R38
R43
N; R50-53

Xn
R: 22
S: (2-)

R: 52/53
S: 61

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

Xn; N
R: 10-22-38-43-48/22-
50/53
S: (2-)24-36/37-60-61

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
0
9

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

422-040-1

—

Xi; R36/38
N; R51-53

Xi; N
R: 36/38-51/53
S: (2-)26-37-61

601-074-00-2

A mixture of: 4-(2,2,3-
trimethylcyclopent-3-en-1-yl)-1-
methyl-2-oxabicyclo[2.2.2]oc-
tane
1-(2,2,3-trimethylcyclopent-3-
en-1-yl)-5-methyl-6-oxabicy-
clo[3.2.1]octane
spiro[cyclohex-3-en-1-yl-
[(4,5,6,6a-tetrahydro-3,6′,6′,6′a-
tetramethyl)-1,3′(3′aH)-
[2H]cyclopenta[b]furan]
spiro[cyclohex-3-en-1-yl-
[4,5,6,6a-tetrahydro-4,6′,6′,6′a-
tetramethyl)-1,3′(3′aH)-
[2H]cyclopenta[b]]furan]

602-093-00-9

α,α,α,4-tetrachlorotoluene
p-chlorobenzotrichloride

E

226-009-1

5216-25-1

602-094-00-4

diphenylether; octabromo
derivate

251-087-9

32536-52-0

Carc.Cat.2; R45
Repr.Cat.3; R62
T; R48/23
Xn; R21/22
Xi; R37/38

Repr.Cat.2; R61
Repr.Cat.3; R62

T
R: 45-21/22-37/38-48/
23-62
S: 53-45

T
R: 61-62
S: 53-45

602-096-00-5 malachite green hydrochloride

[1]
malachite green oxalate
[2]

209-322-8 [1]
219-441-7 [2]

569-64-2 [1]
18015-76-4 [2]

Xn; R22
Xi; R41
Repr. Cat. 3; R63
N; R50-53

Xn; N
R: 22-41-63-50/53
S: (2-)26-36/37-39-46-
60-61

602-097-00-0

1-bromo-9-(4,4,5,5,5-penta-
fluoropentylthio)nonane

422-850-5

148757-89-5

R43
N; R50-53

603-167-00-3

3,3′,5,5′-tetra-tert-butylbiphe-
nyl-2,2′-diol

407-920-5

6390-69-8

R 53

603-168-00-9

3-(2-ethylhexyloxy)propane-1,2-
diol

408-080-2

70445-33-9

Xi; R41
R 52-53

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

R: 53
S: 61

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

L

2
1
6
/
1
1
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

603-169-00-4

(+/-)-trans-4-(4-fluorophenyl)-3-
hydroxymethyl-N-methylpiperi-
dine

415-550-0

109887-53-8

415-990-3

67739-11-1

603-170-00-X

A mixture of: 2-methyl-1-(6-
methylbicyclo[2.2.1]hept-5-en-2-
yl)pent-1-en-3-ol
2-methyl-1-(1-methylbicy-
clo[2.2.1]hept-5-en-2-yl)-pent-1-
en-3-ol
2-methyl-1-(5-methylbicy-
clo[2.2.1]hept-5-en-2-yl)pent-1-
en-3-ol

603-171-00-5

5-thiazolylmethanol

414-780-9

38585-74-9

603-172-00-0 mono-2-[2-(4-diben-

415-180-1

—

zo[b,f][1,4]thiazepin-11-yl)piper-
azinium-1-yl]ethoxy)ethanol
trans-butenedioate

Xn; R22
Xi; R41
N; R51-53

Xi; R36
N; R51-53

Xi; R41
R 52-53

Xn; R22
Xi; R41
N; R51-53

603-173-00-6

4,4-dimethyl-3,5,8-trioxabicy-
clo[5.1.0]octane

421-750-9

57280-22-5

Xi; R36
R 43

603-174-00-1

4-cyclohexyl-2-methyl-2-butanol

420-630-3

83926-73-2

Xi; R41
N; R51-53

603-175-00-7

2-(2-hexyloxyethoxy)ethanol
DEGHE
diethylene glycol monohexyl
ether
3,6-dioxa-1-dodecanol
hexyl carbitol
3,6-dioxadodecan-1-ol

203-988-3

112-59-4

Xn; R21
Xi; R41

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

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

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

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

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

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

Xn
R: 21-41
S: (2-)26-36/37-46

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
1
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

603-176-00-2

1,2-bis(2-methoxyethoxy)ethane
TEGDME
triethylene glycol dimethyl ether
triglyme

203-977-3

112-49-2

R19
Repr. Cat.2; R61
Repr. Cat.3; R62

T
R: 61-19-62
S: 53-45

603-177-00-8

1-ethoxypropan-2-ol
2PG1EE
1-ethoxy-2-propanol
propylene glycol monoethyl
ether
[1]
2-ethoxy-1-methylethyl acetate
2PG1EEA
[2]

216-374-5 [1]
259-370-9 [2]

1569-02-4 [1]
54839-24-6
[2]

R10
R67

R: 10-67
S: (2-)24

603-178-00-3

2-hexyloxyethanol
ethylene glycol monohexyl ether
n-hexylglycol

203-951-1

112-25-4

Xn
R21/22
C; R34

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

603-179-00-9

ergocalciferol
Vitamin D2

200-014-9

50-14-6

T+; R26
T; R24/25-48/25

603-180-00-4

colecalciferol
Vitamin D3

200-673-2

67-97-0

T+; R26
T; R24/25-48/25

603-181-00-X

tert-butyl methyl ether
MTBE
2-methoxy-2-methylpropane

216-653-1

1634-04-4

F; R11
Xi; R38

603-183-00-0

2-[2-(2-butoxyethoxy)ethox-
y]ethanol
TEGBE
triethylene glycol monobutyl
ether
butoxytriethylene glycol

205-592-6

143-22-6

Xi; R41

T+
R: 24/25-26-48/25
S: (1/2-)28-36/37-45

T+
R: 24/25-26-48/25
S: (1/2-)28-36/37-45

F; Xi
R: 11-38
S: (2-)9-16-24

Xi
R: 41
S: (2-)26-39-46

C ≥ 30 %: Xi; R41
20 % ≤ C < 30 %: Xi; R36

L

2
1
6
/
1
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
o
p
e
a
n
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

603-184-00-6

2-(hydroxymethyl)-2-[[2-
hydroxy-3-(isooctadecyloxy)pro-
poxy]methyl]-1,3-propanediol

416-380-1

146925-83-9

N; R50-53

N
R: 50/53
S: 60-61

603-185-00-1

2,4-dichloro-3-ethyl-6-nitrophe-
nol

420-740-1

99817-36-4

T; R25
Xi; R41
R43
N; R50-53

T; N
R: 25-41-43-50/53
S: (1/2-)26-36/37/39-45-
60-61

603-186-00-7

trans-(5RS,6SR)-6-amino-2,2-
dimethyl-1,3-dioxepan-5-ol

419-050-3

79944-37-9

R 43

Xi
R: 43
S: (2-)22-24/25-26-37

603-187-00-2

2-((4,6-bis(4-(2-(1-methylpyridi-
nium-4-yl)vinyl)phenylamino)-
1,3,5-triazin-2-yl)(2-hydro-
xyethyl)amino)ethanol dichloride

603-189-00-3

A mixture of complexes of:
titanium, 2,2′-oxydiethanol,
ammonium lactate, nitrilotris(2-
propanol) and ethylene glycol

603-191-00-4

2-(4,6-bis(2,4-dimethylphenyl)-
1,3,5-triazin-2-yl)-5-(3-((2-ethyl-
hexyl)oxy)-2-hydroxypropoxy)-
phenol

419-360-9

163661-77-6

N; R50-53

405-250-8

—

N; R51-53

419-740-4

137658-79-8

R53

603-195-00-6

2-[4-(4-methoxyphenyl)-6-phe-
nyl-1,3,5-triazin-2-yl]-phenol

430-810-3

154825-62-4

R52-53

N
R: 50/53
S: 60-61

N
R: 51/53
S: 61

R: 53
S: 61

R: 52/53
S: 61

603-196-00-1

2-(7-ethyl-1H-indol-3-yl)ethanol

431-020-1

41340-36-7

Xn; 22-48/22
N; R51-53

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

603-197-00-7

1-(4-chlorophenyl)-4,4-dimethyl-
3-(1,2,4-triazol-1-ylmethyl)pen-
tan-3-ol

403-640-2

107534-96-3

Repr.Cat.3; R63
Xn; R22
N; R51-53

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

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
1
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

603-199-00-8

etoxazol

—

153233-91-1

N; R50-53

604-065-00-1

4,4′,4″-(1-methylpropan-1-yl-3-
ylidene)tris(2-cyclohexyl-5-
methylphenol)

407-460-5

111850-25-0

N; R51-53

604-066-00-7 A mixture of: phenol, 6-(1,1-

414-550-8

—

N; R50-53

C ≥ 0.25 %: N; R50/53
0.025 % ≤ C < 0.25 %: N; R51/53
0.0025 % ≤ C < 0.025 %: R52/53

N
R: 50/53
S: 60-61

N
R: 51/53
S: 61

N
R: 50/53
S: 60-61

dimethylethyl)-4-tetrapropyl-2-
[(2-hydroxy-5-tetra-propylphe-
nyl)methyl (C41-compound) and
methane, 2,2′-bis[6-(1,1-
dimethyl-ethyl)-1-hydroxy-4-tet-
rapropyl-phenyl)]- (C45-
compound)
2,6-bis(1,1-dimethylethyl)-4-
tetra-propyl-phenol and 2-(1,1-
dimethylethyl)-4-tetrapropyl-phe-
nol
2,6-bis[(6-(1,1-dimethylethyl)-1-
hydroxy-4-tetrapropylphenyl)-
methyl]-4-(tetrapropyl)phenol
and 2-[(6-(1,1-dimethylethyl)-1-
hydroxy-4-tetrapropylphenyl-
methyl]-6-[1-hydroxy-4-tetrapro-
pylphenyl)methyl]-4-(tetrapro-
pyl)phenol

A mixture of: 2,2′-[[(2-
hydroxyethyl)imino]bis(methyle-
ne)bis[4-dodecylphenol]
formaldehyde, oligomer with 4-
dodecyl phenol and 2-
aminoethanol(n = 2)
formaldehyde, oligomer with 4-
dodecyl phenol and 2-
aminoethanol(n = 3, 4 and
higher)

604-067-00-2

414-520-4

—

Xi; R38-41
N; R50-53

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

604-068-00-8

(+/-)-4-[2-[[3-(4-hydroxyphenyl)-
1-methylpropyl]amino]-1-hydro-
xyethyl]phenol hydrochloride

415-170-5

99095-19-9

Xn; R20/22
R 43

Xn
R: 20/22-43
S: (2-)24-26-37

L

2
1
6
/
1
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
r
o
p
e
a
n
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

604-069-00-3

2-(1-methylpropyl)-4-tert-butyl-
phenol

421-740-4

51390-14-8

C; R34
N; R51-53

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

222-182-2

3380-34-5

Xi; R36/38
N; R50-53

Xi; N
R: 36/38-50/53
S: 26-39-46-60-61

C ≥ 20 %: Xi, N; R36/38-50/53
0,25 % ≤ C < 20 %: N; R50/53
0,025 % ≤ C < 0,25 %: N; R51/53
0,0025 % ≤ C < 0,025 %: R52/53

421-890-0

—

R43

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

604-070-00-9

605-031-00-9

triclosan
2,4,4′-trichloro-2′-hydroxy-
diphenyl-ether
5-chloro-2-(2,4-dichlorophenox-
y)phenol

A mixture of: 2,2-
dimethoxyethanal (this
component is considered to be
anhydrous in terms of identity,
structure and composition.
However, 2,2-dimethoxyethanal
will exist in a hydrated form.
60 % anhydrous is equivalent to
70.4 % hydrate)
water(Including free water and
water in hydrated 2,2-
dimethoxyethanal)

606-062-00-0

tetrahydrothiopyran-3-carboxal-
dehyde

407-330-8

61571-06-0

Repr.Cat.2; R61
Xi; R41
R 52-53

T
R: 61-41-52/53
S: 53-45-61

606-063-00-6

(E)-3-(2-chlorophenyl)-2-(4-
fluorophenyl)propenal

410-980-5

112704-51-5

Xi; R36
R 43

606-064-00-1

pregn-5-ene-3,20-dione bis(ethy-
lene ketal)

407-450-0

7093-55-2

R 53

606-065-00-7

1-(4-morpholinophenyl)butan-1-
one

413-790-0

—

N; R51-53

606-066-00-2

(E)-5[(4-chlorophenyl)methy-
lene]-2,2-dimethylcyclopenta-
none

410-440-9

131984-21-9

N; R51-53

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

R: 53
S: 61

N
R: 51/53
S: 61

N
R: 51/53
S: 61

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
1
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

414-870-8

96792-67-5

N; R50-53

N
R: 50/53
S: 60-61

606-067-00-8

A mixture of: 1-(2,3,6,7,8,9-
hexahydro-1,1-dimethyl-1H-
benz(g)inden-4-yl)ethanone
1-(2,3,5,6,7,8-hexahydro-1,1-
dimethyl-1H-benz(f)inden-4-
yl)ethanone
1-(2,3,6,7,8,9-hexahydro-1,1-
dimethyl-1H-benz(g)inden-5-
yl)ethanone
1-(2,3,6,7,8,9-hexahydro-3,3-
dimethyl-1H-benz(g)inden-5-
yl)ethanone

606-068-00-3

2,7,11-trimethyl-13-(2,6,6-tri-
methylcyclohex-1-en-1-yl)tride-
cahexaen-2,4,6,8,10,12-al

415-770-7

1638-05-7

Xn; R48/22
R 43
R 52-53

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

606-069-00-9

spiro[1,3-dioxolane-2,5′-
(4′,4′,8′,8′-tetramethyl-hexahy-
dro-3′,9′-methanonaphthalene)]

606-070-00-4

5-(3-butyryl-2,4,6-trimethylphe-
nyl)-2-[1-(ethoxyimino)propyl]-
3-hydroxycyclohex-2-en-1-one

415-460-1

154171-77-4

N; R51-53

N
R: 51/53
S: 24-61

414-790-3

138164-12-2

Repr.Cat.3; R62-63
Xn; R22
Xi; R38
N; R50-53

Xn; N
R: 22-38-62-63-50/53
S: (2-)22-36/37-60-61

606-071-00-X

17-spiro(5,5-dimethyl-1,3-
dioxan-2-yl)androsta-1,4-diene-
3-one

421-050-3

13258-43-0

N; R50-53

606-072-00-5

3-acetyl-1-phenyl-pyrrolidine-
2,4-dione

421-600-2

719-86-8

Xn; R48/22
N; R51-53

N
R: 50/53
S: 22-60-61

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

606-073-00-0

4,4′-bis(dimethylamino)benzo-
phenone
Michler's ketone

202-027-5

90-94-8

Carc.Cat.2; R45
Muta.Cat.3; R68
Xi; R41

T
R: 45-41-68
S: 53-45

606-075-00-1

1-benzyl-5-ethoxyimidazolidine-
2,4-dione

417-340-4

65855-02-9

Xn; R22

Xn
R: 22
S: (2-)22

L

2
1
6
/
1
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

606-076-00-7

1-((2-quinolinyl-carbonyl)oxy)-
2,5-pyrrolidinedione

418-630-3

136465-99-1

Xi; R41
R43

606-077-00-2

(3S,4S)-3-hexyl-4-[(R)-2-hydro-
xytridecyl]-2-oxetanone

418-650-2

104872-06-2

N; R50-53

606-078-00-8

1-octylazepin-2-one

420-040-6

59227-88-2

606-079-00-3

2-n-butyl-benzo[d]isothiazol-3-
one

420-590-7

—

C; R34
R 43
N; R51-53

C; R34
R43
N; R50-53

606-080-00-9

Reaction product of: 3-hydroxy-
5,7-di-tert-butylbenzofuran-2-
one with o-xylene

606-081-00-4

(3β, 5α, 6β)-3-(acetyloxy)-5-
bromo-6-hydroxy-androstan-17-
one

606-082-00-X A mixture of: butan-2-one
oxime
syn-O,O′-di(butan-2-one oxime)-
diethoxysilane

417-100-9

—

R 53

419-790-7

4229-69-0

406-930-7

96-29-7

R43
R52-53

T; R48/22
R43
R52-53

Xi; R36/38
R43
R52-53

606-083-00-5

2-chloro-5-sec-hexadecylhydro-
quinone

407-750-1

—

606-084-00-0

1-(4-methoxy-5-benzofuranyl)-
3-phenyl-1,3-propanedione

414-540-3

484-33-3

N; R50-53

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

N
R: 50/53
S: 60-61

C; N
R: 34-43-51/53
S: (1/2-)26-36/37/39-45-
61

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

R: 53
S: 61

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

T
R: 43-48/25-52/53
S: (1/2-)25-36/37-45-61

Xi
R: 36/38-43-52/53
S: (2-)24-26-37-61

N
R: 50/53
S: 60-61

606-085-00-6

(1R,4S)-2-azabicyclo[2.2.1]hept-
5-en-3-one

418-530-1

79200-56-9

Xn; R22
Xi; R41
R43

Xn
R: 22-41-43
S: (2-)24-26-37/39

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
1
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

606-086-00-1

1-(3,3-dimethylcyclohexyl)pent-
4-en-1-one

422-330-8

56973-87-6

N; R51-53

606-087-00-7

6-ethyl-5-fluoro-4(3H)-pyrimi-
done

422-460-5

137234-87-8

Xn; R22
N; R50-53

606-088-00-2

2,4,4,7-tetramethyl-6-octen-3-
one

422-520-0

74338-72-0

Xi; R38
N; R51-53

606-089-00-8 A mixture of: 1,4-diamino-2-

423-220-2

12223-77-7

R53

chloro-3-phenoxyanthraquinone
1,4-diamino-2,3-bis-phenoxyan-
thraquinone

606-091-00-9

6-chloro-5-(2-chloroethyl)-1,3-
dihydroindol-2-one

421-320-0

118289-55-7

N; R50-53

606-092-00-4 A mixture of: (E)-

422-320-3

111879-80-2

N; R50-53

607-379-00-7

oxacyclohexadec-12-en-2-one
(E)-oxacyclohexadec-13-en-2-one
a) (Z)-oxacyclohexadec-(12)-en-
2-one and b) (Z)-
oxacyclohexadec-(13)-en-2-one

A mixture of: 2-[N-(2-
hydroxyethyl)stearamido]ethyl
stearate
sodium [bis[2-
(stearoyloxy)ethyl]amino]methyl-
sulfonate
sodium [bis(2-
hydroxyethyl)amino]methylsulfo-
nate
N,N-bis(2-hydroxyethyl)steara-
mide

401-230-8

55349-70-7

R52-53

N
R: 51/53
S: 61

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

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

R: 53
S: 61

N
R: 50/53
S: 60-61

N
R: 50/53
S: 60-61

R: 52/53
S: 61

L

2
1
6
/
1
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-380-00-2

A mixture of: ammonium-1,2-
bis(hexyloxycarbonyl)ethanesul-
fonate
ammonium-1-hexyloxycarbonyl-
2-octyloxycarbonylethanesulfo-
nate
ammonium-2-hexyloxycarbonyl-
1-octyloxycarbonylethanesulfo-
nate

407-320-3

—

Xi; R38-41
R 52-53

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

607-381-00-8 mixed triesters of 2,2-

413-710-4

—

R 53

bis(hydroxymethyl)butanol with
C7-alkanoic acids and 2-
ethylhexanoic acid

607-382-00-3

2-((4-amino-2-nitrophenyl)ami-
no)benzoic acid

411-260-3

117907-43-4

415-430-8

86403-32-9

Xi; R41
R 43
R 52-53

Xi; R41
R 43
N; R50-53

R: 53
S: 61

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

Xi; N
R: 41-43-50/53
S: (2-)24-26-37/39-60-
61

607-383-00-9

A mixture of: 2,2,6,6-
tetramethylpiperidin-4-yl-hexa-
decanoate
2,2,6,6-tetramethylpiperidin-4-
yl-octadecanoate

607-384-00-4

A mixture of: esters of C14-C15
branched alcohols with 3,5-di-t-
butyl-4-hydroxyphenyl propionic
acid
C15 branched and linear alkyl
3,5-bis(1,1-dimethylethyl)-4-
hydroxybenzenepropanoate
C13 branched and linear alkyl
3,5-bis(1,1-dimethylethyl)-4-
hydroxybenzenepropanoate

607-385-00-X

Copolymer of vinyl-alcohol and
vinyl acetate partially acetilized
with 4-(2-(4-
formylphenyl)ethenyl)-1-methyl-
pyridinium methylsulfate

413-750-2

171090-93-0

R 53

R: 53
S: 61

414-590-6

125229-74-5

N; R51-53

N
R: 51/53
S: 61

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
1
9

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-386-00-5

A mixture of: tetradecanoic acid
(42.5-47.5 %)
poly(1-7)lactate esters of
tetradecanoic acid (52.5-57.5 %)

607-387-00-0

A mixture of: dodecanoic acid
(35-40 %)
poly(1-7)lactate esters of
dodecanoic acid (60-65 %)

412-580-6

174591-51-6

412-590-0

58856-63-6

607-388-00-6

4-ethylamino-3-nitrobenzoic
acid

412-090-2

2788-74-1

Xi; R38-41
R 43
N; R50-53

Xi; R38-41
R 43
N; R50-53

Xn; R22
R 43
R 52-53

Xi; N
R: 38-41-43-50/53
S: (2-)24-26-37/39-60-
61

Xi; N
R: 38-41-43-50/53
S: (2-)24-26-37/39-60-
61

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

607-389-00-1

trisodium N,N-
bis(carboxymethyl)-3-amino-2-
hydroxypropionate

414-130-4

119710-96-2

Xn; R22

607-390-00-7

1,2,3,4-tetrahydro-6-nitro-qui-
noxaline

414-270-6

41959-35-7

Xn; R22
N; R51-53

607-391-00-2

dimethylcyclopropane-1,1-dicar-
boxylate

414-240-2

6914-71-2

R 52-53

607-392-00-8

2-phenoxyethyl 4-((5-cyano-1,6-
dihydro-2-hydroxy-1,4-dimethyl-
6-oxo-3-pyridinyl)azo)benzoate

414-260-1

88938-37-8

R 53

607-393-00-3

3-(cis-1-propenyl)-7-amino-8-
oxo-5-thia-1-azabicy-
clo[4.2.0]oct-2-ene-2-carboxylic
acid

415-750-8

106447-44-3

R 43

607-394-00-9

5-methylpyrazine-2-carboxylic
acid

413-260-9

5521-55-1

Xi; R41

Xn
R: 22
S: (2-)22

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

R: 52/53
S: 61

R: 53
S: 61

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

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

L

2
1
6
/
1
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-395-00-4

A mixture of: sodium 1-tridecyl-
4-allyl-(2 or 3)-sulfobutanedioate
sodium 1-dodecyl-4allyl-(2 or
3)-sulfobutanedioate

410-230-7

—

C; R34
R 43
N; R51-53

C; N
R: 34-43-51/53
S: (1/2-)26-36/37/39-45-
61

607-396-00-X

bis(1,2,2,6,6-pentamethyl-4-
piperidinyl) 2-(4-
methoxybenzylidene)malonate

607-397-00-5

A mixture of: Ca salicylates
(branched C10-14 and C18-30
alkylated)
Ca phenates (branched C10-14
and C18-30 alkylated)
Ca sulfurized phenates
(branched C10-14 and C18-30
alkylated)

607-398-00-0

ethyl N-(5-chloro-3-(4-
(diethylamino)-2-methylphenyli-
mino)-4-methyl-6-oxo-1,4-cyclo-
hexadienyl)carbamate

414-840-4

147783-69-5

N; R50-53

415-930-6

—

R 43

414-820-5

125630-94-6

N; R50-53

607-399-00-6

2,2-dimethyl 3-methyl-3-butenyl
propanoate

415-610-6

104468-21-5

Xi; R38
R52-53

607-400-00-X methyl 3-

414-400-1

32750-89-3

N; R50-53

[[(dibutylamino)thioxomethyl]-
thio]propanoate

607-401-00-5

ethyl 3-hydroxy-5-oxo-3-
cyclohexene-1-carboxylate

414-450-4

88805-65-6

Xi; R38-41
R 43

607-402-00-0 methyl N-(phenoxycarbonyl)-L-

414-500-5

153441-77-1

R 52-53

valinate

N
R: 50/53
S: 22-60-61

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

N
R: 50/53
S: 60-61

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

N
R: 50/53
S: 60-61

Xi
R: 38-41-43
S: (2-)24-26-37/39

R: 52/53
S: 61

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
2
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

414-810-0

—

Xn; R48/22
Xi; R41
N; R50-53

Xn; N
R: 41-48/22-50/53
S: (2-)22-26-36/39-60-
61

415-190-4

—

R 43

607-403-00-6

A mixture of: bis(1S,2S,4S)-(1-
benzyl-4-tert-butoxycarboxa-
mido-2-hydroxy-5-phenyl)
pentylammonium succinate
isopropyl alcohol

607-404-00-1

A mixture of: ((Z)-3,7-dimethyl-
2,6-octadienyl)oxycarbonylpro-
panoic acid
di-((E)-3,7-dimethyl-2,6-octadie-
nyl) butandioate
di-((Z)-3,7-dimethyl-2,6-octadie-
nyl) butandioate
(Z)-3,7-dimethyl-2,6-octadienyl
butandioate
((E)-3,7-dimethyl-2,6-octadienyl)
oxycarbonylpropanoic acid

607-405-00-7

2-hexyldecyl p-hydroxybenzoate

415-380-7

148348-12-3

N; R51-53

607-406-00-2

potassium 2,5-dichlorobenzoate

415-700-5

—

Xn; R22
Xi; R41

607-407-00-8

ethyl 2-carboxy-3-(2-
thienyl)propionate

415-680-8

143468-96-6

Xi; R38-41
R 43

607-408-00-3

potassium N-(4-
fluorophenyl)glycinate

607-409-00-9

A mixture of: (3R)-[1S-(1α, 2α,
6β-((2S)-2-methyl-1-oxo-
butoxy)-8a.gamma.)hexahydro-
2,6-dimethyl-1-naphthalene]-
3,5-dihydroxyheptanoic acid
inert biomass from Aspergillus
terreus

415-710-1

415-840-7

—

—

Xn; R48/22
Xi; R41
R 43
R 52-53

R 43
R 52-53

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

N
R: 51/53
S: 61

Xn
R: 22-41
S: (2-)26-39

Xi
R: 38-41-43
S: (2-)24-26-37/39

Xn
R: 41-43-48/22-52/53
S: (2-)22-26-36/37/39-
61

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

L

2
1
6
/
1
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-410-00-4 mono[2-(dimethylamino)ethyl]-

415-880-5

—

monohydrogen-2-(hexadec-2-
enyl)butanedioate and/or
mono[2-(dimethylamino)ethyl]-
monohydrogen-3-(hexadec-2-
enyl)butanedioate

Xi; R38-41
R 43
N; R50-53

Xi; N
R: 38-41-43-50/53
S: (2-)24-26-37/39-60-
61

607-411-00-X

oxiranemethanol, 4-
methylbenzene-sulfonate, (S)-

417-210-7

70987-78-9

Carc.Cat.2; R45
Muta.Cat.3; R68
Xi; R41
R43
N; R51-53

607-412-00-5

ethyl 2-(1-
cyanocyclohexyl)acetate

415-970-4

133481-10-4

Xn; R22-48/22
R 52-53

T; N
R: 45-41-43-51/53
S: 53-45-61

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

607-413-00-0

trans-4-phenyl-L-proline

416-020-1

96314-26-0

Repr.Cat.3; R62
R 43

Xn
R: 43-62
S: (2-)22-36/37

607-414-00-6

tris(2-ethylhexyl)-4,4′,4″-(1,3,5-
triazine-2,4,6-triyltriimino)tri-
benzoate

607-415-00-1

poly-(methyl methacrylate)-co-
(butylmethacrylate)-co-(4-acry-
loxybutyl-isopropenyl-.alpha.,.al-
pha.-dimethylbenzyl carbamate)-
co-(maleicanhydride)

607-416-00-7

4-(2-carboxymethylthio)ethoxy-
1-hydroxy-5-isobutyloxycarbo-
nylamino-N-(3-dodecyloxypro-
pyl)-2-naphthamide

402-070-1

88122-99-0

R53

419-590-1

—

F; R11
R 43

420-730-7

—

N; R50-53

607-418-00-8

2-ethylhexyl 4-aminobenzoate

420-170-3

26218-04-2

N; R50-53

R: 53
S: 61

F; Xi
R: 11-43
S: (2-)24-37-43

N
R: 50/53
S: 60-61

N
R: 50/53
S: 60-61

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
2
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-419-00-3

(3′-carboxymethyl-5-(2-(3-ethyl-
3H-benzothiazol-2-ylidene)-1-
methyl-ethylidene)-4,4′-dioxo-
2′-thioxo-(2,5′)bithiazolidinyli-
den-3-yl)-acetic acid

422-240-9

166596-68-5

Xi; R41
R 43

Xi
R: 41-43
S: (2-)26-36/37/39

607-420-00-9

2,2-bis(hydroxymethyl)butanoic
acid

424-090-1

10097-02-6

Xi; R41
R52-53

607-421-00-4

cypermethrin cis/trans +/- 40/60
(RS )-α-cyano-3-phenoxybenzyl
(1RS ,3RS ;1RS ,3SR )-3-(2,2-
dichlorovinyl)-2,2-dimethylcyclo-
propanecarboxylate

257-842-9

52315-07-8

607-422-00-X

α-cypermethrin

257-842-9

67375-30-8

607-423-00-5

esters of mecoprop and of
mecoprop-P

—

—

Xn; R20/22
Xi; R37
N; R50-53

T; R25
Xn; R48/22
Xi; R37
N; R50-53

Xn; R22
R43
N; R50-53

607-424-00-0

trifloxystrobin (ISO)
(E,E )-α-methoxyimino-{2-[[[[1-
[3-(trifluoromethyl)phenyl]ethy-
lidene]amino]oxy]methyl]benze-
neacetic acid methyl ester

—

141517-21-7

R43
N; R50-53

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

Xn; N
R: 20/22-37-50/53
S: (2-)24-36/37/39-60-
61

T; N
R: 25-37-48/22-50/53
S: (2-)36/37/39-45-60-
61

Xn; N
R: 22-43-50/53
S: (2-)13-36/37-60-61

Xi; N
R: 43-50/53
S: (2-)24-37-46-60-61

607-425-00-6 metalaxyl (ISO)

260-979-7

57837-19-1

methyl-N-(2,6-dimethylphenyl)-
N-(methoxyacetyl)-DL-alaninate

Xn; R22
R43
R52-53

Xn
R: 22-43-52/53
S: (2-)13-24-37-46-61

L

2
1
6
/
1
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-426-00-1

1,2-benzenedicarboxylic acid,
dipentylester, branched and
linear
[1]
n-pentyl-isopentylphthalate
[2]
di-n-pentyl phthalate
[3]
diisopentylphthalate
[4]

284-032-2 [1]
- [2]
205-017-9 [3]
210-088-4 [4]

84777-06-0 [1]
- [2]
131-18-0 [3]
605-50-5 [4]

Repr. Cat. 2; R60-61
N; R50

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

607-427-00-7

bromoxynil heptanoate (ISO)
2,6-dibromo-4-cyanophenyl
heptanoate

260-300-4

56634-95-8

Repr.Cat3; R63
Xn; R20/22
R43
N; R50-53

Xn; N
R: 20/22-43-63-50/53
S: (2-)36/37-46-60-61

607-430-00-3

BBP
benzyl butyl phtalate

201-622-7

85-68-7

Repr. Cat.2; R61
Repr. Cat.3; R62
N; R50-53

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

245-387-9

23031-36-9

T; R23
Xn; R22
N; R50-53

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

- [1]
- [2]

87392-12-9 [1]
178961-20-1
[2]

R43
N; R50-53

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

607-431-00-9

prallethrin
ETOC
2-methyl-4-oxo-3-(prop-2-ynyl)-
cyclopent-2-en-1-yl 2,2-
dimethyl-3-(2-methylprop-1-
enyl)cyclopropanecarboxylate

607-432-00-4

S-metolachlor
mixture of (S )-2-chloro-N-(2-
ethyl-6-methyl-phenyl)-N-(2-
methoxy-1-methyl-ethyl)-aceta-
mide (80-100 %)
[1]
S-metolachlor
(R )-2-chloro-N-(2-ethyl-6-
methyl-phenyl)-N-(2-methoxy-1-
methyl-ethyl)-acetamide (0-20 %)
[2]

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
2
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-433-00-X

cypermethrin cis/trans +/- 80/20
(RS )-α-cyano-3-phenoxybenzyl
(1RS ; 3RS ; 1RS , 3SR )-3-(2,2-
dichlorovinyl)-2,2-dimethylcyclo-
propanecarboxylate

257-842-9

52315-07-8

607-434-00-5 mecoprop-P [1] and its salts

240-539-0

16484-77-8

(R )-2-(4-chloro-2-
methylphenoxy)propionic acid

607-435-00-0

2S-isopropyl-5R-methyl-1R-
cyclohexyl 2,2-dihydroxyacetate

416-810-6

111969-64-3

607-436-00-6

2-hydroxy-3-(2-ethyl-4-methyli-
midazoyl)propyl neodecanoate

417-350-9

607-437-00-1

3-(4-aminophenyl)-2-cyano-2-
propenoic acid

417-480-6

607-438-00-7 methyl-2-[(aminosulfonyl)-

419-010-5

methyl]benzoate

—

—

—

Xn; R22
Xi; R37/38
R43
N; R50-53

Xn; R22
Xi; R41
N; R51-53

Xn; R48/22
Xi; R41
N; R51-53

Xi; R38-41
N; R50-53

R43

Xn; R22
Xi; R36

607-439-00-2 methyl tetrahydro-2-

furancarboxylate

420-670-1

37443-42-8

Xi; R41

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

Xn; N
R: 22-41-51/53
S: (2-)13-26-37/39-46-
61

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

Xi; N
R: 38-41-50/53
S: (2-)26-28-37/39-60-
61

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

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

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

607-440-00-8 methyl 2-aminosulfonyl-6-

421-220-7

144740-59-0

(trifluoromethyl)pyridine-3-c
arboxylate

R43
N; R51-53

Xi; N
R: 43-51/53
S: (2-)22-24-37-61

607-441-00-3

3-[3-(2-dodecyloxy-5-methylphe-
nylcarbamoyl)-4-hydroxy-1-
naphthylthio]propionic acid

421-490-6

167684-63-1

R53

R: 53
S: 57-61

L

2
1
6
/
1
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-442-00-9

benzyl [hydroxy-(4-
phenylbutyl)phosphinyl] acetate

416-050-5

87460-09-1

Xi; R41

607-443-00-4

bis(2,4-di-tert-butyl-6-methyl-
phenyl)ethyl phosphate

416-140-4

145650-60-8

R 53

607-444-00-X A mixture of: cis-1,4-

416-230-3

35541-81-2

R 53

dimethylcyclohexyl dibenzoate
trans-1,4-dimethylcyclohexyl
dibenzoate

607-445-00-5

Iron (III) tris(4-
methylbenzenesulfonate)

420-960-8

77214-82-5

Xi; R41

Xi
R: 41
S: (2-)26-36/39

R: 53
S: 61

R: 53
S: 61

Xi
R: 41
S: (2-)24-26-39

607-446-00-0 methyl 2-[4-(2-chloro-4-

416-240-8

155522-12-6

nitrophenylazo)-3-(1-oxopropyl)
amino]phenylaminopropionate

R 43
R 53

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

607-447-00-6

sodium 4-[4-(4-
hydroxyphenylazo)phenyla-
mino]-3-nitrobenzenesulfonate

416-370-5

156738-27-1

R 43
R52-53

607-448-00-1

2,3,5,6-tetrafluorobenzoic acid

416-800-1

652-18-6

Xi; R38-41

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

Xi
R: 38-41
S: (2-)22-26-37/39

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
2
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

417-080-1

—

E; R2
R43
N; R50-53

E; Xi; N
R: 2-43-50/53
S: (2-)24-35-37-60-61

607-449-00-7

A mixture of: 4,4′,4″-[(2,4,6-
trioxo-1,3,5(2H,4H,6H)-triazine-
1,3,5-triyl)tris[methylene(3,5,5-
trimethyl-3,1-cyclohexanediyl)i-
minocarbonyloxy-2,1-ethanediy-
l(ethyl)amino]]trisbenzenediazo-
niumtri[bis(2-methylpropyl)-
naphthalenesulfonate]
4,4′,4″,4‴-[[5,5′-[carbonylbis[i-
mino(1,5,5-trimethyl-3,1-cyclo-
hexanediyl)methylene]]-2,4,6-
trioxo-1,3,5(2H,4H,6H)-triazine-
1,1′,3,3′-tetrayl]tetrakis[methy-
lene(3,5,5-trimethyl-3,1-cyclo-
hexanediyl)iminocarbonyloxy-
2,1-ethanediyl(ethyl)amino]]te-
trakisbenzenediazoniumtetra[-
bis(2-methylpropyl)naphthalene-
sulfonate]

607-450-00-2

2-mercaptobenzothiazolyl-(Z)-
(2-aminothiazol-4-yl)-2-(tert-
butoxycarbonyl) isopropoxyimi-
noacetate

419-040-9

89604-92-2

R 53

R: 53
S: 61

607-451-00-8

4-[4-amino-5-hydroxy-3-(4-(2-
sulfoxyethylsulfonyl)phenylazo)-
2,7-disulfonapht-6-ylazo]-6-[3-
(4-amino-5-hydroxy-3-(4-(2-sul-
foxyethylsulfonyl)phenylazo)-
2,7-disulfonapht-6-ylazo]phenyl-
carbonylamino]benzenesulfonic
acid, sodium salt

417-640-5

161935-19-9

Xi; R41
R43

Xi
R: 41-43
S: (2-)22-24-26-37/39

607-453-00-9

4-benzyl-2,6-dihydroxy-4-aza-
heptylene bis(2,2-
dimethyloctanoate)

418-100-1

172964-15-7

R 43
R 53

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

L

2
1
6
/
1
2
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-454-00-4

A mixture of: trans-2-(1-
methylethyl)-1,3-dioxane-5-car-
boxylic acid;
cis-2-(1-methylethyl)-1,3-diox-
ane-5-carboxylic acid

607-455-00-X

1-amino-4-(3-[4-chloro-6-(2,5-
di-sulfophenylamino)-1,3,5-tria-
zin-2-ylamino]-2,2-dimethyl-
propylamino)-anthraquinone-2-
sulfonic acid, na/li salt

418-170-3

—

Xi; R41
R52-53

Xi
R: 41-52/53
S: (2-)25-26-39-61

419-520-8

172890-93-6

R 43

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

N
R: 51/53
S: 61

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

N
R: 51/53
S: 61

607-456-00-5

3-amino-4-chlorobenzoic acid,
hexadecyl ester

419-700-6

143269-74-3

N; R51-53

420-350-1

172277-97-3

Xi; R41
N; R51-53

420-850-1

—

N; R51-53

607-457-00-0

tetrasodium dihydrogen 1,1″-
dihydroxy-8,8″-[p-phenylbis(i-
mino-{6-[4-(2-aminoethyl)piper-
azin-1-yl]}-1,3,5-triazine-4,2-
diyl-imino)]bis(2,2′-azonaphtha-
lene-1′,3,6-trisulfonate)

607-458-00-6

A mixture of: 2-ethyl-[2,6-
dibromo-4-[1-[3,5-dibromo-4-
(2-hydroxyethoxy)phenyl]-1-
methylethyl]phenoxy]propenoate
2,2′-diethyl-[4,4′-bis(2,6-dibro-
mophenoxy)-1-methylethylidene]
dipropenoate
2,2′-[(1-methylethylidene)-
bis[[2,6-dibromo-4,1-phenyle-
ne)oxy]ethanol]]

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
2
9

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-459-00-1

isopentyl 4-{2-[5-cyano-1,2,3,6-
tetrahydro-1-(2-isopropox-
yethoxy-carbonylmethyl)-4-
methyl-2,6-dioxo-3-pyridylide-
ne]hydrazino}benzoate

418-930-4

—

R 53

R: 53
S: 61

607-460-00-7

3-tridecyloxy-propyl-ammonium
9-octadecenoate

418-990-1

—

Xn; R48/22
Xi; R36/38
N; R50-53

Xn; N
R: 36/38-48/22-50/53
S: (2-)23-26-37/39-60-
61

607-461-00-2

A mixture of: pentasodium 2-{4-
{3-methyl-4-[6-sulfonato-4-(2-
sulfonato-phenylazo)-naphtha-
len-1-ylazo]-phenylamino}-6-[3-
(2-sulfato-ethanesulfonyl)-pheny-
lamino]-1,3,5-triazin-2-yla-
mino}-benzene-1,4-disulfonate
pentasodium 2-{4-{3-methyl-4-
[7-sulfonato-4-(2-sulfonato-phe-
nylazo)-naphthalen-1-ylazo]-
phenylamino}-6-[3-(2-sulfato-
ethanesulfonyl)-phenylamino]-
1,3,5-triazin-2-ylamino}-ben-
zene-1,4-disulfonate

607-462-00-8

A mixture of: 1-hexyl acetate
2-methyl-1-pentyl acetate
3-methyl-1-pentyl acetate;
4-methyl-1-pentyl acetate
other mixed linear and branched
C6-alkyl acetates

421-160-1

—

R 52-53

R: 52/53
S: 61

421-230-1

88230-35-7

N; R51-53

N
R: 51/53
S: 61

L

2
1
6
/
1
3
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-463-00-3

3-(phenothiazin-10-yl)propionic
acid

421-260-5

362-03-8

N; R51-53

607-464-00-9

A mixture of: 7-chloro-1-ethyl-
6-fluoro-1,4-dihydro-4-oxo-qui-
noline-3-carboxylic acid
5-chloro-1-ethyl-6-fluoro-1,4-
dihydro-4-oxo-quinoline-3-car-
boxylic acid

607-465-00-4

tris(2-hydroxyethyl)ammonium
7-{4-[4-(2-cyanoamino-4-
hydroxy-6-oxidopyrimidin-5-yla-
zo)benzamido]-2-ethoxy-pheny-
lazo}naphthalene-1,3-disulfonate

421-280-4

68077-26-9

R 52-53

421-440-3

—

R 52-53

607-466-00-X A mixture of: phenyl 1-(1-[2-

421-480-1

—

N; R51-53

chloro-5-(hexadecyloxycarbonyl)-
phenylcarbamoyl]-3,3-dimethyl-
2-oxobutyl)-1H-2,3,3a,7a-tetra-
hydrobenzotriazole-5-carboxy-
late
phenyl 2-(1-(2-chloro-5-
(hexadecyloxycarbonyl)phenyl-
carbamoyl)-3,3-dimethyl-2-oxo-
butyl)-1H-2,3,3a,7a-tetrahydro-
benzotriazole-5-carboxylate
phenyl 3-(1-(2-chloro-5-
(hexadecyloxycarbonyl)phenyl-
carbamoyl)-3,3-dimethyl-2-oxo-
butyl)-1H-2,3,3a,7a-tetrahydro-
benzotriazole-5-carboxylate

N
R: 51/53
S: 24/25-61

R: 52/53
S: 61

R: 52/53
S: 61

N
R: 51/53
S: 37/39-61

607-467-00-5

1,1,3,3-tetrabutyl-1,3-ditinoxydi-
caprylate

419-430-9

56533-00-7

Xn; R21/22-48/22
C; R34
N; R50-53

C; N
R: 21/22-34-48/22-50/
53
S: (1/2-)26-36/37/39-45-
60-61

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
3
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-468-00-0

A mixture of: monosodium 4-
((4-(5-sulfonato-2-methoxyphe-
nylamino)-6-chloro-1,3,5-tria-
zine-2-yl)amino)-2-((1,4-
dimethyl-6-oxido-2-oxo-5-sulfo-
natomethyl-1,2-dihydropyridine-
3-yl)azo)benzenesulfonate
disodium 4-((4-(5-sulfonato-2-
methoxyphenylamino)-6-chloro-
1,3,5-triazine-2-yl)amino)-2-
((1,4-dimethyl-6-oxido-2-oxo-5-
sulfonatomethyl-1,2-dihydropyri-
dine-3-yl)azo)benzenesulfonate
trisodium 4-((4-(5-sulfonato-2-
methoxyphenylamino)-6-chloro-
1,3,5-triazine-2-yl)amino)-2-
((1,4-dimethyl-6-oxido-2-oxo-5-
sulfonatomethyl-1,2-dihydropyri-
dine-3-yl)azo)benzenesulfonate
tetrasodium 4-((4-(5-sulfonato-2-
methoxyphenylamino)-6-chloro-
1,3,5-triazine-2-yl)amino)-2-
((1,4-dimethyl-6-oxido-2-oxo-5-
sulfonatomethyl-1,2-dihydropyri-
dine-3-yl)azo)benzenesulfonate

607-469-00-6

disodium 7-((4,6-bis(3-
diethylaminopropylamino)-1,3,5-
triazine-2-yl)amino)-4-hydroxy-
3-(4-(4-sulfonatophenylazo)phe-
nylazo)-2-naphthalene sulfonate

607-470-00-1

potassium sodium 6,13-
dichloro-3,10-bis{2-[4-[3-(2-
hydroxysulphonyloxyethanesul-
fonyl)phenylamino]-6-(2,5-disul-
fonatophenylamino)-1,3,5-tria-
zin-2-ylamino]ethylamino}ben-
zo[5,6][1,4]oxazino[2,3-b]phe-
noxazine-4,11-disulfonate

419-450-8

—

R43

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

419-460-2

120029-06-3

R52-53

R: 52/53
S: 61

414-100-0

—

Xi; R41
R52-53

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

L

2
1
6
/
1
3
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-472-00-2

ammonium iron(III)
trimethylenediaminetetraacetate
hemihydrate

400-660-3

111687-36-6

N; R51-53

607-474-00-3

(4-(4-(4-dimethylaminobenzyli-
den-1-yl)-3-methyl-5-oxo-2-pyr-
azolin-1-yl)benzoic acid

410-430-4

117573-89-4

R53

412-940-2

148878-18-6

R43

607-475-00-9

A mixture (50/50) of:
tetrasodium 7-(4-[4-chloro-6-
[methyl-(3-sulfonatophenyl)a-
mino]-1,3,5-triazin-2-ylamino]-
2-ureidophenylazo)naphthalene-
1,3,6-trisulfonate
tetrasodium 7-(4-[4-chloro-6-
[methyl-(4-sulfonatophenyl)a-
mino]-1,3,5-triazin-2-ylamino]-
2-ureidophenylazo)naphthalene-
1,3,6-trisulfonate

N
R: 51/53
S: 61

R: 53
S: 61

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

607-476-00-4

trisodium N,N-
bis(carboxymethyl)-β-alanine

414-070-9

129050-62-0

C; R34
R52-53

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

607-478-00-5

tetramethylammonium hydrogen
phthalate

416-900-5

79723-02-7

T; R25
Xn; R48/22
N; R50

T; N
R: 25-48/22-50
S: (1/2-)25-36-45-61

607-479-00-0

hexadecyl 4-chloro-3-[2-(5,5-
dimethyl-2,4-dioxo-1,3-oxazoli-
din-3-yl)-4,4-dimethyl-3-oxopen-
tamido]benzoate

418-550-9

168689-49-4

R53

607-480-00-6

1,2-benzenedicarboxylic acid
di-C7-11-branched and linear
alkylesters

271-084-6

68515-42-4

Repr. Cat. 2; R61
Repr. Cat. 3; R62

R: 53
S: 61

T
R: 61-62
S: 53-45

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
3
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

402-660-9

—

Repr.Cat.2; R61
R52-53

T
R: 61-52/53
S: 53-45-61

607-487-00-4

A mixture of: disodium 4-(3-
ethoxycarbonyl-4-(5-(3-ethoxy-
carbonyl-5-hydroxy-1-(4-sulfo-
natophenyl)pyrazol-4-yl)penta-
2,4-dienylidene)-4,5-dihydro-5-
oxopyrazol-1-yl)benzenesulfo-
nate
trisodium 4-(3-ethoxycarbonyl-
4-(5-(3-ethoxycarbonyl-5-oxido-
1-(4-sulfonatophenyl)pyrazol-4-
yl)penta-2,4-dienylidene)-4,5-
dihydro-5-oxopyrazol-1-yl)ben-
zenesulfonate

607-488-00-X

ethyl (2-acetylamino-5-fluoro-4-
isothiocyanatophenoxy)acetate

414-210-9

147379-38-2

N; R50-53

607-489-00-5

A mixture of: 2-ethylhexyl
linolenate, linoleate and oleate
2-ethylhexyl epoxyoleate
2-ethylhexyl diepoxylinoleate
2-ethylhexyl triepoxylinolenate

414-890-7

71302-79-9

R43

607-490-00-0

N-[2-hydroxy-3-(C12-16-alkyl-
oxy)propyl]-N-methyl glycinate

415-060-7

—

Xi; R41
R43

607-492-00-1

2-(1-(3′,3′-dimethyl-1′-cyclo-
hexyl)ethoxy)-2-methyl propyl
propanoate

415-490-5

141773-73-1

N; R51-53

607-493-00-7 methyl (3aR,4R,7aR)-2-methyl-

415-670-3

78850-37-0

Xi; R41

4-(1S,2R,3-triacetoxypropyl)-
3a,7a-dihydro-4H-pyrano[3,4-
d]oxazole-6-carboxylate

607-494-00-2

bis(2-ethylhexyl)octylphospho-
nate

417-170-0

52894-02-7

N; R50-53

N
R: 50/53
S: 60-61

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

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

N
R: 51/53
S: 61

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

N
R: 50/53
S: 60-61

L

2
1
6
/
1
3
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-495-00-8

sodium 4-sulfophenyl-6-((1-
oxononyl)amino)hexanoate

417-550-6

168151-92-6

R43

607-496-00-3

2,2′-methylenebis(4,6-di-tert-
butyl-phenyl)-2-ethylhexyl phos-
phite

418-310-3

126050-54-2

R53

607-497-00-9

cerium oxide isostearate

419-760-3

—

R53

607-498-00-4

(E)-3,7-dimethyl-2,6-octadienyl-
hexadecanoate

421-370-3

3681-73-0

Xi; R38
R53

607-499-00-X

bis(dimethyl-(2-hydroxyethyl)am-
monium) 1,2-ethanediyl-bis(2-
hexadecenylsuccinate)

607-500-00-3

calcium 2,2,bis[(5-
tetrapropylene-2-hydroxy)pheny-
l]ethanoate

607-501-00-9

A mixture of:
triphenylthiophosphate and
tertiary butylated phenyl
derivatives

421-660-1

421-670-4

—

—

Xi; R41
R43
N; R51-53

Xi; R38
N; R50-53

421-820-9

—

R53

607-502-00-4

(N-benzyl-N,N,N-tributyl)ammo-
nium 4-dodecylbenzenesulfonate

422-200-0

—

C; R34
Xn; R22
N; R51-53

607-503-00-X

2,4,6-tri-n-propyl-2,4,6-trioxo-
1,3,5,2,4,6-trioxatriphosphori-
nane

422-210-5

68957-94-8

C; R34

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

R: 53
S: 61

R: 53
S: 61

Xi
R: 38-53
S: (2-)37-61

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

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

R: 53
S: 61

C; N
R: 22-34-51/53
S: (1/2-)26-36/37/39-45-
61

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

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
3
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

607-505-00-0

pentasodium 7-(4-(4-(5-amino-
4-sulfonato-2-(4-((2-(sulfonato-
ethoxy)sulfonyl)phenylazo)phe-
nylamino)-6-chloro-1,3,5-tria-
zin-2-yl)amino-2-ureidophenyla-
zo)naphtalene-1,3,6-trisulfonate

607-506-00-6

A mixture of: strontium (4-
chloro-2-((4,5-dihydro-3-methyl-
5-oxo-1-(3-sulfonatophenyl)-1H-
pyrazol-4-yl)azo)-5-methyl)ben-
zenesulfonate
disodium (4-chloro-2-((4,5-
dihydro-3-methyl-5-oxo-1-(3-
sulfonatophenyl)-1H-pyrazol-4-
yl)azo)-5-methyl)benzenesulfo-
nate

607-507-00-1

potassium,sodium 2,4-diamino-
3-[4-(2-sulfonatoethoxysulfonyl)-
phenylazo]-5-[4-(2-sulfona-
toethoxysulfonyl)-2-sulfonato-
phenylazo]-benzenesulfonate

607-508-00-7

disodium 3,3′-
[iminobis[sulfonyl-4,1-pheny-
lene-(5-hydroxy-3-methylpyra-
zole-1,4-diyl)azo-4,1-phenylene-
sulfonylimino-(4-amino-6-hydro-
xypyrimidine-2,5-diyl)azo-4,1-
phenylenesulfonylimino(4-
amino-6-hydroxypyrimidine-2,5-
diyl)azo]bis(benzenesulfonate)]

422-930-1

171599-84-1

R52-53

422-970-8

136248-04-9

N; R51-53

422-980-2

187026-95-5

Xi; R41

423-110-4

—

Xi; R41

R: 52/53
S: 22-61

N
R: 51/53
S: 22-61

Xi
R: 41
S: (2-)22-26-39

Xi
R: 41
S: (2-)22-26-39

607-512-00-9

trisodium 2,4-diamino-3,5-bis-
[4-(2-sulfonatoethoxy)sulfonyl)-
phenylazo]benzenesulfonate

423-970-0

182926-43-8

R52-53

R: 52/53
S: 22-61

L

2
1
6
/
1
3
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

423-200-3

—

Xi; R41
R43
R52-53

Xi
R: 41-43-52/53
S: 22-26-36/37/39-61

607-513-00-4

A mixture of: Trisodium 4-
benzoylamino-6-(6-ethenesulfo-
nyl-1-sulfato-naphthalen-2-
ylazo)-5-hydroxynaphthalene-
2,7-disulfonate
5-(benzoylamino)-4-hydroxy-3-
((1-sulfo-6-((2-(sulfooxy)ethyl)-
sulfonyl)-2-naphtyl)azo)naphtha-
lene-2,7-disulfonic acid sodium
salt
5-(benzoylamino)-4-hydroxy-3-
((1-sulfo-6-((2-(sulfooxy)ethyl)-
sulfonyl)-2-naphtyl)azo)naphtha-
lene-2,7-disulfonic acid

607-515-00-5

A mixture of: disodium
hexyldiphenyl ether disulphonate
disodium dihexyldiphenyl ether
disulphonate

429-650-7

147732-60-3

Xi; R36
N; R51-53

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

607-516-00-0

N,N′-bis(trifluoroacetyl)-S,S′-bis-
L-homocysteine

429-670-6

105996-54-1

Xi; R41
R43

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

607-517-00-6

(S)-α-(acetylthio)benzenepropa-
noic acid

430-300-0

76932-17-7

Xn; R22
Xi; R41
R43

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

607-526-00-5

cartap
1,3-bis(carbamoylthio)-2-
(dimethylamino)propane

—

15263-53-3

N; R50-53

N
R: 50/53
S: 60-61

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
3
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

423-180-6

—

Xn; R48/22

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

607-527-00-0

A mixture of: 1-
(1′H,1′H,2′H,2′H-tridecafluor-
ooctyl)-12-(1″H,1″H,2″H,2″H-tri-
decafluorooctyl)dodecanedioate
1-(1′H,1′H,2′H,2′H-tridecafluor-
ooctyl)-12-(1″H,1″H,2″H,2″H-
heptdecafluorodecyl)dodecane-
dioate
1-(1′H,1′H,2′H,2′H-tridecafluor-
ooctyl)-12-(1″H,1″H,2″H,2″H-
heneicosafluorododecyl)dodeca-
nedioate
1-(1′H,1′H,2′H,2′H-tridecafluor-
ooctyl)-12-(1″H,1″H,2″H,2″H-
pentacosafluorotetradecyl)dode-
canedioate
1-(1′H,1′H,2′H,2′H-heptadeca-
fluorodecyl)-12-
(1″H,1″H,2″H,2″H-heptadeca-
fluorodecyl)dodecanedioate
1-(1′H,1′H,2′H,2′H-heptadeca-
fluorodecyl)-12-
(1″H,1″H,2″H,2″H-heneicosa-
fluorododecyl)dodecanedioate

608-031-00-7

2-benzyl-2-methyl-3-butenitrile

407-870-4

97384-48-0

608-033-00-8 N-butyl-3-(2-chloro-4-nitrophe-

407-970-8

75511-91-0

nylhydrazono)-1-cyano-2-
methylprop-1-ene-1,3-dicarboxi-
mide

Xn; R22
R 52-53

R 43
R 52-53

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

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

608-034-00-3

chlorfenapyr
4-bromo-2-(4-chlorophenyl)-1-
ethoxymethyl-5-trifluoromethyl-
pyrrole-3-carbonitrile

—

122453-73-0

T; R23
Xn; R22
N; R50-53

608-035-00-9

(+/-)-α-[(2-acetyl-5-methylphe-
nyl)-amino]-2,6-dichloroben-
zene-aceto-nitrile

419-290-9

—

R43
R53

T; N
R: 22-23-50/53
S: (1/2-)13-36/37-45-60-
61

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

L

2
1
6
/
1
3
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

608-036-00-4

3-(2-{4-[2-(4-cyanophenyl)vinyl]-
phenyl}vinyl)benzonitrile

419-060-8

79026-02-1

R 53

608-037-00-X A mixture of: (E)-2,12-

422-190-8

124071-40-5

N; R50-53

tridecadiennitrile
(E)-3,12-tridecadiennitrile
(Z)-3,12-tridecadiennitrile

608-038-00-5

2,2,4-trimethyl-4-phenyl-butane-
nitrile

422-580-8

75490-39-0

608-039-00-0

2-phenylhexanenitrile

423-460-8

3508-98-3

Xn; R22
N; R51-53

Xn; R22
N; R50-53

608-040-00-6

4,4′-dithiobis(5-amino-1-(2,6-
dichloro-4-(trifluoromethyl)phe-
nyl)-1H-pyrazole-3-carbonitrile)

608-041-00-1

4′-((2-butyl-4-oxo-1,3-diazaspir-
o[4.4]non-1-ene-3-
yl)methyl)(1,1′-biphenyl)-2-car-
bonitrile

423-490-1

130755-46-3

N; R50-53

423-500-4

138401-24-8

N; R50-53

R: 53
S: 61

N
R: 50/53
S: 60-61

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

Xn; N
R: 22-50/53
S: (2-)23-60-61

N
R: 50/53
S: 60-61

N
R: 50/53
S: 60-61

608-043-00-2

3-(cis-3-hexenyloxy)propanenitril

415-220-6

142653-61-0

T; R23
Xn; R22
N; R50-53

T; N
R: 22-23-50/53
S: (1/2-)13-36/37-45-60-
61

609-064-00-X mesotrione

2-[4-(methylsulfonyl)-2-nitro-
benzoyl]-1,3-cyclohexanedione

609-066-00-0

lithium sodium 3-amino-10-{4-
(10-amino-6,13-dichloro-4,11-
disulfonatobenzo[5,6][1,4]oxa-
zino[2,3-b]phenoxazine-3-yla-
mino)-6-[methyl(2-sulfonato-
ethyl)amino]-1,3,5-triazin-2-yla-
mino}-6,13-dichloroben-
zo[5,6][1,4]oxazino[2,3-b]phe-
noxazine-4,11-disulfonate

—

104206-82-8

N; R50-53

418-870-9

154212-58-5

Xn; R20/21/22-68/20/
21/22

N
R: 50/53
S: 60-61

Xn
R: 20/21/22-68/20/21/
22
S: (2-)36/37

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
3
9

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

609-067-00-6

sodium and potassium 4-(3-
aminopropylamino)-2,6-bis[3-(4-
methoxy-2-sulfophenylazo)-4-
hydroxy-2-sulfo-7-naphthyla-
mino]-1,3,5-triazine

416-280-6

156769-97-0

R 43

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

609-068-00-1 musk xylene

201-329-4

81-15-2

5-tert-butyl-2,4,6-trinitro-m-
xylene

609-070-00-2

1,4-dichloro-2-(1,1,2,3,3,3-hexa-
fluoropropoxy)-5-nitrobenzene

415-580-4

130841-23-5

Carc. Cat. 3; R40
E; R2
N; R50-53

E; Xn; N
R: 2-40-50/53
S: (2-)36/37-46-60-61

Xn; R22
R 43
N; R50-53

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

609-071-00-8

611-099-00-0

611-100-00-4

A mixture of: 2-methylsulfanyl-
4,6-bis-(2-hydroxy-4-methoxy-
phenyl)-1,3,5-triazine
2-(4,6-bis-methylsulfanyl-1,3,5-
triazin-2-yl)-5-methoxy-phenol

(methylenebis(4,1-phenyle-
nazo(1-(3-(dimethylamino)pro-
pyl)-1,2-dihydro-6-hydroxy-4-
methyl-2-oxopyridine-5,3-diyl)))-
1,1′-dipyridinium dichloride
dihydrochloride

potassium sodium 3,3′-(3(or4)-
methyl-1,2-phenylenebis(i-
mino(6-chloro)-1,3,5-triazine-
4,2-diylimino(2-acetamido-5-
methoxy)-4,1-phenylenazo)di-
naphthalene-1,5-disulfonate

611-101-00-X

2′-(4-chloro-3-cyano-5-formyl-
2-thienyl)azo-5′-diethylaminoa-
cetanilide

611-103-00-0

trisodium (1-(3-carboxylato-2-
oxido-5-sulfonatophenylazo)-5-
hydroxy-7-sulfonatonaphthalen-
2-amido)nickel(II)

423-520-3

156137-33-6

R43

401-500-5

—

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

403-810-6

140876-13-7

Xi; R41

405-200-5

104366-25-8

R43

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

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

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

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

407-110-1

—

Xi; R41
R 43
N; R51-53

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

L

2
1
6
/
1
4
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

406-870-1

—

R 43
N; R51-53

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

611-104-00-6

A mixture of: trisodium (2,4(or
2,6 or 4,6)-bis(3,5-dinitro-2-
oxidophenylazo)-5-hydroxyphe-
nolato)(2(or 4or 6)-(3,5-dinitro-
2-oxidophenylazo)-5-hydroxy-
4(or 2or 6)-(4-(4-nitro-2-
sulfonatoanilino)phenylazo)phe-
nolato)ferrate(1-)
trisodium bis(2,4(or 2,6 or 4,6)-
bis(3,5-dinitro-2-oxidopheny-
lazo)-5-hydroxyphenolato)fer-
rate(1-)
trisodium (2,4(or 2,6 or 4,6)-
bis(3,5-dinitro-2-oxidopheny-
lazo)-5-hydroxyphenolato)(2(or
4 or 6)-(3,5-dinitro-2-
oxidophenylazo)-5-hydroxy-4(or
2 or 6)-(4-nitro-2-
sulfonatophenylazo)phenolato)-
ferrate(1-)
trisodium (2,4(or 2,6 or 4,6)-
bis(3,5-dinitro-2-oxidopheny-
lazo)-5-hydroxyphenolato)(2(or
4 or 6)-(3,5-dinitro-2-
oxidophenylazo)-5-hydroxy-4(or
2 or 6)-(3-
sulfonatophenylazo)phenolato)-
ferrate(1-)
disodium 3,3′-(2,4-dihydroxy-
1,3(or 1,5 or 3,5)-
phenylenediazo)dibenzenesulfo-
nate

611-105-00-1

sodium 4-(4-chloro-6-(N-
ethylanilino)-1,3,5-triazin-2-yla-
mino)-2-(1-(2-chlorophenyl)-5-
hydroxy-3-methyl-1H-pyrazol-4-
ylazo)benzenesulfonate

611-106-00-7

hexasodium 4,4′-dihydroxy-
3,3′-bis[2-sulfonato-4-(4-sulfo-
natophenylazo)phenylazo]-
7,7′[p-phenylenebis[imino(6-
chloro-1,3,5-triazine-4,2-diyl)i-
mino]]dinaphthalene-2-sulfonate

407-800-2

136213-75-7

R 43
N; R51-53

Xi; N
R: 43-51/53
S: (2-)22-24-37-61

410-180-6

—

Xi; R41

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

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
4
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

611-107-00-2

potassium sodium 4-(4-chloro-
6-(3,6-disulfonato-7-(5,8-disulfo-
nato-naphthalen-2-ylazo)-8-
hydroxy-naphthalen-1-ylamino)-
1,3,5-triazin-2-ylamino)-5-
hydroxy-6-(4-(2-sulfatoethane-
sulfonyl)-phenylazo)-naphtha-
lene-1,7-disulfonate

412-490-7

—

R 43

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

611-108-00-8

disodium 5-((4-((4-chloro-3-
sulfonatophenyl)azo)-1-naphthy-
l)azo)-8-(phenylamino)-1-
naphthalenesulfonate

413-600-6

6527-62-4

R 52-53

611-109-00-3

Reaction products of: copper(II)
sulfate and tetrasodium 2,4-
bis[6-(2-methoxy-5-sulfonato-
phenylazo)-5-hydroxy-7-sulfo-
nato-2-naphthylamino]-6-(2-
hydroxyethylamino)-1,3,5-tria-
zine (2:1)

407-710-3

—

N; R51-53

R: 52/53
S: 61

N
R: 51/53
S: 61

611-110-00-9

tetra-sodium/lithium 4,4′-bis-(8-
amino-3,6-disulfonato-1-
naphthol-2-ylazo)-3-methylazo-
benzene

408-210-8

124605-82-9

R 43
N; R51-53

Xi; N
R: 43-51/53
S: (2-)24-28-37-61

611-111-00-4

disodium 2-[[4-(2-
chloroethylsulfonyl)phenyl]-[(2-
hydroxy-5-sulfo-3-[3-[2-(2-(sul-
fooxy)ethylsulfonyl)ethylazo]-4-
sulfobenzoato(3-)cuprate(1-)

414-230-8

—

R 43

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

L

2
1
6
/
1
4
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

.

.

1
6
6
2
0
0
4

611-112-00-X

tetrasodium 4-hydroxy-5-[4-[3-
(2-sulfatoethanesulfonyl)phenyla-
mino]-6-morpholin-4-yl-1,3,5-
triazin-2-ylamino]-3-(1-sulfona-
tonaphthalen-2-ylazo)naphtha-
lene-2,7-disulfonate

611-113-00-5

lithium sodium (2-(((5-((2,5-
dichlorophenyl)azo)-2-hydroxy-
phenyl)methylene)amino)ben-
zoato(2-))(2-((4,5-dihydro-3-
methyl-5-oxo-1-phenyl-1H-pyra-
zol-4-yl)azo)-5-sulfobenzoato(3-
)) chromate(2-)

611-114-00-0

lithium sodium (4-((5-chloro-2-
hydroxyphenyl)azo)-2,4-dihydro-
5-methyl-3H-pyrazol-3-onato(2-
))(3-((4,5-dihydro-3-methyl-1-(4-
methylphenyl)-5-oxo-1H-pyra-
zol-4-yl)azo)-4-hydroxy-5-nitro-
benzenesulfonato(3-))chro-
mate(2-)

413-070-6

—

R 43

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

414-280-0

149626-00-6

N; R51-53

N
R: 51/53
S: 24/25-61

414-250-7

149564-66-9

Xn; R22
Xi; R41
R 52-53

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

611-115-00-6

trilithium bis(4-((4-
(diethylamino)-2-hydroxypheny-
l)azo)-3-hydroxy-1-naphthalene-
sulfonato(3-))chromate(3-)

414-290-5

149564-65-8

Xn; R22
R 52-53

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

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n
U
n
o
n

i

L

2
1
6
/
1
4
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

414-620-8

—

Xi; R41
R 43

Xi
R: 41-43
S: (2-)22-24-26-37/39

611-116-00-1

A mixture of: trisodium 5-{4-
chloro-6-[2-(2,6-dichloro-5-cya-
nopyrimidin-4-ylamino)-propyla-
mino]-1,3,5-triazin-2-ylamino}-
4-hydroxy-3-(1-sulfonato-
naphthalene-2-ylazo)-naphtha-
lene-2,7-disulfonate
trisodium 5-{4-chloro-6-[2-(2,6-
dichloro-5-cyanopyrimidin-4-
ylamino)-1-methyl-ethylamino]-
1,3,5-triazin-2-ylamino}-4-
hydroxy-3-(1-sulfonatonaphtha-
lene-2-ylazo)-naphthalene-2,7-
disulfonate
trisodium 5-{4-chloro-6-[2-(4,6-
dichloro-5-cyanopyrimidin-2-
ylamino)-propylamino]-1,3,5-
triazin-2-ylamino}-4-hydroxy-3-
(1-sulfonatonaphthalen-2-ylazo)-
naphthalene-2,7-disulfonate
trisodium 5-{4-chloro-6-[2-(4,6-
dichloro-5-cyanopyrimidin-2-
ylamino)-1-methyl-ethylamino]-
1,3,5-triazin-2-ylamino}-4-
hydroxy-3-(1-sulfonatonaphtha-
len-2-ylazo)-naphthalene-2,7-dis-
ulfonate

611-117-00-7

1,3-bis{6-fluoro-4-[1,5-disulfo-4-
(3-aminocarbonyl-1-ethyl-6-
hydroxy-4-methyl-pyrid-2-on-5-
ylazo)-phenyl-2-ylamino]-1,3,5-
triazin-2-ylamino}propane
lithium-, sodium salt

611-118-00-2

sodium 1,2-bis[4-[4-{4-(4-
sulfophenylazo)-2-sulfopheny-
lazo}-2-ureido-phenyl-amino]-6-
fluoro-1,3,5-triazin-2-ylamino]-
propane, sodium salt

415-100-3

149850-29-3

R 43

413-990-8

149850-31-7

R 43

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

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

L

2
1
6
/
1
4
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

611-119-00-8

611-120-00-3

611-121-00-9

611-122-00-4

611-123-00-X

tetrasodium 4-[4-chloro-6-(4-
methyl-2-sulfophenylamino)-
1,3,5-triazin-2-ylamino]-6-(4,5-
dimethyl-2-sulfophenylazo)-5-
hydroxynaphthalene-2,7-disulfo-
nate

5-{4-[5-amino-2-[4-(2-sulfoxy-
ethylsulfonyl)phenylazo]-4-sulfo-
phenylamino]-6-chloro-1,3,5-
triazin-2-ylamino}-4-hydroxy-3-
(1-sulfo-naphthalen-2-ylazo)-
naphthalene-2,7-disulfonicacid
sodium salt

Main component 6 (isomer):
asym. 1:2 Cr(III)-complex of: A:
3-hydroxy-4-(2-hydroxy-
naphthalene-1-ylazo)naphtha-
lene-1-sulfonic acid, Na-salt and
B: 1-[2-hydroxy-5-(4-methoxy-
phenylazo)phenylazo]naphtha-
lene-2-ol
Main component 8 (isomer):
asym. 1:2 Cr-complex of: A: 3-
hydroxy-4-(2-hydroxy-naphtha-
lene-1-ylazo)-naphthalene-1-sul-
fonic acid, Na-salt and B: 1-[2-
hydroxy-5-(4-methoxy-pheny-
lazo)-phenylazo]-naphthalene-2-
ol

hexasodium (di[N-(3-(4-[5-(5-
amino-3-methyl-1-phenylpyra-
zol-4-yl-azo)-2,4-disulfo-ani-
lino]-6-chloro-1,3,5-triazin-2-
ylamino)phenyl)-sulfamoyl](di-
sulfo)-phthalocyaninato)nickel

3-(2,4-bis(4-((5-(4,6-bis(2-ami-
nopropylamino)-1,3,5-triazin-2-
ylamino)-4-hydroxy-2,7-disulfo-
naphthalen-3-yl)azo)phenyla-
mino)-1,3,5-triazin-6-ylamino)-
propyldiethylammonium lactate

415-400-4

148878-22-2

Xi; R41
R 43

Xi
R: 41-43
S: (2-)22-24-26-37/39

418-340-7

157707-94-3

Xi; R41
R 52-53

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

417-280-9

30785-74-1

Xi; R41
N; R50-53

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

417-250-5

151436-99-6

Xi; R41
R 43

Xi
R: 41-43
S: (2-)22-24-26-37/39

424-310-4

178452-66-9

Xi; R41

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

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
4
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

424-320-9

180778-23-8

Xi; R41
N; R51-53

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

423-940-7

—

Xi; R41
N; R51-53

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

611-124-00-5

611-125-00-0

A mixture of: pentasodium 5-
amino-3-(5-{4-chloro-6-[4-(2-
sulfoxyethoxysulfonato)phenyla-
mino]-1,3,5-triazin-2-ylamino}-
2-sulfonatophenylazo)-6-[5-(2,3-
dibromopropionylamino)-2-sul-
fonatophenylazo]-4-hydroxy-
naphthalene-2,7-disulfonate
pentasodium 5-amino-6-[5-(2-
bromoacryloylamino)-2-sulfona-
tophenylazo]-3-(5-{4-chloro-6-
[4-(2-sulfoxyethoxysulfonato)-
phenylamino]-1,3,5-triazin-2-
ylamino}-2-sulfonatophenylazo)-
4-hydroxynaphthalene-2,7-disul-
fonate
tetrasodium 5-amino-3-[5-{4-
chloro-6-[4-(vinylsulfonyl)pheny-
lamino]-1,3,5-triazin-2-yla-
mino}-2-sulfonatophenylazo]-6-
[5-(2,3-dibromopropionyla-
mino)-2-sulfonatophenylazo]-4-
hydroxynaphthalene-2,7-disulfo-
nate

A mixture of: Disodium 6-[3-
carboxy-4,5-dihydro-5-oxo-4-
sulfonatophenyl)pyrazolin-4-yl-
azo]-3-[2-oxido-4-(ethensulfo-
nyl)-5-methoxyphenylazo]-4-oxi-
donaphthalene-2-sulfonate cop-
per (II) complex
Disodium 6-[3-carboxy-4,5-
dihydro-5-oxo-4-sulfonatophe-
nyl)pyrazolin-4-yl-azo]-3-[2-
oxido-4-(2-hydroxyethylsulfo-
nyl)-5-methoxyphenylazo]-4-oxi-
donaphthalene-2-sulfonate cop-
per (II) complex;

611-126-00-6

2,6-bis-(2-(4-(4-amino-phenyla-
mino)-phenylazo)-1,3-dimethyl-
3H-imidazolium)-4-dimethyla-
mino-1,3,5-triazine, dichloride

424-120-1

174514-06-8

Xi; R41
N; R50-53

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

L

2
1
6
/
1
4
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

611-127-00-1

611-128-00-7

611-129-00-2

611-130-00-8

611-131-00-3

611-132-00-9

pentasodium 4-amino-6-(5-(4-
(2-ethyl-phenylamino)-6-(2-sulfa-
toethanesulfonyl)-1,3,5-triazin-2-
ylamino)-2-sulfonatophenylazo)-
5-hydroxy-3-(4-(2-sulfatoethane-
sulfonyl)phenylazo)naphthalene-
2,7-disulfonate

N,N′-bis{6-chloro-4-[6-(4-vinyl-
sulfonylphenylazo)-2,7-disulfoni-
cacid-5-hydroxynapht-4-yla-
mino]-1,3,5-triazin-2-yl}-N-(2-
hydroxyethyl)ethane-1,2-dia-
mine, sodium salt

A mixture of: 5-[(4-[(7-amino-1-
hydroxy-3-sulfo-2-naphthy-
l)azo]-2,5-diethoxyphenyl)azo]-
2-[(3-phosphonophenyl)azo]ben-
zoic acid
5-[(4-[(7-amino-1-hydroxy-3-
sulfo-2-naphthyl)azo]-2,5-
diethoxyphenyl)azo]-3-[(3-phos-
phonophenyl)azo]benzoic acid

tetra-ammonium 2-[6-[7-(2-
carboxylato-phenylazo)-8-
hydroxy-3,6-disulfonato-1-
naphthylamino]-4-hydroxy-
1,3,5-triazin-2-ylamino]benzoate

2-[2-hydroxy-3-(2-chlorophe-
nyl)carbamoyl-1-naphthylazo]-7-
[2-hydroxy-3-(3-methylphenyl)-
carbamoyl-1-naphthylazo]fluo-
ren-9-one

pentasodium bis{7-[4-(1-butyl-5-
cyano-1,2-dihydro-2-hydroxy-4-
methyl-6-oxo-3-pyridylazo)phe-
nylsulfonylamino]-5′-nitro-3,3′-
disulfonatonaphthalene-2-azo-
benzene-1,2′-diolato} chromate
(III)

423-790-2

—

R 5
Xi; R41
R 43
R 52-53

Xi
R: 5-41-43-52/53
S: (2-)22-26-36/37/39-
41-61

419-500-9

171599-85-2

Xi; R41
R 43

Xi
R: 41-43
S: (2-)22-24-26-37/39

418-230-9

163879-69-4

E; R2
Repr.Cat.3; R62
Xn; R48/22
R 43
N; R51-53

E; Xn; N
R: 2-43-48/22-62-51/53
S: (2-)26-35-36/37-61

418-520-5

183130-96-3

Xi; R36
N; R50-53

Xi; N
R: 36-50/53
S: (2-)26-39-60-61

420-580-2

419-210-2

—

—

Repr.Cat.2; R61
R 53

T
R: 61-53
S: 53-45-61

Xi; R41
R 52-53

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

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
4
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

611-133-00-4

611-134-00-X

611-135-00-5

Product by process iron complex
of azo dyestuffs obtained by
coupling a mixture of diazotized
2-amino-1-hydroxybenzene-4-
sulfanilide and 2-amino-1-
hydroxybenzene-4-sulfonamide
with resorcin, the obtained
mixture being subsequently
submitted to a second coupling
reaction with a mixture of
diazotized 3-aminobenzene-1-
sulfonic acid (metanilic acid) and
4′-amino-4-nitro-1,1′-diphenyla-
mine-2-sulfonic acid and
metallization with ferric
chloride, sodium salt

trisodium 2-{α[2-hydroxy-3-[4-
chloro-6-[4-(2,3-dibromopropio-
nylamino)-2-sulfonatophenyla-
mino]-1,3,5-triazin-2-ylamino]-
5-sulfonatophenylazo]-benzyli-
denehydrazino}-4-sulfonato-
benzoate, copper complex

Reaction product of: 2-[[4-
amino-2-ureidophenylazo]-5-[(2-
(sulfooxy)ethyl)sulfonyl]]benze-
nesulfonic acid with 2,4,6-
trifluoropyrimidine and partial
hydrolysis to the corresponding
vinylsulfonyl derivative,mixed
potassium/sodium salt

611-136-00-0

2-{4-(2-ammoniopropylamino)-
6-[4-hydroxy-3-(5-methyl-2-
methoxy-4-sulfamoylphenylazo)-
2-sulfonatonaphth-7-ylamino]-
1,3,5-triazin-2-ylamino}-2-ami-
nopropyl formate

419-260-5

—

Xi; R41
N; R51-53

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

423-770-3

—

Xi; R41
N; R51-53

Xi; N
R: 41-51/53
S: (2-)22-26-39-61

424-250-9

—

Xi; R41
R52-53

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

424-260-3

—

Repr.Cat.3; R62
Xi; R41
N; R51-53

Xn; N
R: 41-62-51/53
S: (2-)22-26-36/37/39-
61

L

2
1
6
/
1
4
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

611-137-00-6

6-tert-butyl-7-chloro-3-tridecyl-
7,7a-dihydro-1H-pyrazolo[5,1-c]-
1,2,4-triazole

419-870-1

159038-16-1

R 53

R: 53
S: 61

611-138-00-1

2-(4-aminophenyl)-6-tert-butyl-
1H-pyrazolo[1,5-b][1,2,4]tria-
zole

415-910-7

152828-25-6

R43
N; R51-53

Xi; N
R: 43-51/53
S: (2-)22-24-37-61

611-140-00-2

azafenidin

—

68049-83-2

T; R48/22
Repr. Cat. 2; R61
Repr. Cat. 3; R62
N; R50-53

T; N
R: 61-48/22-62-50/53
S: 53-45-60-61

C ≥ 0.025 %: N; R50/53
0.0025 % ≤ C < 0.025 %: N; R51/53
0.00025 % ≤ C < 0.0025 %: R52/53

612-184-00-5

6′-(dibutylamino)-3′-methyl-2′-
(phenylamino)spiro[isobenzo-
furan-1(3H),9-(9H)-xanthen]-3-
one

403-830-5

89331-94-2

R 52-53

R: 52/53
S: 61

612-185-00-0

1-[3-[4-((heptadecafluorononyl)
oxy)-benzamido]propyl]-N,N,N-
trimethylammonium iodide

407-400-8

59493-72-0

Xi; R41
N; R50-53

612-186-00-6

bis(N-(7-hydroxy-8-methyl-5-
phenylphenazin-3-ylidene)di-
methylammonium) sulfate

406-770-8

149057-64-7

612-187-00-1

2,3,4-trifluoroaniline

407-170-9

3862-73-5

Xn; R48/22
Xi; R41
R 43
N; R50-53

Xn; R21/22-48/22
Xi; R38-41
N; R51-53

612-188-00-7

4,4′-(9H-fluoren-9-ylidene)bis(2-
chloroaniline)

407-560-9

107934-68-9

N; R51-53

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

Xn; N
R: 41-43-48/22-50/53
S: (2-)22-26-36/37/39-
60-61

Xn; N
R: 21/22-38-41-48/22-
51/53
S: (2-)23-26-36/37/39-
61

N
R: 51/53
S: 61

612-189-00-2

4-amino-2-(aminomethyl)phenol
dihydrochloride

412-510-4

135043-64-0

Xn; R22
R 43
N; R50-53

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

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
4
9

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

612-190-00-8

4,4′-methylenebis(2-isopropyl-6-
methylaniline)

415-150-6

16298-38-7

Xn; R48/22
N; R51-53

612-191-00-3

Polymer of allylamine
hydrochloride

415-050-2

71550-12-4

Xn; R22
R 43

612-192-00-9

2-isopropyl-4-(N-methyl)amino-
methylthiazole

414-800-6

154212-60-9

612-193-00-4

3-methylaminomethylphenyla-
mine

414-570-7

18759-96-1

Xn; R21/22
Xi; R38-41
N; R51-53

Xn; R21/22
C; R34
R 43
N; R50-53

Xn; R22
Xi; R41
N; R50-53

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

414-670-0

141890-30-4

415-210-1

—

612-194-00-X

2-hydroxy-3-[(2-hydroxyethyl)-
[2-(1-oxotetradecyl)amino]ethy-
l]amino]-N,N,N-trimethyl-1-pro-
panammonium chloride

612-195-00-5

bis[tributyl 4-
(methylbenzyl)ammonium] 1,5-
naphthalenedisulfonate

612-196-00-0

4-chloro-o-toluidine
[1]
4-chloro-o-toluidine hydrochlor-
ide
[2]

612-197-00-6

2,4,5-trimethylaniline
[1]
2,4,5-trimethylaniline hydro-
chloride
[2]

E

E

202-441-6 [1]
221-627-8 [2]

95-69-2 [1]
3165-93-3 [2]

Carc.Cat.2; R45
Muta.Cat.3; R68
T; R23/24/25
N; R50-53

T; N
R: 45-23/24/25-68-50/
53
S: 53-45-60-61

205-282-0 [1]
- [2]

137-17-7 [1]
21436-97-5 [2]

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

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

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

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

Xn; N
R: 21/22-38-41-51/53
S: (2-)26-36/37/39-61

C; N
R: 21/22-34-43-50/53
S: (1/2-)26-36/37/39-45-
60-61

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

Xn; N
R: 20/22-41-50/53
S: (2-)26-36/39-60-61

612-198-00-1

4,4′-thiodianiline and its salts

E

205-370-9

139-65-1

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

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

L

2
1
6
/
1
5
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

612-199-00-7

4,4′-oxydianiline and its salts
p-aminophenyl ether

E

202-977-0

101-80-4

612-200-00-0

2,4-diaminoanisole
4-methoxy-m-phenylenediamine
[1]
2,4-diaminoanisole sulphate
[2]

210-406-1 [1]
254-323-9 [2]

615-05-4 [1]
39156-41-7 [2]

Carc.Cat.2; R45
Muta.Cat.2; R46
Repr.Cat.3; R62
T; R23/24/25
N; R51-53

Carc.Cat.2; R45
Muta.Cat.3; R68
Xn; R22
N; R51-53

612-201-00-6

N,N,N′,N′-tetramethyl-4,4′-
methylendianiline

202-959-2

101-61-1

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

T; N
R: 45-46-23/24/25-62-
51/53
S: 53-45-61

T; N
R: 45-22-68-51/53
S: 53-45-61

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

612-202-00-1

3,4-dichloroaniline

202-448-4

95-76-1

T; R23/24/25
Xi; R41
R43
N; R50-53

T; N
R: 23/24/25-41-43-50/
53
S: (1/2-)26-36/37/39-45-
60-61

612-204-00-2

C.I. Basic Violet 3
4-[4,4′-bis(dimethylamino)
benzhydrylidene]cyclohexa-2,5-
dien-1-ylidene]dimethylammo-
nium chloride

208-953-6

548-62-9

Carc.Cat.3; R40
Xn; R22
Xi; R41
N; R50-53

Xn; N
R: 22-40-41-50/53
S: (2-)26-36/37/39-46-
60-61

612-205-00-8

C.I. Basic Violet 3 with ≥ 0.1 %
of Michler's ketone (EC no. 202-
027-5)

E

208-953-6

548-62-9

Carc.Cat.2; R45
Xn; R22
Xi; R41
N; R50-53

T; N
R: 45-22-41-50/53
S: 53-45-60-61

612-206-00-3

famoxadone
3-anilino-5-methyl-5-(4-phenox-
yphenyl)-1,3-oxazolidine-2,4-
dione

—

131807-57-3

Xn; R48/22
N; R50-53

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

612-209-00-X

6-methoxy-m-toluidine
p-cresidine

E

204-419-1

120-71-8

Carc.Cat.2; R45
Xn; R22

T
R: 45-22
S: 53-45

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
5
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

612-210-00-5

5-nitro-o-toluidine
[1]
5-nitro-o-toluidine hydrochloride
[2]

202-765-8 [1]
256-960-8 [2]

99-55-8 [1]
51085-52-0 [2]

Carc.Cat.3; R40
T; R23/24/25
R52-53

T
R: 23/24/25-40-52/53
S: (1/2-)36/37-45-61

612-211-00-0

N-[(benzotriazole-1-yl)methyl)]-
4-carboxybenzenesulfonamide

416-470-9

—

Xi; R36
N; R51-53

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

612-212-00-6

2,6-dichloro-4-trifluoromethyla-
niline

416-430-0

24279-39-8

Xn; R20/22
Xi; R38
R43
N; R50-53

Xn; N
R: 20/22-38-43-50/53
S: (2-)24-37-60-61

612-213-00-1

isobutylidene-(2-(2-isopropyl-
4,4-dimethyloxazolidine-3-yl)-
1,1-dimethylethyl)amine

419-850-2

148348-13-4

C; R34
R52-53

612-214-00-7

4-(2,2-diphenylethenyl)-N,N-di-
phenylbenzenamine

421-390-2

89114-90-9

R 53

612-215-00-2

3-chloro-2-(isopropylthio)aniline

421-700-6

179104-32-6

612-217-00-3

1-methoxy-2-propylamine

422-550-4

37143-54-7

613-181-00-1

5,5-dimethyl-perhydro-pyrimi-
din-2-one α-(4-
trifluoromethylstyryl)-α-(4-tri-
fluoromethyl)cinnamylidenehy-
drazone

405-090-9

67485-29-4

Xi; R38
N; R51-53

F; R11
C; R34
Xn; R22
R52-53

T; R48/25
Xn; R22
Xi; R36
N; R50-53

C
R: 34-52/53
S: (1/2-)23-26-36/37/39-
45-61

R: 53
S: 61

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

F; C
R: 11-22-34-52/53
S: (1/2-)9-26-36/37/39-
45-61

T; N
R: 22-36-48/25-50/53
S: (1/2-)22-26-36/37-45-
60-61

L

2
1
6
/
1
5
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

613-182-00-7

1-(1-naphthylmethyl)quinoli-
nium chloride

406-220-7

65322-65-8

Carc.Cat.3; R40
Muta.Cat.3; R68
Xn; R22
Xi; R38-41
R 52-53

Xn
R: 22-38-40-41-52/53-
68
S: (2-)22-26-36/37/39-
61

613-183-00-2

A mixture of: 5-(N-
methylperfluorooctylsulfonami-
do)methyl-3-octadecyl-1,3-oxa-
zolidin-2-one
5-(N-methylperfluoroheptylsul-
fonamido)methyl-3-octadecyl-
1,3-oxazolidin-2-one

413-640-4

—

Xn; R48/22
N; R50-53

Xn; N
R: 48/22-50/53
S: (2-)36-60-61

613-184-00-8

nitrilotriethyleneammoniopro-
pane-2-ol 2-ethylhexanoate

413-670-8

—

Xi; R36
R 43

613-185-00-3

2,3,5,6-tetrahydro-2-methyl-2H-
cyclopenta[d]-1,2-thiazol-3-one

407-630-9

82633-79-2

613-186-00-9

(2R,3R)-3-((R)-1-(tert-butyldi-
methylsiloxy)ethyl)-4-oxoazeti-
din-2-yl acetate

408-050-9

76855-69-1

613-188-00-X

1-(3-(4-fluorophenoxy)propyl)-
3-methoxy-4-piperidinone

411-500-7

116256-11-2

613-189-00-5

1,4,7,10-tetrakis(p-toluensulfo-
nyl)-1,4,7,10-tetraazacyclodode-
cane

414-030-0

52667-88-6

T; R25
Xi; R41
R 43
N; R50-53

Xi; R36
R 43
N; R51-53

Xn; R22
Xi; R41
R 43
N; R51-53

R 43
N; R50-53

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

T; N
R: 25-41-43-50/53
S: (1/2-)22-26-36/37/39-
45-60-61

Xi; N
R: 36-43-51/53
S: (2-)24-26-37-61

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

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

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
5
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

613-190-00-0

disodium 1-amino-4-(2-(5-
chloro-6-fluoro-pyrimidin-4-yla-
mino-methyl)-4-methyl-6-sulfo-
phenylamino)-9,10-dioxo-9,10-
dihydro-anthracene-2-sulfonate

414-040-5

149530-93-8

Xn; R22
R 43

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

613-191-00-6

3-ethyl-2-methyl-2-(3-methylbu-
tyl)-1,3-oxazolidine

421-150-7

143860-04-2

Repr.Cat.2; R60
C; R34
N; R50-53

T; N
R: 60-34-50/53
S: 53-45-60-61

613-193-00-7

pentakis[3-(dimethylammonio)-
propylsulfamoyl]-[(6-hydroxy-
4,4,8,8-tetramethyl-4,8-diazo-
niaundecane-1,11-diyldisulfa-
moyl)di[phthalocyaninecop-
per(II)]] heptalactate

613-194-00-2

6,13-dichloro-3,10-bis{2-[4-
fluoro-6-(2-sulfophenylamino)-
1,3,5-triazin-2-ylamino]propyla-
mino}benzo[5,6][1,4]oxa-
zino[2,3-.b.]phenoxazine-4,11-
disulphonic acid, lithium-,
sodium salt.

414-930-3

—

N; R51-53

418-000-8

163062-28-0

Xi; R41

613-195-00-8

2,2-(1,4-phenylene)bis((4H-3,1-
benzoxazine-4-one)

418-280-1

18600-59-4

R 43
R 53

418-380-5

168113-78-8

Xi; R41

613-196-00-3

5-[[4-chloro-6-[[2-[[4-fluoro-6-
[[5-hydroxy-6-[(4-methoxy-2-
sulfophenyl)azo]-7-sulfo-2-
naphthalenyl]amino]-1,3,5-tria-
zin-2-yl]amino]-1-methylethyl]a-
mino]-1,3,5-triazin-2-yl]amino]-
3-[[4-(ethenylsulfonyl)phenyl]
azo]-4-hydroxy-naphtalene-2,7-
disulfonic acid, sodium salt

N
R: 51/53
S: 61

Xi
R: 41
S: (2-)22-26-39

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

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

L

2
1
6
/
1
5
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

613-197-00-9

613-199-00-X

A mixture of: 2,4,6-
tri(butylcarbamoyl)-1,3,5-triazine
2,4,6-tri(methylcarbamoyl)-
1,3,5-triazine
[(2-butyl-4,6-dimethyl)tricarba-
moyl]-1,3,5-triazine
[(2,4-dibutyl-6-methyl)tricarba-
moyl]-1,3,5-triazine

A mixture of: 1,3,5-tris(3-
aminomethylphenyl)-1,3,5-
(1H,3H,5H)-triazine-2,4,6-trione
a mixture of oligomers of 3,5-
bis(3-aminomethylphenyl)-1-
poly[3,5-bis(3-aminomethylphe-
nyl)-2,4,6-trioxo-1,3,5-
(1H,3H,5H)-triazin-1-yl]-1,3,5-
(1H,3H,5H)-triazine-2,4,6-trione

613-200-00-3

Reaction product of: copper,
(29H,31H-phthalocyaninato(2-)-
N29,N30,N31,N32)-, chlorosul-
furic acid and 3-(2-
sulfooxyethylsulfonyl)aniline,
sodium salts

420-390-1

187547-46-2

R 43
N; R51-53

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

421-550-1

—

Carc.Cat.2; R45
Repr.Cat.2; R61
R 43
R 52-53

T
R: 45-61-43-52/53
S: 53-45-61

420-980-7

—

Xi; R41

Xi
R: 41
S: (2-)22-26-39

613-201-00-9

(R)-5-bromo-3-(1-methyl-2-pyr-
rolidinyl methyl)-1H-indole

422-390-5

143322-57-0

Repr.Cat.3; R62
T; R39-48/25
Xn; R20/22
Xi; R41
R 43
N; R50-53

T; N
R: 20/22-39-41-43-48/
25-62-50/53
S: (1/2-)53-45-60-61

613-202-00-4

pymetrozine (ISO)
(E )-4,5-dihydro-6-methyl-4-(3-
pyridylmethyleneamino)-1,2,4-
triazin-3(2H )-one

—

123312-89-0

Carc.Cat3; R40
R52-53

Xn
R: 40-52/53
S: (2-)36/37-61

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
5
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

613-203-00-X

pyraflufen-ethyl
[1]
pyraflufen
[2]

- [1]
- [2]

N; R50-53

129630-19-9
[1]
129630-17-7
[2]

N
R: 50/53
S: 60-61

613-204-00-5

oxadiargyl (ISO)
3-[2,4-dichloro-5-(2-propynyl-
oxy)phenyl]-5-(1,1-dimethy-
lethyl)-1,3,4-oxadiazol-2(3H )-
one
5-tert-butyl-3-[2,4-dichloro-5-
(prop-2-ynyloxy)phenyl]-1,3,4-
oxadiazol-2(3H )-one

254-637-6

39807-15-3

Repr.Cat3; R63
Xn; R48/22
N; R50-53

Xn; N
R: 48/22-63-50/53
S: (2-)36/37-46-60-61

613-205-00-0

propiconazole
(+)-1-[2-(2,4-dichlorophenyl)-4-
propyl-1,3-dioxolan-2-ylmethyl]-
1H-1,2,4-triazole

262-104-4

60207-90-1

Xn; R22
R43
N; R50-53

Xn; N
R: 22-43-50/53
S: (2-)36/37-46-60-61

613-206-00-6

fenamidone (ISO)
(S )-5-methyl-2-methylthio-5-
phenyl-3-phenylamino-3,5-dihy-
droimidazol-4-one

613-207-00-1

imazalil sulphate, aqueous
solution
1- [2-(allyloxy)ethyl-2-(2,4-
dichlorophenyl)]-1H-imidazo-
lium hydrogen sulphate
(±)-1- [2-(allyloxy)ethyl-2-(2,4-
dichlorophenyl)]-1H-imidazo-
lium hydrogen sulphate

—

161326-34-7

N; R50-53

N
R: 50/53
S: 60-61

261-351-5
281-291-3

58594-72-2
83918-57-4

Xn; R22
C; R34
R43
N; R50-53

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

613-208-00-7

imazamox

—

114311-32-9

N; R50-53

N
R: 50/53
S: 60-61

C 50 %: C, Xn, N; R22-34-43-50-53
30 % < C ≤ 50 %: Xn, N; R22-38-41-43-
50-53
25 % ≤ C ≤ 30 %: Xn, N; R22-41-43-50-
53
15 % < C < 25 %: Xi, N; R41-43-51-53
5 % ≤ C ≤ 15 %: Xi, N; R36-43-51-53
2,5 % ≤ C < 5 %: Xi, N; R43-51-53
1 % ≤ C < 2,5 %: Xi; R43-52-53
0,25 % ≤ C < 1 %: R52-53

L

2
1
6
/
1
5
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

613-209-00-2

cis-1-(3-chloropropyl)-2,6-
dimethyl-piperidin hydrochloride

417-430-3

63645-17-0

613-210-00-8

2-(3-chloropropyl)-2,5,5-tri-
methyl-1,3-dioxane

417-650-1

88128-57-8

T; R25
Xn; R48/22
R43
N; R51-53

Xn; R48/22
R52-53

613-211-00-3

N-methyl-4-(p-formylstyryl)pyri-
dinium methylsulfate

418-240-3

74401-04-0

R43
R52-53

613-212-00-9

4-[4-(2-ethylhexyloxy)phe-
nyl](1,4-thiazinane-1,1-dioxide)

418-320-8

133467-41-1

Xn; R22
N; R50-53

613-213-00-4

cis-1-benzoyl-4-[(4-methylsulfo-
nyl)oxy]-L-proline

416-040-0

120807-02-5

R 52-53

613-214-00-X

N,N-di-n-butyl-2-(1,2-dihydro-3-
hydroxy-6-isopropyl-2-quinolyli-
dene)-1,3-dioxoindan-5-carboxa-
mide

416-260-7

147613-95-4

R 53

T; N
R: 25-43-48/22-51/53
S: (1/2-)22-36/37-45-61

Xn
R: 48/22-52/53
S: (2-)23-25-36-61

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

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

R: 52/53
S: 61

R: 53
S: 61

613-215-00-5

2-chloromethyl-3,4-dimethoxy-
pyridinium chloride

416-440-5

72830-09-2

Xn; R21/22-48/22
Xi; R38-41
R43
N; R51-53

Xn; N
R: 21/22-38-41-43-48/
22-51/53
S: (2-)26-36/37/39-61

613-216-00-0

6-tert-butyl-7-(6-diethylamino-2-
methyl-3-pyridylimino)-3-(3-
methylphenyl)pyrazolo[3,2-
c][1,2,4]triazole

613-217-00-6

4-[3-(3,5-di-tert-butyl-4-hydro-
xyphenyl)propionyloxy]-1-[2-[3-
(3,5-di-tert-butyl-4-hydrophe-
nyl)propionyloxy]ethyl]-2,2,6,6-
tetramethylpiperidine

416-490-8

—

N; R50-53

416-770-1

73754-27-5

R 53

N
R: 50/53
S: 60-61

R: 53
S: 61

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
5
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

613-218-00-1

6-hydroxyindole

417-020-4

2380-86-1

613-219-00-7

7a-ethyl-3,5-bis(1-methylethyl)-
2,3,4,5-tetrahydrooxazolo[3,4-
c]-2,3,4,5-tetrahydrooxazole

417-140-7

79185-77-6

Xn; R22
Xi; R41
R43
N; R51-53

Xi; R38
N; R51-53

613-220-00-2

trans-(4S,6S)-5,6-dihydro-6-
methyl-4H-thieno[2,3-b]thio-
pyran-4-ol, 7,7-dioxide

417-290-3

147086-81-5

Xn; R22

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

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

Xn
R: 22
S: (2-)36

613-221-00-8

2-chloro-5-methyl-pyridine

418-050-0

18368-64-4

Xn; R21/22
Xi; R38
R52-53

Xn
R: 21/22-38-52/53
S: (2-)23-25-36/37-61

613-222-00-3

4-(1-oxo-2-propenyl)-morpho-
line

418-140-1

5117-12-4

Xn; R22-48/22
Xi; R41
R43

Xn
R: 22-41-43-48/22
S: (2-)23-26-36/37/39

613-223-00-9

N-isopropyl-3-(4-fluorophenyl)-
1H-indole

418-790-4

93957-49-4

R 53

R: 53
S: 61

613-224-00-4

2,5-dimercaptomethyl-1,4-
dithiane

419-770-8

136122-15-1

613-225-00-X

A mixture of:[2-(anthraquinon-
1-ylamino)-6-[(5-benzoylamino)-
anthraquinone-1-ylamino]-4-
phenyl]-1,3,5-triazine
2,6-bis-[(5-benzoylamino)-
anthraquinon-1-ylamino]-4-phe-
nyl-1,3,5-triazine.

421-290-9

—

Xn; R22
C; R34
R43
N; R50-53

Xn; R48/22
R53

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

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

L

2
1
6
/
1
5
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

613-226-00-5

1-(2-(ethyl(4-(4-(4-(4-(ethyl(2-
pyridinoethyl)amino)-2-methyl-
phenylazo)benzoylamino)-phe-
nylazo)-3-methylphenyl)ami-
no)ethyl-pyridinium dichloride

420-950-3

163831-67-2

Xi; R41
N; R50-53

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

613-227-00-0

(+/-)-[(R*,R*)and(R*,S*)]-6-
fluoro-3,4-dihydro-2-oxiranyl-
2H-1-benzopyran

419-600-2

613-228-00-6

(+/-)-(R*,S*)-6-fluoro-3,4-dihy-
dro-2-oxiranyl-2H-1-benzopyran

419-630-6

—

—

R 43
N; R51-53

N; R51-53

613-230-00-7

florasulam (ISO)
2′,6′,8-trifluoro-5-methoxy-5-
triazolo[1,5-c]
pyrimidine-2-sulfonanilide

—

145701-23-1

N; R50-53

613-233-00-3

4,4′-(oxy-(bismethylene))-bis-
1,3-dioxolane

423-230-7

56552-15-9

Xi; R41

414-420-0

—

Xi; R41

419-640-0

68784-14-5

Xn; R22

614-028-00-1

A mixture of: 2-ethylhexyl
mono-D-glucopyranoside
2-ethylhexyl di-D-
glucopyranoside

614-029-00-7

Constitutional isomers of penta-
O-allyl-β-D-fructofuranosyl-α-D-
glucopyranoside
Constitutional isomers of hexa-
O-allyl-β-D-fructofuranosyl-α-D-
glucopyranoside
Constitutional isomers of hepta-
O-allyl-β-D-fructofuransoyl-α-D-
glucopyranoside

Xi; N
R: 43-51/53
S: (2-)24-28-36/37-61

N
R: 51/53
S: 24-61

N
R: 50/53
S: 60-61

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

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

Xn
R: 22
S: (2-)

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
5
9

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

615-030-00-5

alkali salts, alkali earth salts and
other salts of thiocyanic acid not
mentioned elsewhere in this
Annex

A

—

—

Xn; R20/21/22
R32
R52-53

Xn
R: 20/21/22-32-52/53
S: (2-)13-61

615-031-00-0

thallium salt of thiocyanic acid

A

222-571-7

3535-84-0

615-032-00-6 metal salts of thiocyanic acid

A

—

not mentioned elsewhere in this
Annex

616-092-00-6

Polymeric reaction product of
bicyclo[2.2.1]hepta-2,5-diene,
ethene, 1,4-hexadiene, 1-propene
with N,N-di-2-
propenylformamide

404-035-6

—

—

Xn; R20/21/22
R32
N; R51-53

Xn; N
R: 20/21/22-32-51/53
S: (2-)13-61

Xn; R20/21/22
R32
N; R50-53

Xn; N
R: 20/21/22-32-50/53
S: (2-)13-60-61

R 43
R 53

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

616-093-00-1

Reaction products of: aniline-
terephthalaldehyde-o-toluidine
condensate with maleic
anhydride

406-620-1

129217-90-9

R 43
N; R51-53

616-094-00-7

3,3′-dicyclohexyl-1,1′-methyle-
nebis(4,1-phenylene)diurea

406-370-3

58890-25-8

R 43
R 53

616-095-00-2

3,3′-dioctadecyl-1,1′-methylene-
bis(4,1-phenylene)diurea

406-690-3

43136-14-7

R 53

616-096-00-8

N-(3-hexadecyloxy-2-hydroxy-
prop-1-yl)-N-(2-hydroxyethyl)-
palmitamide

408-110-4

110483-07-3

R 53

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

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

R: 53
S: 61

R: 53
S: 61

L

2
1
6
/
1
6
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

616-097-00-3

N,N′-1,4-phenylenebis(2-((2-
methoxy-4-nitrophenyl)azo)-3-
oxobutanamide

616-098-00-9

1-[4-chloro-3-((2,2,3,3,3-penta-
fluoropropoxy)methyl)phenyl]-5-
phenyl-1H-1,2,4-triazole-3-car-
boxamide

616-099-00-4

2-[4-[(4-hydroxyphenyl)sulfo-
nyl]phenoxy]-4,4-dimethyl-N-[5-
[(methylsulfonyl)amino]-2-[4-
(1,1,3,3-tetramethylbutyl)phe-
noxy]phenyl]-3-oxopentanamide

411-840-6

83372-55-8

R 53

411-750-7

119126-15-7

N; R51-53

414-170-2

135937-20-1

R 53

616-100-00-8

1,3-dimethyl-1,3-bis(trimethylsi-
lyl)urea

414-180-7

10218-17-4

616-101-00-3

(S)-N-tert-butyl-1,2,3,4-tetrahy-
dro-3-isoquinolinecarboxamide

414-600-9

149182-72-9

Xn; R22
Xi; R38

Xn; R22
R 52-53

415-870-0

—

R 43
N; R51-53

616-102-00-9

A mixture of: α-[3-(3-
mercaptopropanoxycarbonylami-
no)methylphenylaminocarbo-
nyl]-ω-[3-(3-mercaptopropanox-
ycarbonylamino)methylphenyla-
minocarbonyloxy]-poly-(oxy-
ethylene-co-oxypropylene)
1,2-(or 1,3-)bis[α-(3-
mercaptopropanoxycarbonylami-
no)methylphenylaminocarbo-
nyl)-ω-oxy-poly(oxyethylene-co-
oxypropylene)]-3-
(or 2-)propanol
1,2,3-tris[α-(3-mercaptopropa-
noxycarbonyl-amino)methylphe-
nylaminocarbonyl)-ω-oxy-poly-
(oxyethylene-co-oxypropylene)]-
propane]

R: 53
S: 61

N
R: 51/53
S: 61

R: 53
S: 61

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

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

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

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
6
1

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

616-103-00-4

(S,S)-trans-4-(acetylamino)-5,6-
dihydro-6-methyl-7,7-dioxo-4H-
thieno[2,3-b]thiopyran-2-sulfo-
namide

616-104-00-X

benalaxyl
methyl N-(2,6-dimethylphenyl)-
N-(phenylacetyl)-DL-alaninate

415-030-3

120298-38-6

R43
N; R50-53

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

275-728-7

71626-11-4

N; R50-53

N
R: 50/53
S: 60-61

616-105-00-5

chlorotoluron
3-(3-chloro-p-tolyl)-1,1-dimethy-
lurea

239-592-2

15545-48-9

Carc. Cat. 3; R40
Repr. Cat. 3; R63
N; R50-53

Xn; N
R: 40-63-50/53
S: (2-)36/37-26-46-60-
61

616-106-00-0

phenmedipham
methyl 3-(3-
methylcarbaniloyloxy)carbanilate
(ISO)

616-108-00-1

iodosulfuron-methyl-sodium

616-109-00-7

sulfosulfuron
1-(4,6-dimethoxypyrimidin-2-
yl)-3-(2-ethylsulfonylimi-
dazo[1,2-a]pyridin-3-yl)sulfony-
lurea

237-199-0

13684-63-4

N; R50-53

—

—

144550-36-7

N; R50-53

141776-32-1

N; R50-53

616-110-00-2

cyclanilide
1-(2,4-dichloroanilinocarbonyl)-
cyclopropanecarboxylic acid

419-150-7

113136-77-9

Xn; R22
N; R51-53

616-111-00-8

fenhexamid
N-(2,3-dichlor-4-hydroxyphe-
nyl)-1-methylcyclohexancarboxa-
mid

422-530-5

126833-17-8

N; R51-53

N
R: 50/53
S: 60-61

N
R: 50/53
S: 60-61

N
R: 50/53
S: 60-61

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

N
R: 51/53
S: 61

L

2
1
6
/
1
6
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

—

144651-06-9

Xn; R48/22
N; R50-53

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

616-115-00-X

N-(3-acetyl-2-hydroxyphenyl)-4-
(4-phenylbutoxy)benzamide

416-150-9

136450-06-1

R 53

616-112-00-3

616-113-00-9

616-114-00-4

oxasulfuron
oxetan-3-yl 2-[(4,6-
dimethylpyrimidin-2-yl)-carba-
moylsulfamoyl]benzoate

desmedipham
ethyl 3-
phenylcarbamoyloxyphenylcar-
bamate

dodecanamide, N,N′-
(9,9′,10,10′-tetrahydro-
9,9′,10,10′-tetraoxo(1,1′-
bianthracene)-4,4′-diyl)bis-

616-116-00-5

616-117-00-0

616-118-00-6

616-119-00-1

616-120-00-7

N-(4-dimethylaminopyridinium)-
3-methoxy-4-(1-methyl-5-
nitroindol-3-ylmethyl)-N-(o-
tolylsulfonyl)benzamidate

N-[2-(3-acetyl-5-nitrothiophen-
2-ylazo)-5-diethylaminopheny-
l]acetamide

N-(2′,6′-dimethylphenyl)-2-
piperidinecarboxamide hydro-
chloride

2-(1-butyl-3,5-dioxo-2-phenyl-
(1,2,4)-triazolidin-4-yl)-4,4-
dimethyl-3-oxo-N-(2-methoxy-5-
(2-(dodecyl-1-sulfonyl))propiony-
lamino)-phenyl)-pentanamide

A mixture of: N-(3-
dimethylamino-4-methyl-phe-
nyl)-benzamide
N-(3-dimethylamino-2-methyl-
phenyl)-benzamide
N-(3-dimethylamino-3-methyl-
phenyl)-benzamide

C ≥ 2,5 %: N; R50/53
0,25 % ≤ C < 2,5 %: N; R51/53
0,025 % ≤ C < 0,25 %: R52/53

N
R: 50/53
S: 60-61

R: 53
S: 22-61

R: 53
S: 61

R: 53
S: 61

237-198-5

13684-56-5

N; R50-53

418-010-2

136897-58-0

R53

R 53

416-790-9

416-860-9

—

—

417-950-0

65797-42-4

Xn; R22
R52-53

418-060-5

118020-93-2

R 53

420-600-1

—

Xn; R48/22
N; R51-53

Repr.Cat.3; R62
R43
N; R50-53

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

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

R: 53
S: 61

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

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

616-121-00-2

2,4-dihydroxy-N-(2-methoxyphe-
nyl)benzamide

419-090-1

129205-19-2

R 43
N; R51-53

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
6
3

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

616-123-00-3

N-[3-[[4-(diethylamino)-2-
methylphenyl]imino]-6-oxo-1,4-
cyclohexadienyl]acetamide

414-740-0

96141-86-5

N; R50-53

N
R: 50/53
S: 60-61

616-124-00-9

lithium bis(trifluoromethylsulfo-
nyl)imide

415-300-0

90076-65-6

T; R24/25
C; R34
R 52-53

T
R: 24/25-34-52/53
S: (1/2-)22-26-36/37/39-
45-61

616-125-00-4

3-cyano-N-(1,1-dimethylethyl)an-
drosta-3,5-diene-17-β-carboxa-
mide

616-127-00-5

A mixture of: N,N′-Ethane-1,2-
diylbis(decanamide)
12-Hydroxy-N-[2-[1-oxydecyl)a-
mino]ethyl]octadecanamide
N,N′-Ethane-1,2-diylbis(12-
hydroxyoctadecanamide)

415-730-9

151338-11-3

N; R50-53

430-050-2

—

R43
N; R51-53

616-128-00-0

N-(2-(1-allyl-4,5-dicyanoimida-
zol-2-ylazo)-5-(dipropylamino)-
phenyl)-acetamide

417-530-7

123590-00-1

R53

616-129-00-6

N,N′-bis(2,2,6,6-tetramethyl-4-
piperidyl)isophthalamide

419-710-0

42774-15-2

Xn; R22
Xi; R36

616-130-00-1

N-(3-(2-(4,4-dimethyl-2,5-dioxo-
imidazolin-1-yl)-4,4-dimethyl-3-
oxo-pentanoylamino)-4-meth-
oxy-phenyl)-octadecanamide

616-132-00-2

N-[4-(4-cyano-2-furfurylidene-
2,5-dihydro-5-oxo-3-furyl)phe-
nyl]butane-1-sulfonamide

421-780-2

150919-56-5

R53

423-250-6

130016-98-7

N; R50-53

N
R: 50/53
S: 60-61

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

R: 53
S: 61

Xn
R: 22-36
S: (2-)22-25-26

R: 53
S: 61

N
R: 50/53
S: 60-61

L

2
1
6
/
1
6
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

616-133-00-8

N-cyclohexyl-S,S-dioxobenzo[b]-
tiophene-2-carboxamide

423-990-1

149118-66-1

Xn; R22
Xi; R41
N; R50-53

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

616-134-00-3

3,3′-bis(dioctyloxyphosphi-
nothioylthio)-N,N′-oxybis(-
methylene)dipropionamide

401-820-5

—

R52-53

616-135-00-9

(3S,4aS,8aS)-2-[(2R,3S)-3-
amino-2-hydroxy-4-phenylbu-
tyl]-N-tert-butyldecahydroisoqui-
noline-3-carboxamide

430-230-0

136522-17-3

Xn; R22
R52-53

R: 52/53
S: 61

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

616-142-00-7

1,3-Bis(vinylsulfonylacetamido)-
propane

428-350-3

93629-90-4

Muta.Cat.3; R68
Xi; R41
R 43
R 52-53

Xn
R: 41-43-68-52/53
S: (2-)22-26-36/37/39-
61

616-143-00-2

N,N′-dihexadecyl-N,N′-bis(2-
hydroxyethyl)propanediamide

422-560-9

149591-38-8

Xn; Repr. Cat. 3; R62
Xi; R36
R53

Xn
R: 62-36-53
S: (2-)26-36/37-61

617-018-00-5

A mixture of: 1-methyl-1-(3-(1-
methylethyl)phenyl)ethyl-1-
methyl-1-phenylethylperoxide,
63 % by weight
1-methyl-1-(4-(1-methylethyl)-
phenyl)ethyl-1-methyl-1-pheny-
lethylperoxide, 31 % by weight

410-840-3

71566-50-2

O; R7
N; R51-53

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

617-019-00-0

6-(phthalimido)peroxyhexanoic
acid

410-850-8

128275-31-0

O; R7
Xi; R41
N; R50

O; Xi; N
R: 7-41-50
S: (2-)3/7-14-26-36/37/
39-61

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
6
5

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

617-020-00-6

1,3-di(prop-2,2-diyl)benzene
bis(neodecanoylperoxide)

420-060-5

117663-11-3

650-042-00-4

Reaction product of:
polyethylene-polyamine-(C16-
C18)-alkylamides with
monothio-(C2)-alkyl phospho-
nates

417-450-2

650-043-00-X

Reaction product of: 3,5-bis-tert-
butylsalicylic acid and
aluminiumsulfate

420-310-3

—

—

650-044-00-5 mixed linear and branched C14-
15 alcohols ethoxylated, reaction
product with epichlorohydrin

420-480-9

158570-99-1

650-045-00-0

Reaction product of: 1,2,3-
propanetricarboxylic acid, 2-
hydroxy, diethyl ester, 1-
propanol and zirconium tetra-n-
propanolate

650-046-00-6

di(tetramethylammo-
nium)(29H,31H-phthalocyanin-
N29,N30,N31,N32)disulfona-
mide disulfonate, cuprate(2-
)complex, derivates

417-110-3

416-180-2

—

—

650-047-00-1

dibenzylphenylsulfonium hexa-
fluoroantimonate

417-760-8

134164-24-2

R10
O; R7
N; R51-53

O; N
R: 7-10-51/53
S: (2-)7-14-36/37/39-47-
61

Xi; R36/38
R43
R52-53

Xi
R: 36/38-43-52/53
S: (2-)24-26-37-61

Xn; R22
N; R50-53

Xi; R38
R43
N; R50-53

F; R11
Xi; R38-41
N; R51-53

Xn; N
R: 22-50/53
S: (2-)22-56-60-61

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

F; Xi; N
R: 11-38-41-51/53
S: (2-)9-16-26-37/39-61

Xn; R22-48/22
N; R51-53

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

T; R48/25
Xn; R22
Xi; R41
R43
N; R51-53

T; N
R: 22-41-43-48/25-51/
53
S: (1/2-)22-26-36/37/39-
45-61

L

2
1
6
/
1
6
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
U
n
o
n

i

.

.

1
6
6
2
0
0
4

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

650-048-00-7

Reaction product of: borax,
hydrogen peroxide, acetic acid
anhydride and acetic acid

650-049-00-2

2-alkoyloxyethyl hydrogen
maleate, where alkoyl represents
(by weight) 70 to 85 %
unsaturated octadecoyl, 0.5 to
10 % saturated octadecoyl, and 2
to 18 % saturated hexadecoyl

420-070-1

417-960-5

—

—

O; R7
Xn; R20/21/22
C; R35
N; R50

Xi; R38-41
R43
N; R50-53

O; C; N
R: 7-20/21/22-35-50
S: (1/2-)3/7-14-26-36/
37/39-45-61

Xi; N
R: 38-41-43-50/53
S: (2-)24-26-37/39-60-
61

650-050-00-8 A mixture of: 1-methyl-3-

423-600-8

—

N; R51-53

hydroxypropyl 3,5-[1,1-
dimethylethyl]-4-hydroxydihy-
dro-cinnamate and/or 3-
hydroxybutyl 3,5-[1,1-
dimethylethyl]-4-hydroxydihy-
drocinnamate
1,3-butanediol bis[3-(3′-(1,1-
dimethylethyl)4′-hydroxy-phe-
nyl)propionate] isomers
1,3-butanediol bis[3-(3′,5′-(1,1-
dimethylethyl)-4′-hydroxyphe-
nyl)propionate] isomers

650-055-00-5

silver sodium zirconium
hydrogenphosphate

422-570-3

—

N; R50-53

N
R: 51/53
S: 61

N
R: 50/53
S: 60-61’

.

.

1
6
6
2
0
0
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
U
n
o
n

i

L

2
1
6
/
1
6
7

Index No

chemical name

Notes related
to substances

EC No

CAS No

Classification

Labelling

Concentration Limits

Notes related
to preparations

ANNEX 1D

‘048-002-00-0

cadmium (non-pyrophoric)
[1]
cadmium oxide (non-
pyrophoric)
[2]

048-011-00-X

cadmium (pyrophoric)

609-006-00-3

4-nitrotoluene

609-065-00-5

2-nitrotoluene

612-039-00-6

2-ethoxyaniline
o-phenetidine

612-207-00-9

4-ethoxyaniline
p-phenetidine

E

E

C

E

C

231-152-8 [1]
215-146-2 [2]

7440-43-9 [1]
1306-19-0 [2]

231-152-8

7440-43-9

202-808-0

99-99-0

201-853-3

88-72-2

202-356-4

94-70-2

205-855-5

156-43-4

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Repr. Cat. 3; R62-63
T; R48/23/25
T+; R26
N; R50-53

Carc. Cat. 2; R45
Muta. Cat. 3; R68
Repr. Cat. 3; R62-63
T; R48/23/25
T+; R26
F; R17
N; R50-53

T; R23/24/25
R33
N; R51/53

Carc. Cat. 2; R45
Muta. Cat. 2; R46
Repr. Cat. 3; R62
Xn; R22
N; R51-53

T; R23/24/25
R33

Muta. Cat. 3; R68
Xn; R20/21/22
Xi; R36
R43

T+; N
R: 45-26-48/23/25-62-
63-68-50/53
S: 53-45-60-61

F; T+; N
R: 45-17-26-48/23/25-
62-63-68-50/53
S: 53-45-7/8-43-60-61

T; N
R: 23/24/25-33-51/53
S: (1/2-)28-37-45-61

T; N
R: 45-46-22-62-51/53
S: 53-45-61

T
R: 23/24/25-33
S: (1/2-)28-36/37-45

Xn
R: 20/21/22-36-43-68
S: (2-)36/37-46’

L

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6
/
1
6
8

E
N

O

f
f
i
c
i
a
l

J
o
u
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n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n
U
n
o
n

i

.

.

1
6
6
2
0
0
4

16.6.2004

EN

Official Journal of the European Union

L 216/169

ANNEX 2A

‘A.21. OXIDIZING PROPERTIES (LIQUIDS)

1.

1.1

METHOD

INTRODUCTION

This test method is designed to measure the potential for a liquid substance to increase the burning rate or
burning intensity of a combustible substance, or to form a mixture with a combustible substance which
spontaneously ignites, when the two are thoroughly mixed. It is based on the UN test for oxidizing liquids
(1) and is equivalent to it. However, as this method A.21 is primarily designed to satisfy the requirements
of Dir 67/548, comparison with only one reference substance is required. Testing and comparison to
additional reference substances may be necessary when the results of the test are expected to be used for
other purposes (1).

This test need not be performed when examination of
reasonable doubt that the substance is incapable of reacting exothermically with a combustible material.

formula establishes beyond

the structural

It is useful to have preliminary information on any potential explosive properties of the substance before
performing this test.

test

This
not
or organic peroxides.

is

applicable

to

solids,

gases,

explosive

or

highly

flammable

substances,

This test may not need be performed when results for the test substance in the UN test for oxidizing
liquids (1) are already available.

1.2

DEFINITIONS AND UNITS

Mean pressure rise time is the mean of the measured times for a mixture under test to produce a
pressure rise from 690 kPa to 2 070 kPa above atmospheric.

1.3

REFERENCE SUBSTANCE

65 % (w/w) aqueous nitric acid (analytical grade) is required as a reference substance (2).

Optionally,
purposes,1 testing of additional reference substances may also be appropriate (3).

if the experimenter foresees that the results of this test may eventually be used for other

1.4

PRINCIPLE OF THE TEST METHOD

The liquid to be tested is mixed in a 1 to 1 ratio, by mass, with fibrous cellulose and introduced into a
pressure vessel. If during mixing or filling spontaneous ignition occurs, no further testing is necessary.

If spontaneous ignition does not occur the full test is carried out. The mixture is heated in a pressure
vessel and the mean time taken for the pressure to rise from 690 kPa to 2 070 kPa above atmospheric is
determined. This is compared with the mean pressure rise time for the 1:1 mixture of the reference
substance(s) and cellulose.

(1) As, for example, in the framework of UN transport regulations.
(2)
(3)

The acid should be titrated before testing to confirm its concentration.
E.g.: 50 % (w/w) perchloric acid and 40 % (w/w) sodium chlorate are used in reference 1.

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1.5

QUALITY CRITERIA

In a series of five trials on a single substance no results should differ by more than 30 % from the
arithmetic mean. Results that differ by more than 30 % from the mean should be discarded, the mixing
and filling procedure improved and the testing repeated.

1.6

DESCRIPTION OF THE METHOD

1.6.1

Preparation

1.6.1.1

Combustible substance

Dried, fibrous cellulose with a fibre length between 50 and 250 μm and a mean diameter of 25 μm (4), is
used as the combustible material. It is dried to constant weight in a layer not more than 25 mm thick at
105 °C for 4 hours and kept in a desiccator, with desiccant, until cool and required for use. The water
content of the dried cellulose should be less than 0.5 % by dry mass (5). If necessary, the drying time
should be prolonged to achieve this (6). The same batch of cellulose is to be used throughout the test.

1.6.1.2

Apparatus

1.6.1.2.1

Pressure vessel

A pressure vessel is required. The vessel consists of a cylindrical steel pressure vessel 89 mm in length and
60 mm in external diameter (see figure 1). Two flats are machined on opposite sides (reducing the cross-
section of the vessel to 50 mm) to facilitate holding whilst fitting up the firing plug and vent plug. The
vessel, which has a bore of 20 mm diameter is internally rebated at either end to a depth of 19 mm and
threaded to accept 1″ British Standard Pipe (BSP) or metric equivalent. A pressure take-off, in the form of a
side arm, is screwed into the curved face of the pressure vessel 35 mm from one end and at 90° to the
machined flats. The socket for this is bored to a depth of 12 mm and threaded to accept the 1/2″ BSP (or
metric equivalent) thread on the end of the side-arm. If necessary, an inert seal is fitted to ensure a gas-
tight seal. The side-arm extends 55 mm beyond the pressure vessel body and has a bore of 6 mm. The
end of the side-arm is rebated and threaded to accept a diaphragm type pressure transducer. Any pressure-
measuring device may be used provided that it is not affected by the hot gases or the decomposition
products and is capable of responding to rates of pressure rise of 690-2 070 kPa in not more than 5 ms.

The end of the pressure vessel farthest from the side-arm is closed with a firing plug which is fitted with
two electrodes, one insulated from, and the other earthed to, the plug body. The other end of the pressure
vessel
is closed by a bursting disk (bursting pressure approximately 2 200 kPa) held in place with a
retaining plug which has a 20 mm bore. If necessary, an inert seal is used with the firing plug to ensure a
gas-tight fit. A support stand (figure 2) holds the assembly in the correct attitude during use. This usually
comprises a mild steel base plate measuring 235 mm x 184 mm x 6 mm and a 185 mm length of square
hollow section (S.H.S.) 70 mm x 70 mm x 4 mm.

A section is cut from each of two opposite sides at one end of the length of S.H.S. so that a structure
having two flat sided legs surmounted by 86 mm length of intact box section results. The ends of these
flat sides are cut to an angle of 60° to the horizontal and welded to the base plate. A slot measuring 22
mm wide x 46 mm deep is machined in one side of the upper end of the base section such that when the
pressure vessel assembly is lowered, firing plug end first, into the box section support, the side-arm is
accommodated in the slot. A piece of steel 30 mm wide and 6 mm thick is welded to the lower internal
face of the box section to act as a spacer. Two 7 mm thumb screws, tapped into the opposite face, serve to
hold the pressure vessel firmly in place. Two 12 mm wide strips of 6 mm thick steel, welded to the side
pieces abutting the base of the box section, support the pressure vessel from beneath.

e.g. Whatman Column Chromatographic Cellulose Powder CF 11, catalogue no 4021 050

(4)
(5) Confirmed by (e.g.) Karl-Fisher titration
(6) Alternatively, this water content can also be achieved by (e.g.) heating at 105 °C under vacuum for 24 h

16.6.2004

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L 216/171

1.6.1.2.2

Ignition System

The ignition system consists of a 25 cm long Ni/Cr wire with a diameter 0.6 mm and a resistance of 3.85
ohm/m. The wire is wound, using a 5 mm diameter rod, in the shape of a coil and is attached to the firing
plug electrodes. The coil should have one of the configurations shown in figure 3. The distance between
the bottom of the vessel and the underside of the ignition coil should be 20 mm. If the electrodes are not
adjustable, the ends of the ignition wire between the coil and the bottom of the vessel should be insulated
by a ceramic sheath. The wire is heated by a constant current power supply able to deliver at least 10 A.

1.6.2

Performance of the test (7)

The apparatus, assembled complete with pressure transducer and heating system but without the bursting
disk in position, is supported firing plug end down. 2.5 g of the liquid to be tested is mixed with 2.5 g of
dried cellulose in a glass beaker using a glass stirring rod (8). For safety, the mixing should be performed
with a safety shield between the operator and mixture. If the mixture ignites during mixing or filling, no
further testing is necessary. The mixture is added, in small portions with tapping, to the pressure vessel
making sure that the mixture is packed around the ignition coil and is in good contact with it. It is
important that the coil
is not distorted during the packing process as this may lead to erroneous
results (9). The bursting disk is placed in position and the retaining plug is screwed in tightly. The charged
vessel is transferred to the firing support stand, bursting disk uppermost, which should be located in a
suitable, armoured fume cupboard or firing cell. The power supply is connected to the external terminals
of the firing plug and 10 A applied. The time between the start of mixing and switching on the power
should not exceed 10 minutes.

The signal produced by the pressure transducer is recorded on a suitable system which allows both
evaluation and the generation of a permanent record of the time pressure profile obtained (e.g. a transient
recorder coupled to a chart recorder). The mixture is heated until the bursting disk ruptures or until at
least 60 s have elapsed. If the bursting disk does not rupture, the mixture should be allowed to cool before
carefully dismantling the apparatus, taking precautions to allow for any pressurization which may occur.
Five trials are performed with the test substance and the reference substance(s). The time taken for the
pressure to rise from 690 kPa to 2 070 kPa above atmospheric is noted. The mean pressure rise time is
calculated.

In some cases, substances may generate a pressure rise (too high or too low), caused by chemical reactions
not characterizing the oxidizing properties of the substance. In these cases, it may be necessary to repeat
the test with an inert substance, e.g. diatomite (kieselguhr), in place of the cellulose in order to clarify the
nature of the reaction.

2.

DATA

Pressure rise times for both the test substance and the reference substance(s).

Pressure rise times for the tests with an inert substance, if performed.

2.1

TREATMENT OF RESULTS

The mean pressure rise times for both the test substance and the reference substances(s) are calculated.

The mean pressure rise time for the tests with an inert substance (if performed) is calculated.

(7) Mixtures of oxidizers with cellulose must be treated as potentially explosive and handled with due care
(8)

In practice this can be achieved by preparing a 1:1 mixture of the liquid to be tested and cellulose in a greater amount than needed
for the trial and transferring 5 ± 0.1 g to the pressure vessel. The mixture is to be freshly prepared for each trial.
In particular, contact between the adjacent turns of the coil must be avoided.

(9)

L 216/172

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Official Journal of the European Union

16.6.2004

Some examples of results are shown in Table 1

Table 1

Examples of results (d)

Substance (c)

Mean pressure rise time for a 1:1
mixture with celulose
(ms)

Ammonium dichromate, saturated aqueous solution

Calcium nitrate, saturated aqueous solution

Ferric nitrate, saturated aqueous solution

Lithium perchlorate, saturated aqueous solution

Magnesium perchlorate, saturated aqueous solution

Nickel nitrate, saturated aqueous solution

Nitric acid, 65 %

Perchloric acid, 50 %

Perchloric acid, 55 %

Potassium nitrate, 30 % aqueous solution

Silver nitrate, saturated aqueous solution

Sodium chlorate, 40 % aqueous solution

Sodium nitrate, 45 % aqueous solution

Inert Substance

Water:cellulose

(a) Mean value from interlaboratory comparative trials
(b) Maximum pressure of 2 070 kPa not reached
(c) Saturated solutions should be prepared at 20 °C
(d) See reference (1) for classification under the UN transport scheme

20 800

6 700

4 133

1 686

777

6 250

4 767 (a)

121 (a)

59

26 690

— (b)

2 555 (a)

4 133

— (b)

3.

3.1

REPORT

TEST REPORT

The test report should include the following information:

— the identity, composition, purity, etc of the substance tested;

— the concentration of the test substance;

— the drying procedure of the cellulose used

— the water content of the cellulose used

— the results of the measurements;

— the results from tests with an inert substance, if any;

— the calculated mean pressure rise times;

— any deviations from this method and the reasons for them;

— all additional information or remarks relevant to the interpretation of the results;

16.6.2004

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Official Journal of the European Union

L 216/173

3.2

INTERPRETATION OF THE RESULTS (10)

The test results are assessed on the basis of:

a) whether the mixture of test substance and cellulose spontaneously ignites; and

b)

the comparison of the mean time taken for the pressure to rise from 690 kPa to 2 070 kPa with that
of the reference substance(s).

A liquid substance is to be considered as an oxidizer when:

a)

a 1:1 mixture, by mass, of the substance and cellulose spontaneously ignites; or

b)

a 1:1 mixture, by mass, of the substance and cellulose exhibits a mean pressure rise time less than or
equal to the mean pressure rise time of a 1:1 mixture, by mass, of 65 % (w/w) aqueous nitric acid and
cellulose.

In order to avoid a false positive result, if necessary, the results obtained when testing the substance with
an inert material should also be considered when interpreting the results.

4.

REFERENCES

(1) Recommendations on the Transport of Dangerous Goods, Manual of Tests and Criteria. 3rd revised
edition. UN Publication No: ST/SG/AC.10/11/Rev. 3, 1999, page 342. Test O.2: Test for oxidizing
liquids.

(10) See reference 1 for interpretation of the results under the UN transport regulations using several reference substances.

L 216/174

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Official Journal of the European Union

16.6.2004

Figure 1

Pressure vessel

(A) Pressure vessel body
(D) Soft lead washer
(G) Pressure transducer head
(K) Earthed electrode
(N) Washer distorting groove

(B) Bursting disk retaining plug
(E) Bursting disc
(H) Washer
(L)

Insulation

(C) Firing plug
(F) Side arm
(J)
(M) Steel cone

Insulated electrode

16.6.2004

EN

Official Journal of the European Union

L 216/175

Figure 2

Support stand

L 216/176

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Official Journal of the European Union

16.6.2004

Figure 3

Ignition System

(A)

Ignition coil

(B)

Insulation

(C) Electrodes

(D) Firing plug

Note: either of these configurations may be used.’

16.6.2004

EN

Official Journal of the European Union

L 216/177

ANNEX 2B

‘B.1 bis. ACUTE ORAL TOXICITY - FIXED DOSE PROCEDURE

1.

METHOD

This test method is equivalent to OECD TG 420 (2001)

1.1

INTRODUCTION

Traditional methods for assessing acute toxicity use death of animals as an endpoint. In 1984, a new
approach to acute toxicity testing was suggested by the British Toxicology Society based on the
administration at a series of fixed dose levels (1). The approach avoided using death of animals as an
endpoint, and relied instead on the observation of clear signs of toxicity at one of a series of fixed dose
levels. Following UK (2) and international (3) in vivo validation studies the procedure was adopted as a
testing method in 1992. Subsequently, the statistical properties of the Fixed Dose Procedure have been
evaluated using mathematical models in a series of studies (4)(5)(6). Together, the in vivo and modelling
studies have demonstrated that the procedure is reproducible, uses fewer animals and causes less suffering
than the traditional methods and is able to rank substances in a similar manner to the other acute toxicity
testing methods.

Guidance on the selection of the most appropriate test method for a given purpose can be found in the
Guidance Document on Acute Oral Toxicity Testing (7). This Guidance Document also contains additional
information on the conduct and interpretation of Testing Method B.1bis.

It is a principle of the method that in the main study only moderately toxic doses are used, and that
administration of doses that are expected to be lethal should be avoided. Also, doses that are known to
cause marked pain and distress, due to corrosive or severely irritant actions, need not be administered.
Moribund animals, or animals obviously in pain or showing signs of severe and enduring distress shall be
humanely killed, and are considered in the interpretation of the test results in the same way as animals
that died on test. Criteria for making the decision to kill moribund or severely suffering animals, and
guidance on the recognition of predictable or impending death, are the subject of a separate Guidance
Document (8).

The method provides information on the hazardous properties and allows the substance to be ranked and
classified according to the Globally Harmonised System (GHS) for the classification of chemicals which
cause acute toxicity (9).

The testing laboratory should consider all available information on the test substance prior to conducting
the study. Such information will include the identity and chemical structure of the substance; its physico-
chemical properties; the results of any other in vitro or in vivo toxicity tests on the substance; toxicological
data on structurally related substances; and the anticipated use(s) of the substance. This information is
necessary to satisfy all concerned that the test is relevant for the protection of human health, and will help
in the selection of an appropriate starting dose.

1.2

DEFINITIONS

Acute oral toxicity: refers to those adverse effects occurring following oral administration of a single
dose of a substance or multiple doses given within 24 hours.

Delayed death: means that an animal does not die or appear moribund within 48 hours but dies later
during the 14-day observation period.

Dose: is the amount of test substance administered. Dose is expressed as weight of test substance per unit
weight of test animal (e.g. mg/kg).

Evident toxicity: is a general term describing clear signs of toxicity following the administration of test
substance (see (3) for examples) such that at the next highest fixed dose either severe pain and enduring
signs of severe distress, moribund status (criteria are presented in the Humane Endpoints Guidance
Document (8)), or probable mortality in most animals can be expected.

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GHS: Globally Harmonised Classification System for Chemical Substances and Mixtures. A joint activity of
OECD (human health and the environment), UN Committee of Experts on Transport of Dangerous Goods
(physical–chemical properties) and ILO (hazard communication) and co-ordinated by the Interorganisation
Programme for the Sound Management of Chemicals (IOMC).

Impending death: when moribund state or death is expected prior to the next planned time of
observation. Signs indicative of
lateral position,
recumbence, and tremor. (See the Humane Endpoint Guidance Document (8) for more details).

this state in rodents could include convulsions,

LD50 (median lethal dose): is a statistically derived single dose of a substance that can be expected to
cause death in 50 per cent of animals when administered by the oral route. The LD50 value is expressed in
terms of weight of test substance per unit weight of test animal (mg/kg).

Limit dose: refers to a dose at an upper limitation on testing (2 000 or 5 000 mg/kg).

Moribund status: being in a state of dying or inability to survive, even if treated. (See the Humane
Endpoint Guidance Document (8) for more details).

Predictable death: presence of clinical signs indicative of death at a known time in the future before the
planned end of the experiment, for example: inability to reach water or food. (See the Humane Endpoint
Guidance Document (8) for more details).

1.3

PRINCIPLE OF THE TEST METHOD

Groups of animals of a single sex are dosed in a stepwise procedure using the fixed doses of 5, 50, 300
and 2 000 mg/kg (exceptionally an additional fixed dose of 5 000 mg/kg may be considered, see section
1.6.2). The initial dose level is selected on the basis of a sighting study as the dose expected to produce
some signs of toxicity without causing severe toxic effects or mortality. Clinical signs and conditions
associated with pain, suffering, and impending death, are described in detail in a separate OECD Guidance
Document (8). Further groups of animals may be dosed at higher or lower fixed doses, depending on the
presence or absence of signs of toxicity or mortality. This procedure continues until the dose causing
evident toxicity or no more than one death is identified, or when no effects are seen at the highest dose or
when deaths occur at the lowest dose.

1.4

DESCRIPTION OF THE TEST METHOD

1.4.1

Selection of animal species

The preferred rodent species is the rat, although other rodent species may be used. Normally females are
used (7). This is because literature surveys of conventional LD50 tests show that usually there is little
difference in sensitivity between the sexes, but in those cases where differences are observed, females are
generally slightly more sensitive (10). However,
toxicokinetic
properties of structurally related chemicals indicates that males are likely to be more sensitive then this sex
should be used. When the test is conducted in males, adequate justification should be provided.

the toxicological or

if knowledge of

Healthy young adult animals of commonly used laboratory strains should be employed. Females should be
nulliparous and non-pregnant. Each animal, at the commencement of its dosing, should be between 8 and
12 weeks old and its weight should fall in an interval within ± 20 % of the mean weight of any previously
dosed animals.

1.4.2

Housing and feeding conditions

The temperature of the experimental animal room should be 22ºC (± 3ºC). Although the relative humidity
should be at least 30 % and preferably not exceed 70 % other than during room cleaning the aim should
be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding,
conventional laboratory diets may be used with an unlimited supply of drinking water. Animals may be
group-caged by dose, but the number of animals per cage must not interfere with clear observations of
each animal.

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1.4.3

Preparation of animals

The animals are randomly selected, marked to permit individual identification, and kept in their cages for
at least 5 days prior to the start of dosing to allow for acclimatisation to the laboratory conditions.

1.4.4

Preparation of doses

In general test substances should be administered in a constant volume over the range of doses to be
tested by varying the concentration of the dosing preparation. Where a liquid end product or mixture is to
be tested however, the use of the undiluted test substance, i.e. at a constant concentration, may be more
relevant to the subsequent risk assessment of that substance, and is a requirement of some regulatory
authorities. In either case, the maximum dose volume for administration must not be exceeded. The
maximum volume of liquid that can be administered at one time depends on the size of the test animal.
In rodents, the volume should not normally exceed 1ml/100g of body weight: however in the case of
aqueous solutions 2 ml/100g body weight can be considered. With respect to the formulation of the
dosing preparation,
the use of an aqueous solution/suspension/emulsion is recommended wherever
possible, followed in order of preference by a solution/suspension/emulsion in oil (e.g. corn oil) and then
possibly solution in other vehicles. For vehicles other than water the toxicological characteristics of the
vehicle should be known. Doses must be prepared shortly prior to administration unless the stability of
the preparation over the period during which it will be used is known and shown to be acceptable.

1.5

PROCEDURE

1.5.1

Administration of doses

The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation
canula. In the unusual circumstance that a single dose is not possible, the dose may be given in smaller
fractions over a period not exceeding 24 hours.

Animals should be fasted prior to dosing (e.g. with the rat, food but not water should be withheld over-
night; with the mouse, food but not water should be withheld for 3-4 hours). Following the period of
fasting, the animals should be weighed and the test substance administered. After the substance has been
administered, food may be withheld for a further 3-4 hours in rats or 1-2 hours in mice. Where a dose is
administered in fractions over a period of time, it may be necessary to provide the animals with food and
water depending on the length of the period.

1.5.2

Sighting study

The purpose of the sighting study is to allow selection of the appropriate starting dose for the main study.
The test substance is administered to single animals in a sequential manner following the flow charts in
Annex 1. The sighting study is completed when a decision on the starting dose for the main study can be
made (or if a death is seen at the lowest fixed dose).

The starting dose for the sighting study is selected from the fixed dose levels of 5, 50, 300 and 2 000 mg/
kg as a dose expected to produce evident toxicity based, when possible, on evidence from in vivo and in
vitro data from the same chemical and from structurally related chemicals.
In the absence of such
information, the starting dose will be 300 mg/kg.

A period of at least 24 hours will be allowed between the dosing of each animal. All animals should be
observed for at least 14 days.

Exceptionally, and only when justified by specific regulatory needs, the use of an additional upper fixed
dose level of 5 000 mg/kg may be considered (see Annex 3). For reasons of animal welfare concern,
testing of animals in GHS Category 5 ranges (2 000-5 000 mg/kg is discouraged and should only be
considered when there is a strong likelihood that the results of such a test have a direct relevance for
protecting human or animal health or the environment.

In cases where an animal tested at the lowest fixed dose level (5mg/kg) in the sighting study dies, the
normal procedure is to terminate the study and assign the substance to GHS Category 1 (as shown in
Annex 1). However, if further confirmation of the classification is required, an optional supplementary
procedure may be conducted, as follows. A second animal is dosed at 5mg/kg. If this second animal dies,
then GHS Category 1 will be confirmed and the study will be immediately terminated. If the second
animal survives, then a maximum of three additional animals will be dosed at 5mg/kg. Because there will
be a high risk of mortality, these animals should be dosed in a sequential manner to protect animal

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welfare. The time interval between dosing each animal should be sufficient to establish that the previous
animal is likely to survive. If a second death occurs, the dosing sequence will be immediately terminated
and no further animals will be dosed. Because the occurrence of a second death (irrespective of the
number of animals tested at the time of termination) falls into outcome A (2 or more deaths), the
classification rule of Annex 2 at the 5mg/kg fixed dose is followed (Category 1 if there are 2 or more
deaths or Category 2 if there is no more than 1 death). In addition, Annex 4 gives guidance on the
classification in the EU system until the new GHS is implemented.

1.5.3

Main study

1.5.3.1

Numbers of animals and dose levels

The action to be taken following testing at the starting dose level is indicated by the flow charts in Annex
2. One of three actions will be required; either stop testing and assign the appropriate hazard classification
class, test at a higher fixed dose or test at a lower fixed dose. However, to protect animals, a dose level that
caused death in the sighting study will not be revisited in the main study (see Annex 2). Experience has
shown that the most likely outcome at the starting dose level will be that the substance can be classified
and no further testing will be necessary.

A total of five animals of one sex will normally be used for each dose level investigated. The five animals
will be made up of one animal from the sighting study dosed at the selected dose level together with an
additional four animals (except, unusually, if a dose level used on the main study was not included in the
sighting study).

The time interval between dosing at each level is determined by the onset, duration, and severity of toxic
signs. Treatment of animals at the next dose should be delayed until one is confident of survival of the
previously dosed animals. A period of 3 or 4 days between dosing at each dose level is recommended, if
needed, to allow for the observation of delayed toxicity. The time interval may be adjusted as appropriate,
e.g., in case of inconclusive response.

When the use of an upper fixed dose of 5 000 mg/kg is considered, the procedure outlined in Annex 3
should be followed (see also section 1.6.2).

1.5.3.2

Limit test

The limit test is primarily used in situations where the experimenter has information indicating that the
test material is likely to be nontoxic, i.e., having toxicity only above regulatory limit doses. Information
about the toxicity of the test material can be gained from knowledge about similar tested compounds or
similar tested mixtures or products, taking into consideration the identity and percentage of components
known to be of toxicological significance. In those situations where there is little or no information about
its toxicity, or in which the test material is expected to be toxic, the main test should be performed.

Using the normal procedure, a sighting study starting dose of 2 000 mg/kg (or exceptionally 5 000 mg/
kg) followed by dosing of a further four animals at this level serves as a limit test for this guideline.

1.6

OBSERVATIONS

Animals are observed individually after dosing at least once during the first 30 minutes, periodically
during the first 24 hours, with special attention given during the first 4 hours, and daily thereafter, for a
total of 14 days, except where they need to be removed from the study and humanely killed for animal
welfare reasons or are found dead. However, the duration of observation should not be fixed rigidly. It
should be determined by the toxic reactions, time of onset and length of recovery period, and may thus be
extended when considered necessary. The times at which signs of toxicity appear and disappear are
important, especially if there is a tendency for toxic signs to be delayed (11). All observations are
systematically recorded, with individual records being maintained for each animal.

Additional observations will be necessary if the animals continue to display signs of toxicity. Observations
should include changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory,
autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention
should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
The principles and criteria summarised in the Humane Endpoints Guidance Document should be taken

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into consideration (8). Animals found in a moribund condition and animals showing severe pain or
enduring signs of severe distress should be humanely killed. When animals are killed for humane reasons
or found dead, the time of death should be recorded as precisely as possible.

1.6.1

Body weight

Individual weights of animals should be determined shortly before the test substance is administered and
at least weekly thereafter. Weight changes should be calculated and recorded. At the end of the test
surviving animals are weighed and then humanely killed.

1.6.2

Pathology

All test animals (including those that die during the test or are removed from the study for animal welfare
reasons) should be subjected to gross necropsy. All gross pathological changes should be recorded for each
animal. Microscopic examination of organs showing evidence of gross pathology in animals surviving 24
or more hours after the initial dosing may also be considered because it may yield useful information.

2.

DATA

Individual animal data should be provided. Additionally, all data should be summarised in tabular form,
showing for each test group the number of animals used, the number of animals displaying signs of
toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of
individual animals, a description and the time course of toxic effects and reversibility, and necropsy
findings.

3.

3.1

REPORTING

Test report

The test report must include the following information, as appropriate:

Test substance:

— physical nature, purity, and, where relevant, physico-chemical properties (including isomerisation);

— identification data, including CAS number.

Vehicle (if appropriate):

— justification for choice of vehicle, if other than water.

Test animals:

— species/strain used;

— microbiological status of the animals, when known;

— number, age and sex of animals (including, where appropriate, a rationale for use of males instead

of females);

— source, housing conditions, diet etc.;

Test conditions:

— details of test substance formulation,

including details of the physical form of the material

administered;

— details of the administration of the test substance including dosing volumes and time of dosing;

— details of food and water quality (including diet type/source, water source);

— the rationale for the selection of the starting dose.

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Results:

— tabulation of response data and dose level for each animal (i.e. animals showing signs of toxicity

including mortality, nature, severity and duration of effects);

— tabulation of body weight and body weight changes;

— individual weights of animals at the day of dosing, in weekly intervals thereafter, and at time of

death or sacrifice;

— date and time of death if prior to scheduled sacrifice;

— time course of onset of signs of toxicity and whether these were reversible for each animal;

— necropsy findings and histopathological findings for each animal, if available.

Discussion and interpretation of results.

Conclusions.

4.

REFERENCES

(1) British Toxicology Society Working Party on Toxicity (1984). Special report: a new approach to the
classification of substances and preparations on the basis of their acute toxicity. Human Toxicol., 3,
85-92.

(2) Van den Heuvel, M.J., Dayan, A.D. and Shillaker, R.O. (1987). Evaluation of the BTS approach to the

testing of substances and preparations for their acute toxicity. Human Toxicol., 6, 279-291.

(3) Van den Heuvel, M.J., Clark, D.G., Fielder, R.J., Koundakjian, P.P., Oliver, G.J.A., Pelling, D., Tomlinson,
N.J. and Walker, A.P. (1990). The international validation of a fixed-dose procedure as an alternative to
the classical LD50 test. Fd. Chem. Toxicol. 28, 469-482.

(4) Whitehead, A. and Curnow, R.N. (1992). Statistical evaluation of the fixed-dose procedure. Fd. Chem.

Toxicol., 30, 313-324.

(5) Stallard, N. and Whitehead, A. (1995). Reducing numbers in the fixed-dose procedure. Human Exptl.

Toxicol. 14, 315-323. Human Exptl. Toxicol.

(6) Stallard, N., Whitehead, A. and Ridgeway, P. (2002). Statistical evaluation of the revised fixed dose

procedure. Hum. Exp. Toxicol., 21, 183-196.

(7) OECD (2001). Guidance Document on Acute Oral Toxicity Testing. Environmental Health and Safety

Monograph Series on Testing and Assessment N. 24. Paris

(8) OECD (2000). Guidance Document on the Recognition, Assessment and Use of Clinical Signs as
Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and
Safety Monograph Series on Testing and Assesment N. 19.

(9) OECD (1998). Harmonised Integrated Hazard Classification for Human Health and Environmental
Effects of Chemical Substances as endorsed by the 28th Joint Meeting of the Chemicals Committee
and the Working Party on Chemicals in November 1998, Part 2, p.11 [http://webnet1.oecd.org/oecd/
pages/home/displaygeneral/0,3380,EN-documents-521-14-no-24-no-0,FF.html].

(10) Lipnick, R.L., Cotruvo, J.A., Hill, R.N., Bruce, R.D., Stitzel, K.A., Walker, A.P., Chu, I., Goddard, M.,
Segal, L., Springer, J.A. and Myers, R.C. (1995). Comparison of the Up-and-Down, Conventional LD50,
and Fixed-Dose Acute Toxicity Procedures. Fd. Chem. Toxicol. 33, 223-231.

(11) Chan P.K and A.W. Hayes (1994) Chapter 16 Acute Toxicity and Eye Irritation . In: Principles and

Methods of Toxicology . 3 rd Edition. A.W. Hayes , Editor. Raven Press, Ltd. New York, USA.

ANNEX 1: FLOW CHART FOR THE SIGHTING STUDY

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ANNEX 3

CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH EXPECTED LD50 VALUES EXCEEDING
2 000 MG/KG WITHOUT THE NEED FOR TESTING.

Criteria for hazard Category 5 are intended to enable the identification of test substances which are of relatively low acute
toxicity hazard but which, under certain circumstances may present a danger to vulnerable populations. These substances
are anticipated to have an oral or dermal LD50 in the range of 2 000-5 000 mg/kg or equivalent doses for other routes.
Test substances could be classified in the hazard category defined by: 2 000 mg/kg < LD50 < 5 000 mg/kg (Category 5 in
the GHS) in the following cases:

a)

if directed to this category by any of the testing schemes of Annex 2, based on mortality incidences

b)

c)

if reliable evidence is already available that indicates the LD50 to be in the range of Category 5 values; or other animal
studies or toxic effects in humans indicate a concern for human health of an acute nature.

through extrapolation, estimation or measurement of data if assignment to a more hazardous class is not warranted,
and

— reliable information is available indicating significant toxic effects in humans, or

— any mortality is observed when tested up to Category 4 values by the oral route, or

— where expert judgement confirms significant clinical signs of toxicity, when tested up to Category 4 values,

except for diarrhoea, piloerection or an ungroomed appearance, or

— where expert judgement confirms reliable information indicating the potential for significant acute effects from

the other animal studies.

TESTING AT DOSES ABOVE 2 000 MG/KG

Exceptionally, and only when justified by specific regulatory needs, the use of an additional upper fixed dose level of
5 000 mg/kg may be considered. Recognising the need to protect animal welfare, testing at 5 000 mg/kg is discouraged
and should only be considered when there is a strong likelihood that the results of such a test would have a direct
relevance for protecting animal or human health (9).

Sighting Study

The decision rules governing the sequential procedure presented in Annex 1 are extended to include a 5 000 mg/kg dose
level. Thus, when a sighting study starting dose of 5 000 mg/kg is used outcome A (death) will require a second animal
to be tested at 2 000 mg/kg; outcomes B and C (evident toxicity or no toxicity) will allow the selection of 5 000 mg/kg
as the main study starting dose. Similarly, if a starting dose other than 5 000 mg/kg is used then testing will progress to
5 000 mg/kg in the event of outcomes B or C at 2 000 mg/kg; a subsequent 5 000 mg/kg outcome A will dictate a main
study starting dose of 2 000 mg/kg and outcomes B and C will dictate a main study starting dose of 5 000 mg/kg.

Main Study

The decision rules governing the sequential procedure presented in Annex 2 are extended to include a 5 000 mg/kg dose
level. Thus, when a main study starting dose of 5 000 mg/kg is used, outcome A (≥2 deaths) will require the testing of a
second group at 2 000 mg/kg; outcome B (evident toxicity and/or ≤1 death) or C (no toxicity) will result in the substance
being unclassified according to GHS. Similarly,
if a starting dose other than 5 000 mg/kg is used then testing will
progress to 5 000 mg/kg in the event of outcome C at 2 000 mg/kg; a subsequent 5 000 mg/kg outcome A will result in
the substance being assigned to GHS Category 5 and outcomes B or C will lead to the substance being unclassified.

TEST METHOD B.1 bis – Guidance on classification according to the EU scheme to cover the transition period until full implementation of the Globally Harmonised Classification System (GHS)
(taken from reference (8))

ANNEX 4

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ANNEX 2C

‘B.1 tris. ACUTE ORAL TOXICITY - ACUTE TOXIC CLASS METHOD

1.

METHOD

This test method is equivalent to OECD TG 423 (2001)

1.1

INTRODUCTION

The acute toxic class method (1) set out in this test is a stepwise procedure with the use of 3 animals of a
single sex per step. Depending on the mortality and/or the moribund status of the animals, on average 2-4
steps may be necessary to allow judgement on the acute toxicity of the test substance. This procedure is
reproducible, uses very few animals and is able to rank substances in a similar manner to the other acute
toxicity testing methods. The acute toxic class method is based on biometric evaluations (2)(3)(4)(5) with
fixed doses, adequately separated to enable a substance to be ranked for classification purposes and hazard
assessment. The method as adopted in 1996 was extensively validated in vivo against LD50 data obtained
from the literature, both nationally (6) and internationally (7).

Guidance on the selection of the most appropriate test method for a given purpose can be found in the
Guidance Document on Acute Oral Toxicity Testing (8). This Guidance Document also contains additional
information on the conduct and interpretation of testing method B.1tris.

Test substances, at doses that are known to cause marked pain and distress due to corrosive or severely
irritant actions, need not be administered. Moribund animals, or animals obviously in pain or showing
signs of severe and enduring distress shall be humanely killed, and are considered in the interpretation of
the test results in the same way as animals that died on test. Criteria for making the decision to kill
moribund or severely suffering animals, and guidance on the recognition of predictable or impending
death, are the subject of a separate Guidance Document (9).

The method uses pre-defined doses and the results allow a substance to be ranked and classified according
to the Globally Harmonised System for the classification of chemicals which cause acute toxicity (10).

In principle, the method is not intended to allow the calculation of a precise LD50, but does allow for the
determination of defined exposure ranges where lethality is expected since death of a proportion of the
animals is still the major endpoint of this test. The method allows for the determination of an LD50 value
only when at least two doses result in mortality higher than 0 % and lower than 100 %. The use of a
selection of pre-defined doses, regardless of test substance, with classification explicitly tied to number of
animals observed in different states improves the opportunity for laboratory to laboratory reporting
consistency and repeatability.

The testing laboratory should consider all available information on the test substance prior to conducting
the study. Such information will include the identity and chemical structure of the substance; its physico-
chemical properties; the result of any other in vivo or in vitro toxicity tests on the substance; toxicological
data on the structurally related substances; and the anticipated use(s) of the substance. This information is
necessary to satisfy all concerned that the test is relevant for the protection of human health and will help
in the selection of the most appropriate starting dose.

1.2

DEFINITIONS

Acute oral toxicity: refers to those adverse effects occurring following oral administration of a single
dose of a substance or multiple doses given within 24 hours.

Delayed death: means that an animal does not die or appear moribund within 48 hours but dies later
during the 14-day observation period.

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Dose: is the amount of test substance administered. Dose is expressed as weight of test substance per unit
weight of test animal (e.g. mg/kg).

GHS: Globally Harmonised Classification System for Chemical Substances and Mixtures. A joint activity of
OECD (human health and the environment), UN Committee of Experts on Transport of Dangerous Goods
(physical–chemical properties) and ILO (hazard communication) and co-ordinated by the Interorganisation
Programme for the Sound Management of Chemicals (IOMC).

Impending death: when moribund state or death is expected prior to the next planned time of
lateral position,
observation. Signs indicative of
recumbence, and tremor (See the Humane Endpoint Guidance Document (9) for more details).

this state in rodents could include convulsions,

LD50 (median lethal oral dose): is a statistically derived single dose of a substance that can be expected to
cause death in 50 per cent of animals when administered by the oral route. The LD50 value is expressed in
terms of weight of test substance per unit weight of test animal (mg/kg).

Limit dose: refers to a dose at an upper limitation on testing (2 000 or 5 000 mg/kg).

Moribund status: being in a state of dying or inability to survive, even if treated (See the Humane
Endpoint Guidance Document (9) for more details).

Predictable death: presence of clinical signs indicative of death at a known time in the future before the
planned end of the experiment; for example: inability to reach water or food. (See the Humane Endpoint
Guidance Document (9) for more details).

1.3

PRINCIPLE OF THE TEST

It is the principle of the test that, based on a stepwise procedure with the use of a minimum number of
animals per step, sufficient information is obtained on the acute toxicity of the test substance to enable its
classification. The substance is administered orally to a group of experimental animals at one of the
defined doses. The substance is tested using a stepwise procedure, each step using three animals of a single
sex (normally females). Absence or presence of compound-related mortality of the animals dosed at one
step will determine the next step, i.e.;

— no further testing is needed,

— dosing of three additional animals, with the same dose

— dosing of three additional animals at the next higher or the next lower dose level.

Details of the test procedure are described in Annex 1. The method will enable a judgement with respect
to classifying the test substance to one of a series of toxicity classes defined by fixed LD50 cut-off values.

1.4

DESCRIPTION OF THE METHOD

1.4.1

Selection of animal species

The preferred rodent species is the rat, although other rodent species may be used. Normally females are
used (9). This is because literature surveys of conventional LD50 tests show that, although there is little
difference in sensitivity between the sexes,
in those cases where differences are observed females are
generally slightly more sensitive (11). However if knowledge of the toxicological or toxicokinetic properties
of structurally related chemicals indicates that males are likely to be more sensitive, then this sex should be
used. When the test is conducted in males adequate justification should be provided.

Healthy young adult animals of commonly used laboratory strains should be employed. Females should be
nulliparous and non-pregnant. Each animal, at the commencement of its dosing, should be between 8 and
12 weeks old and its weight should fall in an interval within ± 20 % of the mean weight of any previously
dosed animals.

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1.4.2

Housing and feeding conditions

The temperature in the experimental animal room should be 22ºC (± 3ºC). Although the relative humidity
should be at least 30 % and preferably not exceed 70 % other than during room cleaning the aim should
be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding,
conventional laboratory diets may be used with an unlimited supply of drinking water. Animals may be
group-caged by dose, but the number of animals per cage must not interfere with clear observations of
each animal.

1.4.3

Preparation of animals

The animals are randomly selected, marked to permit individual identification, and kept in their cages for
at least 5 days prior to dosing to allow for acclimatisation to the laboratory conditions.

1.4.4

Preparation of doses

In general test substances should be administered in a constant volume over the range of doses to be
tested by varying the concentration of the dosing preparation. Where a liquid end product or mixture is to
be tested however, the use of the undiluted test substance, i.e. at a constant concentration, may be more
relevant to the subsequent risk assessment of that substance, and is a requirement of some regulatory
authorities. In either case, the maximum dose volume for administration must not be exceeded. The
maximum volume of liquid that can be administered at one time depends on the size of the test animal.
In rodents, the volume should not normally exceed 1ml/100g of body weight: however in the case of
aqueous solutions 2 ml/100g body weight can be considered. With respect to the formulation of the
dosing preparation,
the use of an aqueous solution/suspension/emulsion is recommended wherever
possible, followed in order of preference by a solution/suspension/emulsion in oil (e.g. corn oil) and then
possibly solution in other vehicles. For vehicles other than water the toxicological characteristics of the
vehicle should be known. Doses must be prepared shortly prior to administration unless the stability of
the preparation over the period during which it will be used is known and shown to be acceptable.

1.5

PROCEDURE

1.5.1

Administration of doses

The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation
canula. In the unusual circumstance that a single dose is not possible, the dose may be given in smaller
fractions over a period not exceeding 24 hours.

Animals should be fasted prior to dosing (e.g. with the rat, food but not water should be withheld over-
night, with the mouse, food but not water should be withheld for 3-4 hours). Following the period of
fasting, the animals should be weighed and the test substance administered. After the substance has been
administered, food may be withheld for a further 3-4 hours in rats or 1-2 hours in mice. Where a dose is
administered in fractions over a period it may be necessary to provide the animals with food and water
depending on the length of the period.

1.5.2

Number of animals and dose levels

Three animals are used for each step. The dose level to be used as the starting dose is selected from one of
four fixed levels, 5, 50, 300 and 2 000 mg/kg body weight. The starting dose level should be that which
is most likely to produce mortality in some of the dosed animals. The flow charts of Annex 1 describe the
procedure that should be followed for each of the starting doses. In addition, Annex 4 gives guidance on
the classification in the EU system until the new GHS is implemented.

When available information suggests that mortality is unlikely at the highest starting dose level (2 000
mg/kg body weight), then a limit test should be conducted. When there is no information on a substance
to be tested, for animal welfare reasons it is recommended to use the starting dose of 300 mg/kg body
weight.

The time interval between treatment groups is determined by the onset, duration, and severity of toxic
signs. Treatment of animals at the next dose, should be delayed until one is confident of survival of the
previously dosed animals.

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Exceptionally, and only when justified by specific regulatory needs, the use of additional upper dose level
of 5 000 mg/kg body weight may be considered (see Annex 2). For reasons of animal welfare concern,
testing of animals in GHS Category 5 ranges (2 000-5 000 mg/kg) is discouraged and should only be
considered when there is a strong likelihood that the results of such a test would have a direct relevance
for protecting human or animal health or the environment.

1.5.3

Limit test

The limit test is primarily used in situations where the experimenter has information indicating that the
test material is likely to be nontoxic, i.e., having toxicity only above regulatory limit doses. Information
about the toxicity of the test material can be gained from knowledge about similar tested compounds or
similar tested mixtures or products, taking into consideration the identity and percentage of components
known to be of toxicological significance. In those situations where there is little or no information about
its toxicity, or in which the test material is expected to be toxic, the main test should be performed.

A limit test at one dose level of 2 000 mg/kg body weight may be carried out with six animals (three
animals per step). Exceptionally a limit test at one dose level of 5000 mg/kg may be carried out with three
animals (see Annex 2). If test substance-related mortality is produced, further testing at the next lower
level may need to be carried out.

1.6

OBSERVATIONS

Animals are observed individually after dosing at least once during the first 30 minutes, periodically
during the first 24 hours, with special attention given during the first 4 hours, and daily thereafter, for a
total of 14 days, except where they need to be removed from the study and humanely killed for animal
welfare reasons or are found dead. However, the duration of observation should not be fixed rigidly. It
should be determined by the toxic reactions, time of onset and length of recovery period, and may thus be
extended when considered necessary. The times at which signs of toxicity appear and disappear are
important, especially if there is a tendency for toxic signs to be delayed (12). All observations are
systematically recorded with individual records being maintained for each animal.

Additional observations will be necessary if the animals continue to display signs of toxicity. Observations
should include changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory,
autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention
should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
The principles and criteria summarised in the Humane Endpoints Guidance Document (9) should be taken
into consideration. Animals found in a moribund condition and animals showing severe pain or enduring
signs of severe distress should be humanely killed. When animals are killed for humane reasons or found
dead, the time of death should be recorded as precisely as possible.

1.6.1

Body weight

Individual weights of animals should be determined shortly before the test substance is administered, and
at least weekly thereafter. Weight changes should be calculated and recorded. At the end of the test
surviving animals are weighed and humanely killed.

1.6.2

Pathology

All test animals (including those that die during the test or are removed from the study for animal welfare
reasons) should be subjected to gross necropsy. All gross pathological changes should be recorded for each
animal. Microscopic examination of organs showing evidence of gross pathology in animals surviving 24
or more hours may also be considered because it may yield useful information.

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2.

DATA

Individual animal data should be provided. Additionally, all data should be summarised in tabular form,
showing for each test group the number of animals used, the number of animals displaying signs of
toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of
individual animals, a description and the time course of toxic effects and reversibility, and necropsy
findings.

3.

3.1

REPORTING

Test report

The test report must include the following information, as appropriate:

Test substance:

— physical nature, purity, and, where relevant, physico-chemical properties (including isomerisation);

— identification data, including CAS number.

Vehicle (if appropriate)

— justification for choice of vehicle, if other than water.

Test animals:

— species/strain used;

— microbiological status of the animals, when known;

— number, age, and sex of animals (including, where appropriate, a rationale for the use of males

instead of females);

— source, housing conditions, diet etc.;

Test conditions:

— details of

test substance formulation including details of the physical form of the material

administered;

— details of the administration of the test substance including dosing volumes and time of dosing;

— details of food and water quality (including diet type/source, water source);

— the rationale for the selection of the starting dose.

Results:

— tabulation of response data and dose level for each animal (i.e. animals showing signs of toxicity

including mortality; nature, severity, and duration of effects);

— tabulation of body weight and body weight changes;

— individual weights of animals at the day of dosing, in weekly intervals thereafter, and at the time

of death or sacrifice

— date and time of death if prior to scheduled sacrifice

— time course of onset of signs of toxicity, and whether these were reversible for each animal;

— necropsy findings and histopathological findings for each animal, if available.

Discussion and interpretation of results.

Conclusions.

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4.

REFERENCES

(1) Roll R., Höfer-Bosse Th. And Kayser D.
Chemicals. Toxicol. Lett., Suppl. 31, 86

(1986). New Perspectives in Acute Toxicity Testing of

(2) Roll R., Riebschläger M., Mischke U. and Kayser D. (1989). Neue Wege zur Bestimmung der akuten

Toxizität von Chemikalien. Bundesgesundheitsblatt 32, 336-341.

(3) Diener W., Sichha L., Mischke U., Kayser D. and Schlede E. (1994). The Biometric Evaluation of the

Acute-Toxic-Class Method (Oral). Arch. Toxicol. 68, 559-610

(4) Diener W., Mischke U., Kayser D. and Schlede E. (1995). The Biometric Evaluation of the OECD

Modified Version of the Acute-Toxic-Class Method (Oral). Arch. Toxicol. 69, 729-734.

(5) Diener W., and Schlede E. (1999) Acute Toxicity Class Methods: Alterations to LD/LC50 Tests. ALTEX

16, 129-134

(6) Schlede E., Mischke U., Roll R. and Kayser D. (1992). A National Validation Study of the Acute-Toxic-

Class Method – An Alternative to the LD50 Test. Arch. Toxicol. 66, 455-470.

(7) Schlede E., Mischke U., Diener W. and Kayser D. (1994). The International Validation Study of the

Acute-Toxic-Class Method (Oral). Arch. Toxicol. 69, 659-670.

(8) OECD (2001) Guidance Document on Acute Oral Toxicity Testing. Environmental Health and Safety

Monograph Series on Testing and Assessment N. 24. Paris.

(9) OECD (2000) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as
Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and
Safety Monograph Series on Testing and Assessment N 19.

(10) OECD (1998) Harmonized Integrated Hazard Classification System For Human Health And
Environmental Effects Of Chemical Substances as endorsed by the 28th Joint Meeting of the Chemicals
Committee and the Working Party on Chemicals in November 1998, Part 2, p. 11 [http://
webnet1.oecd.org/oecd/pages/home/displaygeneral/0,3380,EN-documents-521-14-no-24-no-0,FF.html]

(11) Lipnick R L, Cotruvo, J A, Hill R N, Bruce R D, Stitzel K A, Walker A P, Chu I; Goddard M, Segal L,
Springer J A and Myers R C (1995) Comparison of the Up-and Down, Conventional LD50, and Fixed
Dose Acute Toxicity Procedures. Fd. Chem. Toxicol 33, 223-231.

(12) Chan P.K. and A.W. Hayes. (1994 ). Chap. 16. Acute Toxicity and Eye Irritancy. Principles and Methods

of Toxicology. Third Edition. A.W. Hayes, Editor. Raven Press, Ltd., New York, USA.’

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ANNEX 1

PROCEDURE TO BE FOLLOWED FOR EACH OF THE STARTING DOSES

GENERAL REMARKS

For each starting dose, the respective testing schemes as included in this Annex outline the procedure to be followed.

— Annex 1 a: Starting dose is 5 mg/kg bw

— Annex 1 b: Starting dose is 50 mg/kg bw

— Annex 1 c: Starting dose is: 300 mg/kg bw

— Annex 1 d: Starting dose is: 2 000 mg/kg bw

Depending on the number of humanely killed or dead animals, the test procedure follows the indicated arrows.

ANNEX 1 A

TEST PROCEDURE WITH A STARTING DOSE OF 5 MG/KG BODY WEIGHT

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TEST PROCEDURE WITH A STARTING DOSE OF 2 000 MG/KG BODY WEIGHT

ANNEX 1 D

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CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH EXPECTED LD50 VALUES EXCEEDING
2 000 MG/KG WITHOUT THE NEED FOR TESTING

ANNEX 2

Criteria for hazard Category 5 are intended to enable the identification of test substances which are of relatively low acute
toxicity hazard but which, under certain circumstances may present a danger to vulnerable populations. These substances
are anticipated to have an oral or dermal LD50 in the range of 2 000-5 000 mg/kg or equivalent doses for other routes.
The test substance should be classified in the hazard category defined by: 2 000 mg/kg < LD50 < 5 000 mg/kg (Category
5 in the GHS) in the following cases:

a)

If directed to this category by any of the testing schemes of Annex 1a-1d , based on mortality incidences;

b)

c)

if reliable evidence is already available that indicates the LD50 to be in the range of Category 5 values; or other animal
studies or toxic effects in humans indicate a concern for human health of an acute nature.

through extrapolation, estimation or measurement of data if assignment to a more hazardous class is not warranted,
and

— reliable information is available indicating significant toxic effects in humans, or

— any mortality is observed when tested up to Category 4 values by the oral route, or

— where expert judgement confirms significant clinical signs of toxicity, when tested up to Category 4 values,

except for diarrhoea, piloerection or an ungroomed appearance, or

— where expert judgement confirms reliable information indicating the potential for significant acute effects from

the other animal studies.

TESTING AT DOSES ABOVE 2 000 MG/KG

Recognising the need to protect animal welfare, testing of animals in Category 5 (5 000 mg/kg) ranges is discouraged and
should only be considered when there is a strong likelihood that results of such a test have a direct relevance for
protecting human or animal health (10). No further testing should be conducted at higher dose levels.

When testing is required a dose of 5 000 mg/kg, only one step (i.e. three animals) is required. If the first animal dosed
dies, then dosing proceeds at 2 000 mg/kg in accordance with the flow charts in Annex 1. If the first animal survives,
two further animals are dosed. If only one of the three animals dies, the LD50 value is expected to exceed 5 000 mg/kg. If
both animals die, then dosing proceeds at 2 000 mg/kg.

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TEST METHOD B.1 tris: Guidance on classififcation according to EU scheme to cover the transition period until
full implementation of the Globally Harmonised Classification System (GHS) (taken from reference (8))

ANNEX 3

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ANNEX 2D

‘B. 4. ACUTE TOXICITY: DERMAL IRRITATION/CORROSION

1.

METHOD

This method is equivalent to the OECD TG 404 (2002).

1.1

INTRODUCTION

In the preparation of this updated method special attention was given to possible improvements in
relation to animal welfare concerns and to the evaluation of all existing information on the test substance
in order to avoid unnecessary testing in laboratory animals. This method includes the recommendation
that prior to undertaking the described in vivo test for corrosion/irritation of the substance, a weight-of-
the-evidence analysis be performed on the existing relevant data. Where insufficient data are available, they
can be developed through application of sequential
testing (1). The testing strategy includes the
performance of validated and accepted in vitro tests and is provided as an Annex to this method. In
addition, where appropriate, the successive, instead of simultaneous, application of the three test patches
to the animal in the initial in vivo test is recommended.

In the interest of both sound science and animal welfare, in vivo testing should not be undertaken until all
available data relevant to the potential dermal corrosivity/irritation of the substance have been evaluated in
a weight-of-the-evidence analysis. Such data will include evidence from existing studies in humans and/or
laboratory animals, evidence of corrosivity/irritation of one or more structurally related substances or
mixtures of such substances, data demonstrating strong acidity or alkalinity of the substance (2)(3), and
results from validated and accepted in vitro or ex vivo tests (4)(5)(5a). This analysis should decrease the need
for in vivo testing for dermal corrosivity/irritation of substances for which sufficient evidence already exists
from other studies as to those two endpoints.

A preferred sequential testing strategy, which includes the performance of validated and accepted in vitro or
ex vivo tests for corrosion/irritation, is included as an Annex to this Method. The strategy was developed at,
and unanimously recommended by the participants of, an OECD workshop (6), and has been adopted as
the recommended testing strategy in the Globally Harmonised System for the Classification of Chemical
Substances (GHS) (7). It is recommended that this testing strategy be followed prior to undertaking in vivo
testing. For new substances it is the recommended stepwise testing approach for developing scientifically
sound data on the corrosivity/irritation of the substance. For existing substances with insufficient data on
dermal corrosion/irritation, the strategy should be used to fill missing data gaps. The use of a different
testing strategy or procedure, or a decision not to use a stepwise testing approach, should be justified.

If a determination of corrosivity or irritation cannot be made using a weight-of-the-evidence analysis,
consistent with the sequential testing strategy, an in vivo test should be considered (see Annex).

1.2

DEFINITIONS

Dermal irritation: is the production of reversible damage of the skin following the application of a test
substance for up to 4 hours.

Dermal corrosion: is the production of irreversible damage of the skin; namely, visible necrosis through
the epidermis and into the dermis, following the application of a test substance for up to four hours.
Corrosive reactions are typified by ulcers, bleeding, bloody scabs, and, by the end of observation at 14
days, by discoloration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology
should be considered to evaluate questionable lesions.

1.3

PRINCIPLE OF THE TEST METHOD

The substance to be tested is applied in a single dose to the skin of an experimental animal; untreated skin
areas of the test animal serve as the control. The degree of irritation/corrosion is read and scored at
specified intervals and is further described in order to provide a complete evaluation of the effects. The
duration of the study should be sufficient to evaluate the reversibility or irreversibility of the effects
observed.

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Animals showing continuing signs of severe distress and/or pain at any stage of the test should be
humanely killed, and the substance assessed accordingly. Criteria for making the decision to humanely kill
moribund and severely suffering animals can be found in reference (8).

1.4

DESCRIPTION OF THE TEST METHOD

1.4.1

Preparation for the in vivo test

1.4.1.1

Selection of animal species

The albino rabbit is the preferable laboratory animal, and healthy young adult rabbits are used. A rationale
for using other species should be provided.

1.4.1.2

Preparation of the animals

Approximately 24 hours before the test, fur should be removed by closely clipping the dorsal area of the
trunk of the animals. Care should be taken to avoid abrading the skin, and only animals with healthy,
intact skin should be used.

Some strains of rabbit have dense patches of hair that are more prominent at certain times of the year.
Such areas of dense hair growth should not be used as test sites.

1.4.1.3

Housing and feeding conditions

Animals should be individually housed. The temperature of the experimental animal room should be 20°C
(± 3°C) for rabbits. Although the relative humidity should be at least 30 % and preferably not exceed 70 %,
other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence
being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an
unrestricted supply of drinking water.

1.4.2

Test procedure

1.4.2.1

Application of the test substance

The test substance should be applied to a small area (approximately 6 cm2) of skin and covered with a
gauze patch, which is held in place with non-irritating tape. In cases in which direct application is not
possible (e.g., liquids or some pastes), the test substance should first be applied to the gauze patch, which
is then applied to the skin. The patch should be loosely held in contact with the skin by means of a
suitable semi-occlusive dressing for the duration of the exposure period. If the test substance is applied to
the patch, it should be attached to the skin in such a manner that there is good contact and uniform
distribution of the substance on the skin. Access by the animal to the patch and ingestion or inhalation of
the test substance should be prevented.

if
Liquid test substances are generally used undiluted. When testing solids (which may be pulverised,
considered necessary), the test substance should be moistened with the smallest amount of water (or,
where necessary, of another suitable vehicle) sufficient to ensure good skin contact. When vehicles other
than water are used, the potential influence of the vehicle on irritation of the skin by the test substance
should be minimal, if any.

At the end of the exposure period, which is normally 4 hours, residual test substance should be removed,
where practicable, using water or an appropriate solvent without altering the existing response or the
integrity of the epidermis.

1.4.2.2

Dose level

A dose of 0.5 ml. of liquid or 0.5 g of solid or paste is applied to the test site.

1.4.2.3

Initial test (In vivo dermal irritation/corrosion test using one animal)

It is strongly recommended that the in vivo test be performed initially using one animal, especially when
the substance is suspected to have corrosion potential. This is in accordance with the sequential testing
strategy (see Annex 1).

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When a substance has been judged to be corrosive on the basis of a weight-of-the-evidence analysis, no
further animal testing is needed. For most substances suspected of being corrosive, further in vivo testing is
normally not necessary. However,
in those cases where additional data are felt warranted because of
insufficient evidence, limited animal testing may be carried out using the following approach: Up to three
tests patches are applied sequentially to the animal. The first patch is removed after three minutes. If no
the
serious skin reaction is observed, a second patch is applied and removed after one hour.
observations at this stage indicate that exposure can humanely be allowed to extend to four hours, a third
patch is applied and removed after four hours, and the response is graded.

If

If a corrosive effect is observed after any of the three sequential exposures, the test is immediately
terminated. If a corrosive effect is not observed after the last patch is removed, the animal is observed for
14 days, unless corrosion develops at an earlier time point.

In those cases in which the test substance is not expected to produce corrosion but may be irritating, a
single patch should be applied to one animal for four hours.

1.4.2.4

Confirmatory test (In vivo dermal irritation test with additional animals)

If a corrosive effect is not observed in the initial test, the irritant or negative response should be confirmed
using up to two additional animals, each with one patch, for an exposure period of four hours. If an
irritant effect is observed in the initial test, the confirmatory test may be conducted in a sequential
manner, or by exposing two additional animals simultaneously. In the exceptional case, in which the initial
test is not conducted, two or three animals may be treated with a single patch, which is removed after four
hours. When two animals are used,
if both exhibit the same response, no further testing is needed.
Otherwise, the third animal is also tested. Equivocal responses may need to be evaluated using additional
animals.

1.4.2.5

Observation period

The duration of the observation period should be sufficient to evaluate fully the reversibility of the effects
observed. However, the experiment should be terminated at any time that the animal shows continuing
signs of severe pain or distress. To determine the reversibility of effects, the animals should be observed up
to 14 days after removal of the patches. If reversibility is seen before 14 days, the experiment should be
terminated at that time.

1.4.2.6

Clinical observations and grading of skin reactions

All animals should be examined for signs of erythema and oedema, and the responses scored at 60
minutes, and then at 24, 48 and 72 hours after patch removal. For the initial test in one animal, the test
site is also examined immediately after the patch has been removed. Dermal reactions are graded and
recorded according to the grades in the Table below. If there is damage to skin which cannot be identified
as irritation or corrosion at 72 hours, observations may be needed until day 14 to determine the
local toxic effects, such as
reversibility of the effects. In addition to the observation of irritation, all
defatting of the skin, and any systemic adverse effects (e.g., effects on clinical signs of toxicity and body
weight), should be fully described and recorded. Histopathological examination should be considered to
clarify equivocal responses.

The grading of skin responses is necessarily subjective. To promote harmonisation in grading of skin
response and to assist testing laboratories and those involved in making and interpreting the observations,
the personnel performing the observations need to be adequately trained in the scoring system used (see
Table below). An illustrated guide for grading skin irritation and other lesions could be helpful (9).

2.

2.1

DATA

PRESENTATION OF RESULTS

Study results should be summarised in tabular form in the final test report and should cover all items
listed in section 3.1.

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2.2

EVALUATION OF RESULTS

The dermal irritation scores should be evaluated in conjunction with the nature and severity of lesions,
and their reversibility or lack of reversibility. The individual scores do not represent an absolute standard
for the irritant properties of a material, as other effects of the test material are also evaluated. Instead,
individual scores should be viewed as reference values, which need to be evaluated in combination with all
other observations from the study.

Reversibility of dermal lesions should be considered in evaluating irritant responses. When responses such
as alopecia (limited area), hyperkeratosis, hyperplasia and scaling, persist to the end of
the 14-day
observation period, the test substance should be considered an irritant.

3.

3.1

REPORTING

TEST REPORT

The test report must include the following information:

Rationale for in vivo testing: weight-of-evidence analysis of pre-existing test data,
from sequential testing strategy:

including results

— description of relevant data available from prior testing;

— data derived at each stage of testing strategy;

— description of in vitro tests performed, including details of procedures, results obtained with test/

reference substances;

— weight-of-the-evidence analysis for performing in vivo study

Test substance:

— identification data (e.g., CAS number; source; purity; known impurities; lot number);

— physical nature and physicochemical properties (e.g. pH, volatility, solubility, stability);

— if mixture, composition and relative percentages of components.

Vehicle:

— identification, concentration (where appropriate), volume used;

— justification for choice of vehicle.

Test animals:

— species/strain used, rationale for using animals other than albino rabbit;

— number of animals of each sex;

— individual animal weights at start and conclusion of test;

— age at start of study;

— source of animals, housing conditions, diet, etc.

Test conditions:

— technique of patch site preparation;

— details of patch materials used and patching technique;

— details of test substance preparation, application, and removal.

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Results:

— tabulation of irritation/corrosion response scores for each animal at all time points measured;

— descriptions of all lesions observed;

— narrative description of nature and degree of

irritation or corrosion observed, and any

histopathological findings;

— description of other adverse local (e.g., defatting of skin) and systemic effects in addition to

dermal irritation or corrosion.

Discussion of results

4.

REFERENCES

(1) Barratt, M.D., Castell, J.V., Chamberlain, M., Combes, R.D., Dearden, J.C., Fentem, J.H., Gerner, I.,
Giuliani, A., Gray, T.J.B., Livingston, D.J., Provan, W.M., Rutten, F.A.J.J.L., Verhaar, H.J.M., Zbinden, P.
(1995) The Integrated Use of Alternative Approaches for Predicting Toxic Hazard. ECVAM Workshop
Report 8. ATLA 23, 410-429.

(2) Young, J.R., How, M.J., Walker, A.P., Worth W.M.H. (1988) Classification as Corrosive or Irritant to
Skin of Preparations Containing Acidic or Alkaline Substance Without Testing on Animals. Toxicol. In
Vitro, 2, 19-26.

(3) Worth, A.P., Fentem, J.H., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J., Liebsch, M.
(1998) Evaluation of the proposed OECD Testing Strategy for skin corrosion. ATLA 26, 709-720.

(4) ECETOC (1990) Monograph No. 15, “Skin Irritation”, European Chemical

Industry, Ecology and

Toxicology Centre, Brussels.

(5) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J., Holzhutter,
H.G. and Liebsch, M. (1998) The ECVAM international validation study on in vitro tests for skin
corrosivity. 2. Results and evaluation by the Management Team. Toxicology in Vitro 12, pp. 483-524.

(5a) Testing Method B.40 Skin Corrosion.

(6) OECD (1996) OECD Test Guidelines Programme: Final Report of

the OECD Workshop on
Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Held
in Solna, Sweden, 22 - 24 January 1996 (http://www1.oecd.org/ehs/test/background.htm).

(7) OECD (1998) Harmonized Integrated Hazard Classification System for Human Health and
Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of
the
Chemicals Committee and the Working Party on Chemicals, November 1998 (http://www1.oecd.org/
ehs/Class/HCL6.htm).

(8) OECD (2000). Guidance Document on the Recognition, Assessment and Use of Clinical Signs as
Humane Endpoints for Experimental Animals Used in Safety Evaluation. OECD Environmental Health
and Safety Publications. Series on Testing and Assessment No. 19 (http://www1.oecd.org/ehs/test/
monos.htm).

(9) EPA (1990). Atlas of Dermal Lesions, (20T-2004). United States Environmental Protection Agency,

Office of Pesticides and Toxic Substances, Washington, DC, August 1990.
[Available from OECD Secretariat upon request].

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TABLE I: GRADING OF SKIN REACTIONS

Erythema and Eschar Formation

No erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 0

Very slight erythema (barely perceptible)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 1

Well defined erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 2

Moderate to severe erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 3

Severe erythema (beef redness) to eschar formation preventing grading of erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . … 4

Oedema Formation

Maximum possible: 4

No oedema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 0

Very slight oedema (barely perceptible) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 1

Slight oedema (edges of area well defined by definite raising)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 2

Moderate oedema (raised approximately 1 mm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 3

Severe oedema (raised more than 1 mm and extending beyond area of exposure) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 4

Maximum possible: 4

Histopathological examination may be carried out to clarify equivocal responses.

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ANNEX

A Sequential Testing Strategy for Dermal Irritation and Corrosion

GENERAL CONSIDERATIONS

In the interest of sound science and animal welfare, it is important to avoid the unnecessary use of animals and to
minimise any testing that is likely to produce severe responses in animals. All information on a substance relevant to its
potential skin corrosivity/irritancy should be evaluated prior to considering in vivo testing. Sufficient evidence may already
exist to classify a test substance as to its dermal corrosion or irritation potential without the need to conduct testing in
laboratory animals. Therefore, utilising a weight-of-the-evidence analysis and a sequential testing strategy, will minimise
the need for in vivo testing, especially if the substance is likely to produce severe reactions.

It is recommended that a weight-of-the-evidence analysis be used to evaluate existing information regarding the skin
irritation and corrosion of substances to determine whether additional studies, other than in vivo dermal studies, should
be performed to help characterise such potential. Where further studies are needed, it is recommended that the sequential
testing strategy be utilised to develop the relevant experimental data. For substances which have no testing history, the
sequential testing strategy should be utilised to develop the data set needed to evaluate its dermal corrosion/irritation
potential. The testing strategy described in this Annex was developed at an OECD workshop (1) and was later affirmed
and expanded in the Harmonised Integrated Hazard Classification System for Human Health and Environmental Effects of
Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on
Chemicals, in November 1998 (2).

Although this sequential testing strategy is not an integral part of testing method B.4, it expresses the recommended
approach for the determination of skin irritation/corrosion characteristics. This approach represents both best practice
and an ethical benchmark for in vivo testing for skin irritation/corrosion. The testing method provides guidance for the
conduct of the in vivo test and summarises the factors that should be addressed before initiating such a test. The strategy
provides an approach for the evaluation of existing data on the skin irritation/corrosion properties of test substances and
a tiered approach for the generation of relevant data on substances for which additional studies are needed, or for which
no studies have been performed. It also recommends the performance of validated and accepted in vitro or ex vivo tests for
skin corrosion/irritation under specific circumstances.

DESCRIPTION OF THE EVALUATION AND TESTING STRATEGY

Prior to undertaking tests as part of the sequential testing strategy (Figure), all available information should be evaluated
to determine the need for in vivo skin testing. Although significant information might be gained from the evaluation of
single parameters (e.g., extreme pH), the totality of existing information should be considered. All relevant data on the
effects of the substance in question, or its analogues, should be evaluated in making a weight-of-the-evidence decision,
and a rationale for the decision should be presented. Primary emphasis should be placed upon existing human and animal
data on the substance, followed by the outcome of in vitro or ex vivo testing. In vivo studies of corrosive substances should
be avoided whenever possible. The factors considered in the testing strategy include:

Evaluation of existing human and animal data (Step 1). Existing human data, e.g. clinical or occupational studies and case
reports, and/or animal test data, e.g. from single or repeated dermal exposure toxicity studies, should be considered first,
because they provide information directly related to effects on the skin. Substances with known irritancy or corrosivity,
and those with clear evidence of non-corrosivity or non-irritancy, need not be tested in in vivo studies.

Analysis of structure activity relationships (SAR) (Step 2). The results of testing of structurally related substances should be
considered, if available. When sufficient human and/or animal data are available on structurally related substances or
mixtures of such substances to indicate their skin corrosion/irritancy potential, it can be presumed that the test substance
being evaluated will produce the same responses. In those cases, the test substance may not need to be tested. Negative
data from studies of structurally related substances or mixtures of such substances do not constitute sufficient evidence of
non-corrosivity/non-irritancy of a substance under the sequential testing strategy. Validated and accepted SAR approaches
should be used to identify both dermal corrosion and irritation potential.

Physicochemical properties and chemical reactivity (Step 3). Substances exhibiting pH extremes such as ≤2.0 and ≥11.5 may
have strong local effects. If extreme pH is the basis for identifying a substance as corrosive to skin, then its acid/alkali
reserve (or buffering capacity) may also be taken into consideration (3)(4). If the buffering capacity suggests that a
substance may not be corrosive to the skin, then further testing should be undertaken to confirm this, preferably by the
use of a validated and accepted in vitro or ex vivo test (see steps 5 and 6).

Dermal toxicity (Step 4). If a chemical has proven to be very toxic by the dermal route, an in vivo dermal irritation/
corrosion study may not be practicable because the amount of test substance normally applied could exceed the very
toxic dose and, consequently result in the death or severe suffering of the animals. In addition, when dermal toxicity

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studies utilising albino rabbits have already been performed up to the limit dose level of 2 000 mg/kg body weight or
higher, and no dermal irritation or corrosion has been seen, additional testing for skin irritation/corrosion may not be
needed. A number of considerations should be borne in mind when evaluating acute dermal toxicity in previously
performed studies. For example, reported information on dermal lesions may be incomplete. Testing and observations
may have been made on a species other than the rabbit, and species may differ widely in sensitivity of their responses.
Also the form of test substance applied to animals may not have been suitable for assessment of skin irritation/corrosion
(e.g., dilution of substances for testing dermal toxicity (5). However, in those cases in which well-designed and conducted
dermal toxicity studies have been performed in rabbits, negative findings may be considered sufficient evidence that the
substance is not corrosive or irritating.

Results from in vitro or ex vivo tests (Steps 5 and 6). Substances that have demonstrated corrosive or severe irritant properties
in a validated and accepted in vitro or ex vivo test (6)(7) designed for the assessment of these specific effects, need not be
tested in animals. It can be presumed that such substances will produce similar severe effects in vivo.

In vivo test in rabbits (Steps 7 and 8). Should a weight-of the-evidence decision be made to conduct in vivo testing, it should
begin with an initial test using one animal. If the results of this test indicate the substance to be corrosive to the skin,
further testing should not be performed. If a corrosive effect is not observed in the initial test, the irritant or negative
response should be confirmed using up to two additional animals for an exposure period of four hours. If an irritant
effect is observed in the initial test, the confirmatory test may be conducted in a sequential manner, or by exposing the
two additional animals simultaneously.

REFERENCES

(1) OECD (1996). Test Guidelines Programme: Final Report on the OECD Workshop on Harmonization of Validation
and Acceptance Criteria for Alternative Toxicological Test Methods. Held on Solna, Sweden, 22 – 24 January 1996
(http://www1.oecd.org/ehs/test/background.htm).

(2) OECD (1998). Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of
Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on
Chemicals, November 1998 (http://www1.oecd.org/ehs/Class/HCL6.htm).

(3) Worth, A.P., Fentem J.H., Balls M., Botham P.A., Curren R.D., Earl L.K., Esdail D.J., Liebsch M. (1998). An Evaluation

of the Proposed OECD Testing Strategy for Skin Corrosion. ATLA 26, 709-720.

(4) Young,

J.R., How, M.J., Walker, A.P., Worth, W.M.H.

(1988). Classification as Corrosive or Irritant to Skin of

Preparations Containing Acidic or Alkaline Substances, Without Testing on Animals. Toxic In Vitro, 2 (1) pp 19-26.

(5) Patil, S.M., Patrick, E., Maibach, H.I. (1996) Animal, Human, and In Vitro Test Methods for Predicting Skin Irritation,
in: Francis N. Marzulli and Howard I. Maibach (editors): Dermatotoxicology. Fifth Edition ISBN 1-56032-356-6,
Chapter 31, 411-436.

(6) Testing Method B.40.

(7) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Edsail, D.J., Holzhutter, H.G. and Liebsch,
M. (1998) The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation
by the Management Team. Toxicology in Vitro 12, pp. 483–524.

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TESTING AND EVALUATION STRATEGY FOR DERMAL IRRITATION /CORROSION

FIGURE

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’

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ANNEX 2E

‘B. 5. ACUTE TOXICITY: EYE IRRITATION/CORROSION

1.

METHOD

This method is equivalent to the OECD TG 405 (2002)

1.1

INTRODUCTION

animals

and thereby

In the preparation of this updated method special attention was given to possible improvements through
the evaluation of all existing information on the test substance in order to avoid unnecessary testing in
the
laboratory
recommendation that prior to undertaking the described in vivo test for acute eye irritation/corrosion, a
weight-of-the-evidence analysis be performed (1) on the existing relevant data. Where insufficient data are
available, it is recommended that they be developed through application of sequential testing (2)(3). The
testing strategy includes the performance of validated and accepted in vitro tests and is provided as an
Annex to the testing method. In addition, the use of an in vivo dermal irritation/corrosion test to predict
eye corrosion prior to consideration of an in vivo eye test is recommended.

concerns. This method includes

animal welfare

address

In the interest of both sound science and animal welfare, in vivo testing should not be considered until all
available data relevant to the potential eye corrosivity/irritation of the substance has been evaluated in a
weight-of-the-evidence analysis. Such data will include evidence from existing studies in humans and/or
laboratory animals, evidence of corrosivity/irritation of one or more structurally related substances or
mixtures of such substances, data demonstrating high acidity or alkalinity of the substance (4)(5), and
results from validated and accepted in vitro or ex vivo tests for skin corrosion and irritation (6)(6a). The
studies may have been conducted prior to, or as a result of, a weight-of-the-evidence analysis.

For certain substances, such an analysis may indicate the need for in vivo studies of the ocular corrosion/
irritation potential of the substance. In all such cases, before considering the use of the in vivo eye test,
preferably a study of the in vivo dermal effects of the substance should be conducted first and evaluated in
accordance with testing method B.4 (7). The application of a weight-of-the-evidence analysis and the
sequential testing strategy should decrease the need for in vivo testing for eye corrosivity/irritation of
substances for which sufficient evidence already exists from other studies. If a determination of eye
corrosion or irritation potential cannot be made using the sequential testing strategy, even after the
performance of an in vivo study of dermal corrosion and irritation, an in vivo eye corrosion/irritation test
may be performed.

A preferred sequential testing strategy, which includes the performance of validated in vitro or ex vivo tests
for corrosion/irritation, is included in the Annex to this testing method. The strategy was developed at,
and unanimously recommended by the participants of, an OECD workshop (8), and has been adopted as
the recommended testing strategy in the Globally Harmonised System for the Classification of Chemical
Substances (GHS) (9). It is recommended that this testing strategy be followed prior to undertaking in vivo
testing. For new substances it is the recommended stepwise testing approach for developing scientifically
sound data on the corrosivity/irritation of the substance. For existing substances with insufficient data on
skin and eye corrosion/irritation, the strategy should be used to fill missing data gaps. The use of a
different testing strategy or procedure, or the decision not to use a stepwise testing approach, should be
justified.

1.2

DEFINITIONS

Eye irritation: is the production of changes in the eye following the application of a test substance to the
anterior surface of the eye, which are fully reversible within 21 days of application.

Eye corrosion:
is the production of tissue damage in the eye, or serious physical decay of vision,
following application of a test substance to the anterior surface of the eye, which is not fully reversible
within 21 days of application

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1.3

PRINCIPLE OF THE TEST METHOD

The substance to be tested is applied in a single dose to one of the eyes of the experimental animal; the
untreated eye serves as the control. The degree of eye irritation/corrosion is evaluated by scoring lesions of
conjunctiva, cornea, and iris, at specific intervals. Other effects in the eye and adverse systemic effects are
also described to provide a complete evaluation of the effects. The duration of the study should be
sufficient to evaluate the reversibility or irreversibility of the effects.

Animals showing continuing signs of severe distress and/or pain at any stage of the test should be
humanely killed, and the substance assessed accordingly. Criteria for making the decision to humanely kill
moribund and severely suffering animals can be found in reference (10).

1.4

DESCRIPTION OF THE TEST METHOD

1.4.1

Preparation for the in vivo test

1.4.1.1

Selection of species

The albino rabbit is the preferable laboratory animal, and healthy young adult animals are used. A
rationale for using other strains or species should be provided.

1.4.1.2

Preparation of animals

Both eyes of each experimental animal provisionally selected for testing should be examined within 24
hours before testing starts. Animals showing eye irritation, ocular defects, or pre-existing corneal injury
should not be used.

1.4.1.3

Housing and feeding conditions

Animals should be individually housed. The temperature of the experimental animal room should be 20°C
(± 3°C) for rabbits. Although the relative humidity should be at least 30 % and preferably not exceed 70 %,
other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence
being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an
unrestricted supply of drinking water.

1.4.2

Test procedure

1.4.2.1

Application of the test substance

The test substance should be placed in the conjunctival sac of one eye of each animal after gently pulling
the lower lid away from the eyeball. The lids are then gently held together for about one second in order
to prevent loss of the material. The other eye, which remains untreated, serves as a control.

1.4.2.2

Irrigation

The eyes of the test animals should not be washed for at least 24 hours following instillation of the test
substance, except for solids (see section 1.4.2.3.2), and in case of immediate corrosive or irritating effects.
At 24 hours a washout may be used if considered appropriate.

Use of a satellite group of animals to investigate the influence of washing is not recommended unless it is
scientifically justified. If a satellite group is needed, two rabbits should be used. Conditions of washing
should be carefully documented, e.g., time of washing; composition and temperature of wash solution;
duration, volume, and velocity of application.

1.4.2.3

Dose level

1.4.2.3.1

Testing of liquids

For testing liquids, a dose of 0.1 ml is used. Pump sprays should not be used for instilling the substance
directly into the eye. The liquid spray should be expelled and collected in a container prior to instilling 0.1
ml into the eye.

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1.4.2.3.2

Testing of solids

When testing solids, pastes, and particulate substances, the amount used should have a volume of 0.1 ml
or a weight of not more than 100 mg. The test material should be ground to a fine dust. The volume of
solid material should be measured after gently compacting it, e.g., by tapping the measuring container. If
the solid test substance has not been removed from the eye of
the test animal by physiological
mechanisms at the first observation time point of 1 hour after treatment, the eye may be rinsed with
saline or distilled water.

1.4.2.3.3

Testing of aerosols

It is recommended that all pump sprays and aerosols be collected prior to instillation into the eye. The
one exception is for substances in pressurised aerosol containers, which cannot be collected due to
vaporisation. In such cases, the eye should be held open, and the test substance administered to the eye in
a simple burst of about one second, from a distance of 10 cm directly in front of the eye. This distance
may vary depending on the pressure of the spray and its contents. Care should be taken not to damage
the eye from the pressure of the spray. In appropriate cases, there may be a need to evaluate the potential
for “mechanical” damage to the eye from the force of the spray.

An estimate of the dose from an aerosol can be made by simulating the test as follows: the substance is
sprayed on to weighing paper through an opening the size of a rabbit eye placed directly before the paper.
The weight increase of the paper is used to approximate the amount sprayed into the eye. For volatile
substances, the dose may be estimated by weighing a receiving container before and after removal of the
test material.

1.4.2.4

Initial test (In vivo eye irritation/corrosion test using one animal)

As articulated in the sequential testing strategy (see Annex 1), it is strongly recommended that the in vivo
test be performed initially using one animal.

If the results of this test indicate the substance to be corrosive or a severe irritant to the eye using the
procedure described, further testing for ocular irritancy should not be performed.

1.4.2.5

Local anaesthetics

Local anaesthetics may be used on a case-by-case basis. If the weight-of-the-evidence analysis indicates that
the substance has the potential to cause pain, or initial testing shows that a painful reaction will occur, a
local anaesthetic may be used prior to instillation of the test substance. The type, concentration, and dose
of the local anaesthetic should be carefully selected to ensure that differences in reaction to the test
substance will not result from its use. The control eye should be similarly anaesthetised.

1.4.2.6

Confirmatory test (In vivo eye irritation test with additional animals)

If a corrosive effect is not observed in the initial test, the irritant or negative response should be confirmed
using up to two additional animals. If a severe irritant effect is observed in the initial test indicating a
possible strong (irreversible) effect in the confirmatory testing, it is recommended that the confirmatory
test be conducted in a sequential manner in one animal at a time, rather than exposing the two additional
animals simultaneously. If the second animal reveals corrosive or severe irritant effects, the test is not
continued. Additional animals may be needed to confirm weak or moderate irritant responses.

1.4.2.7

Observation period

the observation period should be sufficient

The duration of
to evaluate fully the magnitude and
reversibility of the effects observed. However, the experiment should be terminated at any time that the
animal shows continuing signs of severe pain or distress (9). To determine reversibility of effects, the
animals should be observed normally for 21 days post administration of the test substance. If reversibility
is seen before 21 days, the experiment should be terminated at that time.

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1.4.2.7.1

Clinical observations and grading of eye reactions

The eyes should be examined at 1, 24, 48, and 72 hours after test substance application. Animals should
be kept on test no longer than necessary once definitive information has been obtained. Animals showing
continuing severe pain or distress should be humanely killed without delay, and the substance assessed
accordingly. Animals with the following eye lesions post-instillation should be humanely killed: corneal
perforation or significant corneal ulceration including staphyloma; blood in the anterior chamber of the
eye; grade 4 corneal opacity which persists for 48 hours; absence of a light reflex (iridial response grade 2)
which persists for 72 hours; ulceration of the conjunctival membrane; necrosis of the conjuctivae or
nictitating membrane; or sloughing. This is because such lesions generally are not reversible

Animals that do not develop ocular lesions may be terminated not earlier than 3 days post instillation.
Animals with mild to moderate lesions should be observed until the lesions clear, or for 21 days, at which
time the study is terminated. Observations should be performed at 7, 14, and 21 days in order to
determine the status of the lesions, and their reversibility or irreversibility.

The grades of ocular reaction (conjunctivae, cornea and iris) should be recorded at each examination
(Table I). Any other lesions in the eye (e.g. pannus, staining) or adverse systemic effects should also be
reported.

Examination of reactions can be facilitated by use of a binocular loupe, hand slit-lamp, biomicroscope, or
other suitable device. After recording the observations at 24 hours, the eyes may be further examined with
the aid of fluorescein.

The grading of ocular responses is necessarily subjective. To promote harmonisation of grading of ocular
response and to assist testing laboratories and those involved in making and interpreting the observations,
the personnel performing the observations need to be adequately trained in the scoring system used.

DATA

EVALUATION OF RESULTS

The ocular irritation scores should be evaluated in conjunction with the nature and severity of lesions, and
their reversibility or lack of reversibility. The individual scores do not represent an absolute standard for
the irritant properties of a material, as other effects of the test material are also evaluated. Instead,
individual scores should be viewed as reference values and are only meaningful when supported by a full
description and evaluation of all observations.

REPORTING

TEST REPORT

The test report must include the following information:

Rationale for in vivo testing: weight-of-the-evidence analysis of pre-existing test data, including results
from sequential testing strategy

— description of relevant data available from prior testing;

— data derived in each step of testing strategy;

— description of in vitro tests performed, including details of procedures, results obtained with test/

reference substances;

— description of in vivo dermal irritation/corrosion study performed, including results obtained;

— weight-of-the-evidence analysis for performing in vivo study

2.

2.2

3.

3.1

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Test substance:

— identification data (e.g., CAS number, source, purity, known impurities, lot number);

— physical nature and physicochemical properties (e.g. pH, volatility, solubility, stability, reactivity

with water);

— in case of a mixture, composition and relative percentages of components;

— if local anaesthetic is used, identification, purity, type, dose, and potential interaction with test

substance.

Vehicle:

— identification, concentration (where appropriate), volume used;

— justification for choice of vehicle.

Test animals:

— species/strain used, rationale for using animals other than albino rabbit;

— age of each animal at start of study;

— number of animals of each sex in test and control groups (if required);

— individual animal weights at start and conclusion of test;

— source, housing conditions, diet, etc.

Results:

— description of method used to score irritation at each observation time (e.g., hand slitlamp,

biomicroscope, fluorescein);

— tabulation of irritant/corrosive response data for each animal at each observation time up to

removal of each animal from the test;

— narrative description of the degree and nature of irritation or corrosion observed;

— description of any other lesions observed in the eye (e.g., vascularization, pannus formation,

adhesions, staining);

— description of non-ocular local and systemic adverse effects, and histopathological findings, if any.

Discussion of results.

3.2

INTERPRETATION OF THE RESULTS

Extrapolation of the results of eye irritation studies in laboratory animals to humans is valid only to a
limited degree. In many cases the albino rabbit is more sensitive than humans to ocular irritants or
corrosives.

Care should be taken in the interpretation of data to exclude irritation resulting from secondary infection.

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4.

REFERENCES

(1) Barratt, M.D., Castell, J.V., Chamberlain, M., Combes, R.D., Dearden, J.C., Fentem, J.H., Gerner, I.,
Giuliani, A., Gray, T.J.B., Livingston, D.J., Provan, W.M., Rutten, F.A.J.J.L., Verhaar, H.J.M., Zbinden, P.
(1995) The Integrated Use of Alternative Approaches for Predicting Toxic Hazard. ECVAM Workshop
Report 8. ATLA 23, 410- 429.

(2) de Silva, O., Cottin, M., Dami, N., Roguet, R., Catroux, P., Toufic, A., Sicard, C., Dossou, K.G., Gerner,
I., Schlede, E., Spielmann, H., Gupta, K.C., Hill, R.N. (1997) Evaluation of Eye Irritation Potential:
Statistical Analysis and Tier Testing Strategies. Food Chem. Toxicol 35, 159-164.

(3) Worth A.P. and Fentem J.H. (1999) A general approach for evaluating stepwise testing strategies ATLA

27, 161-177.

(4) Young, J.R., How, M.J., Walker, A.P., Worth W.M.H. (1988) Classification as Corrosive or Irritant to
Skin of Preparations Containing Acidic or Alkaline Substance Without Testing on Animals. Toxicol. In
Vitro, 2, 19 - 26.

(5) Neun, D.J. (1993) Effects of Alkalinity on the Eye Irritation Potential of Solutions Prepared at a Single

pH. J. Toxicol. Cut. Ocular Toxicol. 12, 227 - 231.

(6) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Edsaile, D.J., Holzhutter,
H.G. and Liebsch, M. (1998) The ECVAM international validation study on in vitro tests for skin
corrosivity. 2. Results and evaluation by the Management Team. Toxicology in Vitro 12, pp. 483–524.

(6a) Testing Method B.40 Skin Corrosion.

(7) Testing method B.4. Acute toxicity: dermal irritation/corrosion.

(8) OECD (1996) OECD Test Guidelines Programme: Final Report of

the OECD Workshop on
Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Held
in Solna, Sweden, 22-24 January 1996 (http://www.oecd.org/ehs/test/background.htm).

(9) OECD (1998) Harmonized Integrated Hazard Classification System for Human Health and
Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of
the
Chemicals Committee and the Working Party on Chemicals, November 1998 (http://www.oecd.org/
ehs/Class/HCL6.htm).

(10) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints
for Experimental Animals Used in Safety Evaluation. OECD Environmental Health and Safety
Publications. Series on Testing and Assessment No. 19 (http://www.oecd.org/ehs/test/monos.htm).

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TABLE I: GRADING OF OCULAR LESIONS

Cornea

Opacity: degree of density (readings should be taken from most dense area) (*)

No ulceration or opacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 0

Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible . . . … 1

Easily discernible translucent area; details of iris slightly obscured . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 2

Nacrous area; no details of iris visible; size of pupil barely discernible . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 3

Opaque cornea; iris not discernible through the opacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 4

Maximum possible: 4

(*) The area of corneal opacity should be noted

Iris

Normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 0

Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; or injection; iris reactive to
light (a sluggish reaction is considered to be an effect)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …

1

Hemorrhage, gross destruction, or no reaction to light

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 2

Conjunctivae

Maximum possible: 2

Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris)

Normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 0

Some blood vessels hyperaemic (injected)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 1

Diffuse, crimson colour; individual vessels not easily discernible . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 2

Diffuse beefy red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 3

Chemosis

Swelling (refers to lids and/or nictating membranes)

Maximum possible: 3

Normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 0

Some swelling above normal

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 1

Obvious swelling, with partial eversion of lids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 2

Swelling, with lids about half closed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 3

Swelling, with lids more than half closed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 4

Maximum possible: 4

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ANNEX

A Sequential Testing Strategy for Eye Irritation and Corrosion

GENERAL CONSIDERATIONS

In the interests of sound science and animal welfare, it is important to avoid the unnecessary use of animals, and to
minimise testing that is likely to produce severe responses in animals. All information on a substance relevant to its
potential ocular irritation/corrosivity should be evaluated prior to considering in vivo testing. Sufficient evidence may
already exist to classify a test substance as to its eye irritation or corrosion potential without the need to conduct testing
in laboratory animals. Therefore, utilizing a weight-of-the-evidence analysis and sequential testing strategy will minimise
the need for in vivo testing, especially if the substance is likely to produce severe reactions.

It is recommended that a weight-of-the-evidence analysis be used to evaluate existing information pertaining to eye
irritation and corrosion of substances and to determine whether additional studies, other than in vivo eye studies, should
be performed to help characterise such potential. Where further studies are needed, it is recommended that the sequential
testing strategy be utilised to develop the relevant experimental data. For substances which have no testing history, the
sequential testing strategy should be utilised to develop the data needed to evaluate its eye corrosion/irritation. The testing
strategy described in this Annex was developed at an OECD workshop (1). It was subsequently affirmed and expanded in
the Harmonised Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical
Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, in
November 1998 (2).

Although this testing strategy is not an integrated part of testing method B.5, it expresses the recommended approach for
the determination of eye irritation/corrosion properties. This approach represents both best practice and an ethical
benchmark for in vivo testing for eye irritation/corrosion. The Testing method provides guidance for the conduct of the in
vivo test and summarises the factors that should be addressed before considering such a test. The sequential testing
strategy provides a weight-of-the-evidence approach for the evaluation of existing data on the eye irritation/corrosion
properties of substances and a tiered approach for the generation of relevant data on substances for which additional
studies are needed or for which no studies have been performed. The strategy includes the performance first of validated
and accepted in vitro or ex vivo tests and then of testing method B.4 skin irritation/corrosion studies under specific
circumstances (3)(4).

DESCRIPTION OF THE STEPWISE TESTING STRATEGY

Prior to undertaking tests as part of the sequential testing strategy (Figure), all available information should be evaluated
to determine the need for in vivo eye testing. Although significant information might be gained from the evaluation of
single parameters (e.g., extreme pH), the totality of existing information should be assessed. All relevant data on the effects
of the substance in question, and its structural analogues, should be evaluated in making a weight-of-the-evidence
decision, and a rationale for the decision should be presented. Primary emphasis should be placed upon existing human
and animal data on the substance, followed by the outcome of in vitro or ex vivo testing. In vivo studies of corrosive
substances should be avoided whenever possible. The factors considered in the testing strategy include:

Evaluation of existing human and animal data (Step 1). Existing human data, e.g. clinical and occupational studies, and case
reports, and/or animal test data from ocular studies should be considered first, because they provide information directly
related to effects on the eyes. Thereafter, available data from human and/or animal studies investigating dermal corrosion/
irritation should be evaluated. Substances with known corrosivity or severe irritancy to the eye should not be instilled
into the eyes of animals, nor should substances showing corrosive or irritant effects to the skin; such substances should
be considered to be corrosive and/or irritating to the eyes as well. Substances with sufficient evidence of non-corrosivity
and non-irritancy from previously performed ocular studies should also not be tested in in vivo eye studies.

Analysis of structure activity relationships (SAR) (Step 2). The results of testing of structurally related chemicals should be
considered, if available. When sufficient human and/or animal data are available on structurally related substances or
mixtures of such substances to indicate their eye corrrosion/irritancy potential, it can be presumed that the test substance
will produce the same responses. In those cases, the substance may not need to be tested. Negative data from studies of
structurally related substances or mixtures of such substances do not constitute sufficient evidence of non-corrosivity/
non-irritancy of a substance under the sequential testing strategy. Validated and accepted SAR approaches should be used
to identify the corrosion and irritation potential for both dermal and ocular effects.

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Physicochemical properties and chemical reactivity (Step 3). Substances exhibiting pH extremes such as ≤2.0 or ≥11.5 may have
strong local effects. If extreme pH is the basis for identifying a substance as corrosive or irritant to the eye, then its acid/
alkaline reserve (buffering capacity) may also be taken into consideration (5)(6). If the buffering capacity suggests that a
substance may not be corrosive to the eye, then further testing should be undertaken to confirm this, preferably by the
use of a validated and accepted in vitro or ex vivo test (see section step 5 and 6).

Consideration of other existing information (Step 4). All available information on systemic toxicity via the dermal route should
be evaluated at this stage. The acute dermal toxicity of the test substance should also be considered. If the test substance
has been shown to be very toxic by the dermal route, it may not need to be tested in the eye. Although there is not
necessarily a relationship between acute dermal toxicity and eye irritation/corrosion, it can be assumed that if an agent is
very toxic via the dermal route, it will also exhibit high toxicity when instilled into the eye. Such data may also be
considered between Steps 2 and 3.

Results from in vitro or ex vivo tests (Steps 5 and 6). Substances that have demonstrated corrosive or severe irritant properties
in an in vitro or ex vivo test (7)(8) that has been validated and accepted for the assessment specifically of eye or skin
corrosivity/irritation, need not be tested in animals. It can be presumed that such substances will produce similar severe
effects in vivo. If validated and accepted in vitro/ex vivo tests are not available, one should bypass Steps 5 and 6 and
proceed directly to Step 7.

Assessment of in vivo dermal irritancy or corrosivity of the substance (Step 7). When insufficient evidence exists with which to
perform a conclusive weight-of-the-evidence analysis of the potential eye irritation/corrosivity of a substance based upon
data from the studies listed above, the in vivo skin irritation/corrosion potential should be evaluated first, using testing
method B.4 (4) and its accompanying Annex (9). If the substance is shown to produce corrosion or severe skin irritation,
it should be considered to be a corrosive eye irritant unless other information supports an alternative conclusion. Thus,
an in vivo eye test would not need to be performed. If the substance is not corrosive or severely irritating to the skin, an
in vivo eye test should be performed.

In vivo test in rabbits (Steps 8 and 9): In vivo ocular testing should begin with an initial test using one animal. If the
results of this test indicate the substance to be a severe irritant or corrosive to the eyes, further testing should not be
performed. If that test does not reveal any corrosive or severe irritant effects, a confirmatory test is conducted with two
additional animals.

REFERENCES

(1) OECD (1996) OECD Test Guidelines Programme: Final Report of the OECD Workshop on Harmonization of
Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Held in Solna, Sweden, 22-24 January
1996 (http://www1.oecd.org/ehs/test/background.htm).

(2) OECD (1998) Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of
Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on
Chemicals, November 1998 (http://www1.oecd.org/ehs/Class/HCL6.htm).

(3) Worth, A.P. and Fentem J.H. (1999). A General Approach for Evaluating Stepwise Testing Strategies. ATLA 27, 161-

177.

(4) Testing method B.4. Acute Toxicity: dermal irritation/corrosion.

(5) Young,

J.R., How, M.J., Walker, A.P., Worth W.M.H.

(1988) Classification as Corrosive or Irritant

to Skin of

Preparations Containing Acidic or Alkaline Substance Without Testing on Animals. Toxicol. In Vitro, 2, 19-26.

(6) Neun, D.J. (1993) Effects of Alkalinity on the Eye Irritation Potential of Solutions Prepared at a Single pH. J. Toxicol.

Cut. Ocular Toxicol. 12, 227-231.

(7) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Edsail, D.J., Holzhutter, H.G. and Liebsch,
M. (1998) The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation
by the Management Team. Toxicology in Vitro 12, pp. 483–524.

(8) Testing Method B.40 Skin Corrosion.

(9) Annex to Testing method B.4: A Sequential Testing Strategy for Skin Irritation and Corrosion.

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TESTING AND EVALUATION STRATEGY FOR EYE IRRITATION/CORROSION

FIGURE

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’

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ANNEX 2F

‘B.31. PRENATAL DEVELOPMENTAL TOXICITY STUDY

1.

METHOD

This method is a replicate of OECD TG 414 (2001).

1.1

INTRODUCTION

This method for developmental toxicity testing is designed to provide general information concerning the
effects of prenatal exposure on the pregnant test animal and on the developing organism in utero; this
may include assessment of maternal effects as well as death, structural abnormalities, or altered growth in
the foetus. Functional deficits, although an important part of development, are not an integral part of this
test method. They may be tested for in a separate study or as an adjunct to this study using the test
method for developmental neurotoxicity. For information on testing for functional deficiencies and other
postnatal effects the Test Method for the two-generation reproductive toxicity study and the developmental
neurotoxicity study should be consulted as appropriate.

This test method may require specific adaptation in individual cases on the basis of specific knowledge on
e.g. physicochemical or toxicological properties of the test substance. Such adaptation is acceptable, when
convincing scientific evidence suggests that the adaptation will lead to a more informative test. In such a
case, this scientific evidence should be carefully documented in the study report.

1.2

DEFINITIONS

Developmental toxicology: the study of adverse effects on the developing organism that may result from
exposure prior to conception, during prenatal development, or postnatally to the time of sexual
maturation. The major manifestations of developmental toxicity include 1) death of the organism, 2)
structural abnormality, 3) altered growth, and 4) functional deficiency. Developmental toxicology was
formerly often referred to as teratology.

Adverse effect: any treatment-related alteration from baseline that diminishes an organism’s ability to
survive, reproduce or adapt to the environment. Concerning developmental toxicology, taken in its widest
sense it includes any effect which interferes with normal development of the conceptus, both before and
after birth.

Altered growth: an alteration in offspring organ or body weight or size.

Alterations (anomalies): structural alterations in development that include both malformations and
variations (28).

Malformation/Major Abnormality: Structural change considered detrimental to the animal (may also be
lethal) and is usually rare.

Variation/Minor Abnormality: Structural change considered to have little or no detrimental effect on the
animal; may be transient and may occur relatively frequently in the control population.

Conceptus: the sum of derivatives of a fertilised ovum at any stage of development from fertilisation until
birth including the extra-embryonic membranes as well as the embryo or foetus.

Implantation (nidation): attachment of the blastocyst to the epithelial lining of the uterus, including its
penetration through the uterine epithelium, and its embedding in the endometrium.

Embryo: the early or developing stage of any organism, especially the developing product of fertilisation
of an egg after the long axis appears and until all major structures are present.

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Embryotoxicity: detrimental
embryo.

to the normal structure, development, growth, and/or viability of an

Foetus: the unborn offspring in the post-embryonic period.

Foetotoxicity: detrimental to the normal structure, development, growth, and/or viability of a foetus.

Abortion: the premature expulsion from the uterus of the products of conception: of the embryo or of a
nonviable foetus.

Resorption: a conceptus which, having implanted in the uterus, subsequently died and is being, or has
been resorbed.

Early resorption: evidence of implantation without recognisable embryo/foetus

Late resorption: dead embryo or foetus with external degenerative changes

NOAEL: abbreviation for no-observed-adverse-effect level and is the highest dose or exposure level where
no adverse treatment-related findings are observed.

1.3

REFERENCE SUBSTANCE

None.

1.4

PRINCIPLE OF THE TEST METHOD

Normally, the test substance is administered to pregnant animals at least from implantation to one day
prior to the day of scheduled kill, which should be as close as possible to the normal day of delivery
without risking loss of data resulting from early delivery. The test method is not intended to examine
solely the period of organogenesis, (e.g. days 5-15 in the rodent, and days 6-18 in the rabbit) but also
effects from preimplantation, when appropriate, through the entire period of gestation to the day before
caesarean section. Shortly before caesarean section,
the uterine contents are
examined, and the foetuses are evaluated for externally visible anomalies and for soft tissue and skeletal
changes.

the females are killed,

1.5

DESCRIPTION OF THE TEST METHOD

1.5.1

Selection of animal species

It is recommended that testing be performed in the most relevant species, and that laboratory species and
strains which are commonly used in prenatal developmental toxicity testing be employed. The preferred
rodent species is the rat and the preferred non-rodent species is the rabbit. Justification should be provided
if another species is used.

1.5.2

Housing and feeding conditions

The temperature in the experimental animal room should be 22 ºC (± 3º) for rodents and 18 ºC (± 3º) for
rabbits. Although the relative humidity should be at least 30 % and preferably not exceed 70 % other than
during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence being 12
hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited
supply of drinking water.

Mating procedures should be carried out in cages suitable for the purpose. While individual housing of
mated animals is preferred, group housing in small numbers is also acceptable.

1.5.3

Preparation of the animals

Healthy animals, which have been acclimated to laboratory conditions for at least 5 days and have not
been subjected to previous experimental procedures, should be used. The test animals should be
characterised as to species, strain, source, sex, weight and/or age. The animals of all test groups should, as
nearly as practicable, be of uniform weight and age. Young adult nulliparous female animals should be

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used at each dose level. The females should be mated with males of the same species and strain, and the
mating of siblings should be avoided. For rodents day 0 of gestation is the day on which a vaginal plug
and/or sperm are observed; for rabbits day 0 is usually the day of coitus or of artificial insemination, if
this technique is used. Mated females should be assigned in an unbiased manner to the control and
treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are
minimised. Each animal should be assigned a unique identification number. Mated females should be
assigned in an unbiased manner to the control and treatment groups, and if the females are mated in
batches, the animals in each batch should be evenly distributed across the groups. Similarly, females
inseminated by the same male should be evenly distributed across the groups.

1.6

PROCEDURE

1.6.1

Number and sex of animals

Each test and control group should contain a sufficient number of females to result in approximately 20
female animals with implantation sites at necropsy. Groups with fewer than 16 animals with implantation
sites may be inappropriate. Maternal mortality does not necessarily invalidate the study providing it does
not exceed approximately 10 %.

1.6.2

Preparation of doses

If a vehicle or other additive is used to facilitate dosing, consideration should be given to the following
characteristics: effects on the absorption, distribution, metabolism, and retention or excretion of the test
substance; effects on the chemical properties of the test substance which may alter its toxic characteristics;
and effects on the food or water consumption or the nutritional status of the animals. The vehicle should
neither be developmentally toxic nor have effects on reproduction.

1.6.3

Dosage

Normally, the test substance should be administered daily from implantation (e.g., day 5 post mating) to
the day prior to scheduled caesarean section. If preliminary studies, when available, do not indicate a high
potential for preimplantation loss, treatment may be extended to include the entire period of gestation,
from mating to the day prior to scheduled kill. It is well known that inappropriate handling or stress
during pregnancy can result in prenatal loss. To guard against prenatal loss from factors which are not
treatment-related, unnecessary handling of pregnant animals as well as stress from outside factors such as
noise should be avoided.

At least three dose levels and a concurrent control should be used. Healthy animals should be assigned in
an unbiased manner to the control and treatment groups. The dose levels should be spaced to produce a
gradation of toxic effects. Unless limited by the physical/chemical nature or biological properties of the test
substance, the highest dose should be chosen with the aim to induce some developmental and/or maternal
toxicity (clinical signs or a decrease in body weight) but not death or severe suffering. At least one
intermediate dose level should produce minimal observable toxic effects. The lowest dose level should not
produce any evidence of either maternal or developmental toxicity. A descending sequence of dose levels
should be selected with a view to demonstrating any dosage-related response and no-observed-adverse-
effect level (NOAEL). Two- to four-fold intervals are frequently optimal for setting the descending dose
levels, and the addition of a fourth test group is often preferable to using very large intervals (e.g. more
than a factor of 10) between dosages. Although establishment of a maternal NOAEL is the goal, studies
which do not establish such a level may also be acceptable (1).

Dose levels should be selected taking into account any existing toxicity data as well as additional
information on metabolism and toxicokinetics of the test substance or related materials. This information
will also assist in demonstrating the adequacy of the dosing regimen.

A concurrent control group should be used. This group should be a sham-treated control group or a
vehicle-control group if a vehicle is used in administering the test substance. All groups should be
administered the same volume of either test substance or vehicle. Animals in the control group(s) should
be handled in an identical manner to test group animals. Vehicle control groups should receive the vehicle
in the highest amount used (as in the lowest treatment group).

1.6.4

Limit test

If a test at one dose level of at least 1 000 mg/kg body weight/day by oral administration, using the
procedures described for this study, produces no observable toxicity in either pregnant animals or their
progeny and if an effect would not be expected based upon existing data (e.g., from structurally and/or
metabolically related compounds), then a full study using three dose levels may not be considered

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necessary. Expected human exposure may indicate the need for a higher oral dose level to be used in the
limit test. For other types of administration, such as inhalation or dermal application, the physico-chemical
properties of the test substance often may indicate and limit the maximum attainable level of exposure
(for example, dermal application should not cause severe local toxicity).

1.6.5

Administration of doses

substance or vehicle is usually administered orally by intubation.

route of
The test
administration is used,
the tester should provide justification and reasoning for its selection, and
appropriate modifications may be necessary (2)(3)(4). The test substance should be administered at
approximately the same time each day.

If another

The dose to individual animals should normally be based on the most recent individual body weight
determination. However, caution should be exercised when adjusting the dose during the last trimester of
pregnancy. Existing data should be used for dose selection to prevent excess maternal toxicity. However, if
excess toxicity is noted in the treated dams, those animals should be humanely killed. If several pregnant
animals show signs of excess toxicity, consideration should be given to terminating that dose group. When
the substance is administered by gavage, this should preferably be given as a single dose to the animals
liquid that can be
using a stomach tube or a suitable intubation canula. The maximum volume of
administered at one time depends on the size of the test animal. The volume should not exceed 1 ml/100
g body weight, except in the case of aqueous solutions where 2 ml/100 g body weight may be used. When
corn oil is used as a vehicle, the volume should not exceed 0.4 ml/100 g body weight. Variability in test
volume should be minimised by adjusting the concentrations to ensure a constant volume across all dose
levels.

1.6.6

Observations of the dams

Clinical observations should be made and recorded at least once a day, preferably at the same time(s) each
day taking into consideration the peak period of anticipated effects after dosing. The condition of the
animals should be recorded including mortality, moribundity, pertinent behavioural changes, and all signs
of overt toxicity.

1.6.7

Body weight and food consumption

Animals should be weighed on day 0 of gestation or no later than day 3 of gestation if time-mated
animals are supplied by an outside breeder, on the first day of dosing, at least every 3 days during the
dosing period and on the day of scheduled kill.

Food consumption should be recorded at three-day intervals and should coincide with days of body
weight determination.

1.6.8

Post-mortem examination

Females should be killed one day prior to the expected day of delivery. Females showing signs of abortion
or premature delivery prior to scheduled kill should be killed and subjected to a thorough macroscopic
examination.

At the time of termination or death during the study, the dam should be examined macroscopically for
any structural abnormalities or pathological changes. Evaluation of the dams during caesarean section and
subsequent foetal analyses should be conducted preferably without knowledge of treatment group in order
to minimise bias.

1.6.9

Examination of uterine contents

Immediately after termination or as soon as possible after death, the uteri should be removed and the
pregnancy status of the animals ascertained. Uteri that appear non gravid should be further examined (e.g.
by ammonium sulphide staining for rodents and Salewski staining or a suitable alternative method for
rabbits) to confirm the non-pregnant status (5).

Gravid uteri including the cervix should be weighed. Gravid uterine weights should not be obtained from
animals found dead during the study.

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The number of corpora lutea should be determined for pregnant animals.

The uterine contents should be examined for numbers of embryonic or foetal deaths and viable foetuses.
The degree of resorption should be described in order to estimate the relative time of death of the
conceptus (see section 1.2).

1.6.10

Examination of foetuses

The sex and body weight of each foetus should be determined.

Each foetus should be examined for external alterations (6).

(8)

(7)

Foetuses should be examined for skeletal and soft tissue alterations (e.g. variations and malformations or
anomalies)
(24).
Categorisation of foetal alterations is preferable but not required. When categorisation is done, the criteria
for defining each category should be clearly stated. Particular attention should be paid to the reproductive
tract which should be examined for signs of altered development.

(12 )

(20)

(22)

(23)

(21)

(15)

(13)

(10)

(18)

(17)

(14)

(11)

(16)

(19)

(9)

For rodents, approximately one-half of each litter should be prepared and examined for skeletal alterations.
The remainder should be prepared and examined for soft tissue alterations, using accepted or appropriate
serial sectioning methods or careful gross dissection techniques.

For non-rodents, e.g. rabbits, all foetuses should be examined for both soft tissue and skeletal alterations.
The bodies of these foetuses are evaluated by careful dissection for soft tissue alterations, which may
include procedures to further evaluate internal cardiac structure (25). The heads of one-half of the foetuses
examined in this manner should be removed and processed for evaluation of soft tissue alterations
(including eyes, brain, nasal passages and tongue), using standard serial sectioning methods (26) or an
equally sensitive method. The bodies of these foetuses and the remaining intact foetuses should be
processed and examined for skeletal alterations, utilising the same methods as described for rodents.

2.

2.1

DATA

TREATMENT OF RESULTS

Data shall be reported individually for the dams as well as for their offspring and summarised in tabular
form, showing for each test group and each generation the number of animals at the start of the test, the
number of animals found dead during the test or killed for humane reasons, the time of any death or
humane kill, the number of pregnant females, the number of animals showing signs of toxicity, a
description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic
effects, the types of embryo/foetal observations, and all relevant litter data.

Numerical results should be evaluated by an appropriate statistical method using the litter as the unit for
data analysis. A generally accepted statistical method should be used; the statistical methods should be
selected as part of the design of the study and should be justified. Data from animals that do not survive
to the scheduled kill should also be reported. These data may be included in group means where relevant.
Relevance of the data obtained from such animals, and therefore inclusion or exclusion from any group
mean(s), should be justified and judged on an individual basis.

2.2

EVALUATION OF RESULTS

The findings of the Prenatal Developmental Toxicity Study should be evaluated in terms of the observed
effects. The evaluation will include the following information:

— maternal and embryo/foetal test results, including the evaluation of the relationship, or lack thereof,
between the exposure of the animals to the test substance and the incidence and severity of all
findings;

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— criteria used for categorising foetal external, soft tissue, and skeletal alterations if categorisation has

been done;

— when appropriate, historical control data to enhance interpretation of study results;

— the numbers used in calculating all percentages or indices;

— adequate statistical analysis of the study findings, when appropriate, which should include sufficient
information on the method of analysis, so that an independent reviewer/statistician can re-evaluate
and reconstruct the analysis;

In any study which demonstrates the absence of any toxic effects, further investigations to establish
absorption and bioavailability of the test substance should be considered.

2.3

INTERPRETATION OF RESULTS

A prenatal developmental toxicity study will provide information on the effects of repeated exposure to a
substance during pregnancy on the dams and on the intrauterine development of their progeny. The
results of the study should be interpreted in conjunction with the findings from subchronic, reproduction,
toxicokinetic and other studies. Since emphasis is placed on both general toxicity in terms of maternal
toxicity and on developmental toxicity endpoints, the results of the study will allow to a certain extent for
the discrimination between developmental effects occurring in the absence of general toxicity and those
which are only induced at levels that are also toxic to the maternal animal (27).

3.

3.1

REPORTING

TEST REPORT

The test report must include the following specific information:

Test substance:

— physical nature and, where relevant, physiochemical properties;

— identification including CAS number if known/established;

— purity.

Vehicle (if appropriate):

— justification for choice of vehicle, if other than water.

Test animals:

— species and strain used;

— number and age of animals;

— source, housing conditions, diet, etc.;

— individual weights of animals at the start of the test.

Test conditions:

— rationale for dose level selection;

— details of

test substance formulation/diet preparation, achieved concentration, stability and

homogeneity of the preparation;

— details of the administration of the test substance;

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— conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg

body weight/day), if applicable;

— environmental conditions;

— details of food and water quality.

Results:

Maternal toxic response data by dose, including but not limited to:

— the number of animals at the start of the test, the number of animals surviving, the number

pregnant, and the number aborting, number of animals delivering early;

— day of death during the study or whether animals survived to termination;

— data from animals that do not survive to the scheduled kill should be reported but not included

in the inter-group statistical comparisons;

— day of observation of each abnormal clinical sign and its subsequent course;

— body weight, body weight change and gravid uterine weight, including, optionally, body weight

change corrected for gravid uterine weight;

— food consumption and, if measured, water consumption;

— necropsy findings, including uterine weight;

— NOAEL values for maternal and developmental effects should be reported.

Developmental endpoints by dose for litters with implants, including:

— number of corpora lutea;

— number of implantations, number and percent of live and dead foetuses and resorptions;

— number and percent of pre- and post-implantation losses.

Developmental endpoints by dose for litters with live foetuses, including:

— number and percent of live offspring;

— sex ratio;

— foetal body weight, preferably by sex and with sexes combined;

— external, soft tissue, and skeletal malformations and other relevant alterations;

— criteria for categorisation if appropriate;

— total number and percent of

alteration, as well as the types and incidences of
alterations.

foetuses and litters with any external, soft tissue, or skeletal
individual anomalies and other relevant

Discussion of results.

Conclusions.

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4.

REFERENCES

(1) Kavlock R.J. et al. (1996) A Simulation Study of the Influence of Study Design on the Estimation of

Benchmark Doses for Developmental Toxicity. Risk Analysis 16; 399-410.

(2)

(1990) Proceedings of the Workshop on the Acceptability and
Kimmel, C.A. and Francis, E.Z.
Interpretation of Dermal Developmental Toxicity Studies. Fundamental and Applied Toxicology 14; 386-
398.

(3) Wong, B.A., et al.

(1997) Developing Specialized Inhalation Exposure Systems

to Address

Toxicological Problems. CIIT Activities 17; 1-8.

(4) US Environmental Protection Agency (1985) Subpart E-Specific Organ/Tissue Toxicity, 40 CFR

798.4350: Inhalation Developmental Toxicity Study.

(5)

(6)

(7)

(8)

Salewski, E. (1964) Faerbermethode zum Makroskopischen Nachweis von Implantations Stellen am
Uterusder Ratte. Naunyn‑Schmeidebergs Archiv fur Pharmakologie und Experimentelle Pathologie 247:367.

Edwards, J.A.
Teratology. D.H.M. Woolam (ed.) Vol. 3. Academic Press, NY.

(1968) The external Development of the Rabbit and Rat Embryo. In Advances in

Inouye, M. (1976) Differential Staining of Cartilage and Bone in Fetal Mouse Skeleton by Alcian Blue
and Alizarin Red S. Congenital Anomalies 16; 171-173.

Igarashi, E. et al. (1992) Frequency Of Spontaneous Axial Skeletal Variations Detected by the Double
Staining Techniquefor Ossified and Cartilaginous Skeleton in Rat Foetuses. Congenital Anomalies 32;
:381-391.

(9)

Kimmel, C.A. et al. (1993) Skeletal Development Following Heat Exposure in the Rat. Teratology
47:229-242.

(10) Marr, M.C. et al.

(1988) Comparison of Single and Double Staining for Evaluation of Skeletal

Development: The Effects of Ethylene Glycol (EG) in CD Rats. Teratology 37; 476.

(11) Barrow, M.V. and Taylor, W.J. (1969) A Rapid Method for Detecting Malformations in Rat Foetuses.

Journal of Morphology 127:291-306.

(12) Fritz, H. (1974) Prenatal Ossification in Rabbits ss Indicative of Foetal Maturity. Teratology 11; 313-

320.

(13) Gibson,

J.P. et al.
Pharmacology 9; :398-408.

(1966) Use of

the Rabbit

in Teratogenicity Studies. Toxicology and Applied

(14) Kimmel, C.A. and Wilson,
Teratology 8; 309-316.

J.G.

(1973) Skeletal Deviation in Rats: Malformations or Variations?

(15) Marr, M.C. et al.

(1992) Developmental Stages of the CD (Sprague-Dawley) Rat Skeleton after

Maternal Exposure to Ethylene Glycol. Teratology 46; 169-181.

(16) Monie, I.W. et al. (1965) Dissection Procedures for Rat Foetuses Permitting Alizarin Red Staining of
Skeleton and Histological Study of Viscera. Supplement to Teratology Workshop Manual, pp. 163-173.

(17) Spark, C. and Dawson, A.B. (1928) The Order and Time of appearance of Centers of Ossification in
the Fore and Hind Limbs of the Albino Rat, with Special Reference to the Possible Influence of the
Sex Factor. American Journal of Anatomy 41; 411-445.

(18) Staples, R.E. and Schnell, V.L. (1964) Refinements in Rapid Clearing Technique in the KOH-Alizarin

Red S Method for Fetal Bone. Stain Technology 39; 61-63.

(19) Strong, R.M. (1928) The Order Time and Rate of Ossification of the Albino Rat (Mus Norvegicus

Albinus) Skeleton. American Journal of Anatomy 36; 313-355.

(20) Stuckhardt, J.L. and Poppe, S.M. (1984) Fresh Visceral Examination of Rat and Rabbit Foetuses Used

in Teratogenicity Testing. Teratogenesis, Carcinogenesis, and Mutagenesis 4; 181-188.

(21) Walker, D.G. and Wirtschafter, Z.T. (1957) The Genesis of the Rat Skeleton. Thomas, Springfield, IL.

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(22) Wilson, J.G. (1965) Embryological Considerations in Teratology. In Teratology: Principles and Techniques,

Wilson J.G. and Warkany J. (eds). University of Chicago, Chicago, IL, pp 251-277.

(23) Wilson, J.G. and Fraser, F.C. (eds). (1977) Handbook of Teratology, Vol. 4. Plenum, NY.

(24) Varnagy, L. (1980) Use of Recent Fetal Bone Staining Techniques in the Evaluation of Pesticide

Teratogenicity. Acta Vet. Acad. Sci. Hung. 28; 233-239.

(25) Staples, R.E. (1974) Detection of visceral Alterations in Mammalian Foetuses. Teratology 9; 37-38.

(26) Van Julsingha, E.B. and C.G. Bennett (1977) A Dissecting Procedure for the Detection of Anomalies in
the Rabbit Foetal Head. In: Methods in Prenatal Toxicology Neubert, D., Merker, H.J. and Kwasigroch,
T.E. (eds.). University of Chicago, Chicago, IL, pp. 126-144.

(27) US Environmental Protection Agency (1991) Guidelines for Developmental Toxicity Risk Assessment.

Federal Register 56; 63798-63826.

(28) Wise, D.L. et al.

(1997) Terminology of Developmental Abnormalities in Common Laboratory

Mammals (Version 1) Teratology 55; 249-292.’

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ANNEX 2G

‘B.35. TWO-GENERATION REPRODUCTION TOXICITY STUDY

1.

METHOD

This method is a replicate of the OECD TG 416 (2001).

1.1

INTRODUCTION

function,

including gonadal

two-generation reproduction testing is designed to provide general

This method for
information
concerning the effects of a test substance on the integrity and performance of the male and female
reproductive systems,
the oestrus cycle, mating behaviour, conception,
gestation, parturition, lactation, and weaning, and the growth and development of the offspring. The study
may also provide information about the effects of the test substance on neonatal morbidity, mortality, and
preliminary data on prenatal and postnatal developmental toxicity and serve as a guide for subsequent
tests. In addition to studying growth and development of the F1 generation, this test method is also
intended to assess the integrity and performance of the male and female reproductive systems as well as
growth and development of the F2 generation. For further information on developmental toxicity and
functional deficiencies, either additional study segments can be incorporated into this protocol, consulting
the methods for developmental toxicity and/or developmental neurotoxicity as appropriate, or these
endpoints could be studied in separate studies, using the appropriate test methods.

1.2

PRINCIPLE OF THE TEST METHOD

and in tissue preparation and detailed histopathology.

The test substance is administered in graduated doses to several groups of males and females. Males of the
least one complete spermatogenetic cycle
P generation should be dosed during growth and for at
(approximately 56 days in the mouse and 70 days in the rat) in order to elicit any adverse effects on
spermatogenesis. Effects on sperm are determined by a number of sperm parameters (e.g., sperm
morphology and motility)
If data on
spermatogenesis are available from a previous repeated dose study of sufficient duration, e.g. a 90-day
study, males of the P generation need not be included in the evaluation. It is recommended, however, that
samples or digital recordings of sperm of the P generation are saved, to enable later evaluation. Females of
the P generation should be dosed during growth and for several complete oestrus cycles in order to detect
any adverse effects on oestrus cycle normality by the test substance. The test substance is administered to
parental (P) animals during their mating, during the resulting pregnancies, and through the weaning of
their Fl offspring. At weaning the administration of the substance is continued to Fl offspring during their
growth into adulthood, mating and production of an F2 generation, until the F2 generation is weaned.

Clinical observations and pathological examinations are performed on all animals for signs of toxicity with
special emphasis on effects on the integrity and performance of the male and female reproductive systems
and on the growth and development of the offspring.

1.3

DESCRIPTION OF THE TEST METHOD

1.3.1

Selection of animal species

The rat is the preferred species for testing. If other species are used, justification should be given and
appropriate modifications will be necessary. Strains with low fecundity or well-known high incidence of
developmental defects should not be used. At the commencement of the study, the weight variation of
animals used should be minimal and not exceed 20 % of the mean weight of each sex.

1.3.2

Housing and feeding conditions

The temperature in the experimental animal room should be 22 °C (± 3°). Although the relative humidity
should be at least 30 % and preferably not exceed 70 % other than during room cleaning, the aim should

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be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding,
conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet
may be influenced by the need to ensure a suitable admixture of a test substance when administered by
this method.

Animals may be housed individually or be caged in small groups of the same sex. Mating procedures
should be carried out in cages suitable for the purpose. After evidence of copulation, mated females shall
be single-caged in delivery or maternity cages. Mated rats may also be kept in small groups and separated
one or two days prior to parturition. Mated animals shall be provided with appropriate and defined
nesting materials when parturition is near.

1.3.3

Preparation of animals

Healthy young animals, which have been acclimated to laboratory conditions for at least 5 days and have
not been subjected to previous experimental procedures, should be used. The test animals should be
characterised as to species, strain, source, sex, weight and/or age. Any sibling relationships among the
animals should be known so that mating of siblings is avoided. The animals should be randomly assigned
to the control and treated groups (stratification by body weight is recommended). Cages should be
arranged in such a way that possible effects due to cage placement are minimised. Each animal should be
assigned a unique identification number. For the P generation, this should be done before dosing starts.
For the F1 generation, this should be done at weaning for animals selected for mating. Records indicating
the litter of origin should be maintained for all selected F1 animals. In addition, individual identification of
pups as soon after birth as possible is recommended when individual weighing of pups or any functional
tests are considered.

Parental (P) animals shall be about 5 to 9 weeks old at the start of dosing. The animals of all test groups
shall, as nearly as practicable, be of uniform weight and age.

1.4

PROCEDURE

1.4.1

Number and sex of animals

Each test and control group should contain a sufficient number of animals to yield preferably not less than
20 pregnant females at or near parturition. For substances that cause undesirable treatment related effects
(e.g. sterility, excessive toxicity at the high dose), this may not be possible. The objective is to produce
enough pregnancies to assure a meaningful evaluation of the potential of the substance to affect fertility,
pregnancy and maternal behaviour and suckling, growth and development of the F1 offspring from
conception to maturity, and the development of their offspring (F2) to weaning. Therefore, failure to
achieve the desired number of pregnant animals (i.e. 20) does not necessarily invalidate the study and
should be evaluated on a case-by-case basis.

1.4.2

Preparation of Doses

It is recommended that the test substance be administered orally (by diet, drinking water or gavage) unless
another route of administration (e.g. dermal or inhalation) is considered more appropriate.

Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that,
followed by
wherever possible,
consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles.
For vehicles other than water, the toxic characteristics of the vehicle must be known. The stability of the
test substance in the vehicle should be determined.

solution/suspension be considered first,

the use of an aqueous

1.4.3

Dosage

At least three dose levels and a concurrent control shall be used. Unless limited by the physical‑chemical
nature or biological effects of the test substance, the highest dose level should be chosen with the aim to
induce toxicity but not death or severe suffering. In case of unexpected mortality, studies with a mortality
rate of less than approximately 10 percent in the parental (P) animals would normally still be acceptable.
A descending sequence of dose levels should be selected with a view to demonstrating any dosage related
effect and no‑observed‑adverse-effects levels (NOAEL). Two to four fold intervals are frequently optimal for
setting the descending dose levels and addition of a fourth test group is often preferable to using very
large intervals (e.g. more than a factor of 10) between dosages. For the dietary studies the dose interval

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should be not more than 3 fold. Dose levels should be selected taking into account any existing toxicity
data, especially results from repeated dose studies. Any available information on metabolism and kinetics
of the test compound or related materials should also be considered. In addition, this information will also
assist in demonstrating the adequacy of the dosing regimen.

The control group shall be an untreated group or a vehicle‑control group if a vehicle is used in
administering the test substance. Except for treatment with the test substance, animals in the control
group should be handled in an identical manner to the test group subjects. If a vehicle is used, the control
group shall receive the vehicle in the highest volume used. If a test substance is administered in the diet,
and causes reduced dietary intake or utilisation, then the use of a pair-fed control group may be
considered necessary. Alternatively data from controlled studies designed to evaluate the effects of
decreased food consumption on reproductive parameters may be used in lieu of a concurrent pair-fed
control group.

Consideration should be given to the following characteristics of vehicle and other additives: effects on the
absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties
of
the test substance which may alter its toxic characteristics; and effects on the food or water
consumption or the nutritional status of the animals.

1.4.4

Limit test

If an oral study at one dose level of at least 1 000 mg/kg body weight/day or, for dietary or drinking water
administration, an equivalent percentage in the diet or drinking water using the procedures described for
this study, produces no observable toxic effects in either parental animals or their offspring and if toxicity
would not be expected based upon data from structurally and /or metabolically related compounds, then a
full study using several dose levels may not be considered necessary. The limit test applies except when
to be used. For other types of
human exposure indicates the need for a higher oral dose level
administration, such as inhalation or dermal application, the physical-chemical properties of the test
substance, such as solubility, often may indicate and limit the maximum attainable level of exposure.

1.4.5

Administration of doses

The animals should be dosed with the test substance on a 7‑days‑a‑week basis. The oral route of
administration (diet, drinking water, or gavage) is preferred. If another route of administration is used,
justification shall be provided, and appropriate modifications may be necessary. All animals shall be dosed
by the same method during the appropriate experimental period. When the test substance is administered
by gavage, this should be done using a stomach tube. The volume of liquid administered at one time
should not exceed 1 ml/l00 g body weight (0.4 ml/100 g body weight is the maximum for corn oil),
except in the case of aqueous solutions where 2 ml/100 g body weight may be used. Except for irritant 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. In gavage studies, the pups will normally only receive test substance indirectly through the milk,
until direct dosing commences for them at weaning. In diet or drinking water studies, the pups will
additionally receive test substance directly when they commence eating for themselves during the last
week of the lactation period.

For substances administered via the diet or drinking water, it is important to ensure that the quantities of
the test substance involved do not interfere with normal nutrition or water balance. When the test
substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level
in terms of the body weight of the animal may be used; the alternative used must be specified. For a
substance administered by gavage, the dose should be given at similar times each day, and adjusted at least
weekly to maintain a constant dose level in terms of animal body weight. Information regarding placental
distribution should be considered when adjusting the gavage dose based on weight.

1.4.6

Experimental schedules

Daily dosing of the parental (P) males and females shall begin when they are 5 to 9 weeks old. Daily
dosing of the F1 males and females shall begin at weaning; it should be kept in mind that in cases of test
substance administration via diet or drinking water, direct exposure of the F1 pups to the test substance
may already occur during the lactation period. For both sexes (P and F1), dosing shall be continued for at
least 10 weeks before the mating period. Dosing is continued in both sexes during the 2 week mating
period. Males should be humanely killed and examined when they are no longer needed for assessment of
reproductive effects. For parental (P) females, dosing should continue throughout pregnancy and up to the

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weaning of the F1 offspring. Consideration should be given to modifications in the dosing schedule based
on available information on the test substance, including existing toxicity data, induction of metabolism or
bioaccumulation. The dose to each animal should normally be based on the most recent individual body
weight determination. However, caution should be exercised when adjusting the dose during the last
trimester of pregnancy.

Treatment of the P and F1 males and females shall continue until termination. All P and F1 adult males
and females should be humanely killed when they are no longer needed for assessment of reproductive
effects. F1 offspring not selected for mating and all F2 offspring should be humanely killed after weaning.

1.4.7

Mating procedure

1.4.7.1

Parental (P) mating

For each mating, each female shall be placed with a single male from the same dose level (1:1 mating)
until copulation occurs or 2 weeks have elapsed. Each day, the females shall be examined for presence of
sperm or vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal plug or sperm are found. In
case pairing is unsuccessful, re‑mating of
the same group could be
considered. Mating pairs should be clearly identified in the data. Mating of siblings should be avoided.

females with proven males of

1.4.7.2

F1 mating

For mating the F1 offspring, at least one male and one female should be selected at weaning from each
litter for mating with other pups of the same dose level but different litter, to produce the F2 generation.
Selection of pups from each litter should be random when no significant differences are observed in body
weight or appearance between the litter mates.
the best
representatives of each litter should be selected. Pragmatically, this is best done on a body weight basis but
it may be more appropriate on the basis of appearance. The F1 offspring should not be mated until they
have attained full sexual maturity.

In case these differences are observed,

Pairs without progeny should be evaluated to determine the apparent cause of the infertility. This may
involve such procedures as additional opportunities to mate with other proven sires or dams, microscopic
examination of the reproductive organs, and examination of the oestrous cycles or spermatogenesis.

1.4.7.3

Second mating

In certain instances, such as treatment‑related alterations in litter size or the observation of an equivocal
effect in the first mating, it is recommended that the P or F1 adults be remated to produce a second litter.
It is recommended to remate females or males, which have not produced a litter with proven breeders of
the opposite sex. If production of a second litter is deemed necessary in either generation, animals should
be remated approximately one week after weaning of the last litter.

1.4.7.4

Litter size

Animals shall be allowed to litter normally and rear their offspring to weaning. Standardisation of litter
sizes is optional. When standardisation is done, the method used should be described in detail.

1.5

OBSERVATIONS

1.5.1

Clinical observations

A general clinical observation should be made each day and, and in the case of gavage dosing its timing
should take into account the anticipated peak period of effects after dosing. Behavioural changes, signs of
difficult or prolonged parturition and all signs of toxicity should be recorded. An additional, more detailed
examination of each animal should be conducted on at least a weekly basis and could conveniently be
performed on an occasion when the animal is weighed. Twice daily, during the weekend once daily when
appropriate, all animals should be observed for morbidity and mortality.

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1.5.2

Body weight and food/water consumption of parent animals

Parental animals (P and Fl) shall be weighed on the first day of dosing and at least weekly thereafter.
Parental females (P and F1) shall be weighed at a minimum on gestation days 0, 7, 14, and 20 or 21, and
during lactation on the same days as the weighing of litters and on the day the animals are killed. These
observations should be reported individually for each adult animal. During the premating and gestation
periods food consumption shall be measured weekly at a minimum. Water consumption shall be
measured weekly at a minimum if the test substance is administered in the water.

1.5.3

Oestrus cycle

Estrous cycle length and normality are evaluated in P and F1 females by vaginal smears prior to mating,
and optionally during mating, until evidence of mating is found. When obtaining vaginal/cervical cells,
care should be taken to avoid disturbance of mucosa and subsequently, the induction of pseudopregnancy
(1).

1.5.4

Sperm parameters

For all P and F1 males at termination, testis and epididymis weight shall be recorded and one of each
organ reserved for histopathological examination (see section 1.5.7, 1.5.8.1). Of a subset of at least ten
males of each group of P and F1 males, the remaining testes and epididymides should be used for
enumeration of homogenisation-resistant spermatids and cauda epididymal sperm reserves, respectively.
For this same subset of males, sperm from the cauda epididymides or vas deferens should be collected for
evaluation of sperm motility and sperm morphology. If treatment-related effects are observed or when
there is evidence from other studies of possible effects on spermatogenesis, sperm evaluation should be
conducted in all males in each dose group; otherwise enumeration may be restricted to control and high-
dose P and F1 males.

The total number of homogenisation-resistant testicular spermatids and cauda epididymal sperm should be
enumerated (2)(3). Cauda sperm reserves can be derived from the concentration and volume of sperm in
the suspension used to complete the qualitative evaluations, and the number of sperm recovered by
subsequent mincing and/or homogenising of
the remaining cauda tissue. Enumeration should be
performed on the selected subset of males of all dose groups immediately after killing the animals unless
video or digital recordings are made, or unless the specimens are freezed and analysed later. In these
instances, the controls and high dose group may be analysed first. If no treatment-related effects (e.g.,
effects on sperm count, motility, or morphology) are seen the other dose groups need not be analysed.
When treatment-related effects are noted in the high-dose group, then the lower dose groups should also
be evaluated.

Epididymal (or ductus deferens) sperm motility should be evaluated or video taped immediately after
sacrifice. Sperm should be recovered while minimising damage, and diluted for motility analysis using
acceptable methods (4). The percentage of progressively motile sperm should be determined either
subjectively of objectively. When computer-assisted motion analysis is performed (5)(6)(7)(8)(9)(10) the
derivation of progressive motility relies on user-defined thresholds for average path velocity and
straightness or linear index. If samples are videotaped (11) or the images are otherwise recorded at the
time of necropsy, subsequent analysis of only control and high-dose P and F1 males may be performed
unless treatment-related effects are observed; in that case, the lower dose groups should also be evaluated.
In the absence of a video or digital image, all samples in all treatment groups should be analysed at
necropsy.

A morphological evaluation of an epididymal (or vas deferens) sperm sample should be performed. Sperm
(at least 200 per sample) should be examined as fixed, wet preparations (12) and classified as either
normal or abnormal. Examples of morphologic sperm abnormalities would include fusion, isolated heads,
and misshapen heads and/or tails. Evaluation should be performed on the selected subset of males of all
dose groups either immediately after killing the animals, or, based on the video or digital recordings, at a

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later time. Smears, once fixed, can also be read at a later time. In these instances, the controls and high
dose group may be analysed first. If no treatment-related effects (e.g., effects on sperm morphology) are
seen the other dose groups need not be analysed. When treatment-related effects are noted in the high-
dose group, then the lower dose groups should also be evaluated.

If any of the above sperm evaluation parameters have already been examined as part of a systemic toxicity
study of at least 90 days, they need not necessarily be repeated in the two-generation study.
It is
recommended, however, that samples or digital recordings of sperm of the P generation are saved, to
enable later evaluation, if necessary.

1.5.5

Offspring

Each litter should be examined as soon as possible after delivery (lactation day 0) to establish the number
and sex of pups, stillbirths, live births, and the presence of gross anomalies. Pups found dead on day 0, if
not macerated, should preferably be examined for possible defects and cause of death and preserved. Live
pups should be counted and weighed individually at birth (lactation day 0) or on day 1, and on regular
weigh days thereafter, e.g., on days 4, 7, 14, and 21 of lactation. Physical or behavioural abnormalities
observed in the dams or offspring should be recorded.

Physical development of the offspring should be recorded mainly by body weight gain. Other physical
parameters (e.g. ear and eye opening, tooth eruption, hair growth) may give supplementary information,
but these data should preferably be evaluated in the context of data on sexual maturation (e.g. age and
body weight at vaginal opening or balano-preputial separation) (13). Functional investigations (e.g. motor
activity, sensory function, reflex ontogeny) of the F1 offspring before and/or after weaning, particularly
those related to sexual maturation, are recommended if such investigations are not included in separate
studies. The age of vaginal opening and preputial separation should be determined for F1 weanlings
selected for mating. Anogenital distance should be measured at postnatal day 0 in F2 pups if triggered by
alterations in F1 sex ratio or timing of sexual maturation.

Functional observations may be omitted in groups that otherwise reveal clear signs of adverse effects (e.g.,
significant decrease in weight gain, etc.). If functional investigations are made, they should not be done on
pups selected for mating.

1.5.6

Gross necropsy

At the time of termination or death during the study, all parental animals (P and F1), all pups with
external abnormalities or clinical signs, as well as one randomly selected pup/sex/litter from both the F1
and F2 generation, shall be examined macroscopically for any structural abnormalities or pathological
changes. Special attention should be paid to the organs of
the reproductive system. Pups that are
humanely killed in a moribund condition and dead pups, when not macerated, should be examined for
possible defects and/or cause of death and preserved.

The uteri of all primiparous females should be examined,
histopathological evaluation, for the presence and number of implantation sites.

in a manner which does not compromise

1.5.7

Organ weights

At the time of termination, body weight and the weight of the following organs of all P and F1 parental
animals shall be determined (paired organs should be weighed individually):

— Uterus, ovaries;

— Testes, epididymides (total and cauda);

— Prostate;

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— Seminal vesicles with coagulating glands and their fluids and prostate (as one unit);

— Brain, liver, kidneys, spleen, pituitary, thyroid and adrenal glands and known target organs.

Terminal body weights should be determined for F1 and F2 pups that are selected for necropsy. The
following organs from the one randomly selected pup/sex/litter (see section 1.5.6) shall be weighed: Brain,
spleen and thymus.

Gross necropsy and organ weight results should be assessed in context with observations made in other
repeated dose studies, when feasible.

1.5.8

Histopathology

1.5.8.1

Parental Animals

The following organs and tissues of parental (P and F1) animals, or representative samples thereof, shall be
fixed and stored in a suitable medium for histopathological examination.

— Vagina, uterus with cervix, and ovaries (preserved in appropriate fixative);

— One testis (preserved in Bouin’s or comparable fixative), one epididymis, seminal vesicles, prostate,

and coagulating gland;

— Previously identified target organ(s) from all P and F1 animals selected for mating.

Full histopathology of the preserved organs and tissues listed above should be performed for all high dose
and control P and F1 animals selected for mating. Examination of the ovaries of the P animals is optional.
Organs demonstrating treatment-related changes should also be examined in the low- and mid-dose
groups to aid in the elucidation of the NOAEL. Additionally, reproductive organs of the low-and mid-dose
animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire, or deliver healthy
offspring, or for which oestrus cyclicity or sperm number, motility, or morphology were affected, should
be subjected to histopathological evaluation. All gross lesions such as atrophy or tumours shall be
examined.

Detailed testicular histopathological examination (e.g. using Bouin’s fixative, paraffin embedding and
transverse sections of 4-5 μm thickness) should be conducted in order to identify treatment-related effects
such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of
spermatogenic cells into the lumen (14). Examination of the intact epididymis should include the caput,
corpus, and cauda, which can be accomplished by evaluation of a longitudinal section. The epididymis
should be evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and
phagocytosis of sperm. PAS and haematoxylin staining may be used for examination of
the male
reproductive organs.

The postlactational ovary should contain primordial and growing follicles as well as the large corpora lutea
of lactation. Histopathological examination should detect qualitative depletion of the primordial follicle
population. A quantitative evaluation of primordial follicles should be conducted for F1 females; the
number of animals, ovarian section selection, and section sample size should be statistically appropriate
for the evaluation procedure used. Examination should include enumeration of the number of primordial
follicles, which can be combined with small growing follicles, for comparison of treated and control
ovaries (15) (16) (17) (18) (19).

1.5.8.2

Weanlings

Grossly abnormal tissue and target organs from all pups with external abnormalities or clinical signs, as
well as from the one randomly selected pup/sex/litter from both the F1 and F2 generation which have not
been selected for mating, shall be fixed and stored in a suitable medium for histopathological examination.
Full histopathological characterisation of preserved tissue should be performed with special emphasis on
the organs of the reproductive system.

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2.

2.1

DATA

TREATMENT OF RESULTS

Data shall be reported individually and summarised in tabular form, showing for each test group and each
generation the number of animals at the start of the test, the number of animals found dead during the
test or killed for humane reasons, the time of any death or humane kill, the number of fertile animals, the
number of pregnant females, the number of animals showing signs of toxicity, a description of the signs of
toxicity observed, including time of onset, duration, and severity of any toxic effects, the types of parental
and offspring observations, the types of histopathological changes, and all relevant litter data.

Numerical results should be evaluated by an appropriate, generally accepted statistical method;
the
statistical methods should be selected as part of the design of the study and should be justified. Dose-
for analysing data. The report should include sufficient
response statistical models may be useful
information on the method of analysis and the computer program employed, so that an independent
reviewer/statistician can re-evaluate and reconstruct the analysis.

2.2

EVALUATION OF RESULTS

The findings of this two-generation reproduction toxicity study should be evaluated in terms of the
observed effects including necropsy and microscopic findings. The evaluation will include the relationship,
or lack thereof, between the dose of the test substance and the presence or absence, incidence and severity
of abnormalities, including gross lesions, identified target organs, affected fertility, clinical abnormalities,
affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic
effects. The physico-chemical properties of the test substance, and when available, toxicokinetics data
should be taken into consideration when evaluating test results.

A properly conducted reproduction toxicity test should provide a satisfactory estimation of a no‑effect
level and an understanding of adverse effects on reproduction, parturition,
lactation, postnatal
development including growth and sexual development.

2.3

INTERPRETATION OF RESULTS

A two-generation reproduction toxicity study will provide information on the effects of repeated exposure
to a substance during all phases of the reproductive cycle. In particular, the study provides information on
the reproductive parameters, and on development, growth, maturation and survival of offspring. The
results of the study should be interpreted in conjunction with the findings from subchronic, prenatal
developmental and toxicokinetic and other available studies. The results of this study can be used in
assessing the need for further testing of a chemical. Extrapolation of the results of the study to man is
valid to a limited degree. They are best used to provide information on no-effect-levels and permissible
human exposure (20) (21) (22) (23).

3.

3.1

REPORTING

TEST REPORT

The test report must include the following information:

Test substance:

— physical nature and, where relevant, physicochemical properties;

— identification data;

— purity.

Vehicle (if appropriate):

— justification for choice of vehicle if other than water.

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Test animals:

— species/strain used;

— number, age and sex of animals;

— source, housing conditions, diet, nesting materials, etc.;

— individual weights of animals at the start of the test.

Test conditions:

— rationale for dose level selection;

— details of test substance formulation/diet preparation, achieved concentrations;

— stability and homogeneity of the preparation;

— details of the administration of the test substance;

— conversion from diet/drinking water test substance concentration (ppm) to the achieved dose

(mg/kg body weight/day), if applicable;

— details of food and water quality.

Results:

— food consumption, and water consumption if available, food efficiency (body weight gain per
gram of food consumed), and test material consumption for P and F1 animals, except for the
period of cohabitation and for at least the last third of lactation;

— absorption data (if available);

— body weight data for P and F1 animals selected for mating;

— litter and pup weight data;

— body weight at sacrifice and absolute and relative organ weight data for the parental animals;

— nature, severity and duration of clinical observations (whether reversible or not);

— time of death during the study or whether animals survived to termination;

— toxic response data by sex and dose,

including indices of mating,

fertility, gestation, birth,

viability, and lactation; the report should indicate the numbers used in calculating these indices;

— toxic or other effects on reproduction, offspring, post‑natal growth, etc.;

— necropsy findings;

— detailed description of all histopathological findings;

— number of P and F1 females cycling normally and cycle length;

— total

cauda

epididymal

sperm number, percent progressively motile

sperm, percent

morphologically normal sperm, and percent of sperm with each identified abnormality;

— time-to-mating, including the number of days until mating ;

— gestation length;

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— number of implantations, corpora lutea, litter size;

— number of live births and post‑implantation loss;

— number of pups with grossly visible abnormalities, if determined the number of runts should be

reported;

— data on physical landmarks in pups and other post natal developmental data; physical landmarks

evaluated should be justified;

— data on functional observations in pups and adults, as applicable;

— statistical treatment of results, where appropriate.

Discussion of results.

Conclusions, including NOAEL values for maternal and offspring effects.

4.

REFERENCES

(1)

Sadleir, R.M.F.S.
Fertilization, C.R. Auston and R.V. Short (eds.), Cambridge, New York.

(1979). Cycles and Seasons,

In: Reproduction in Mammals:

I. Germ Cells and

(2) Gray, L.E. et al.,

(1989). A Dose‑Response Analysis of Methoxychlor‑Induced Alterations of

Reproductive Development and Function in the Rat. Fundamental and Applied Toxicology 12:92‑108.

(3) Robb, G.W. et al., (1978). Daily Sperm Production and Epididymal Sperm Reserves of Pubertal and

Adult Rats. Journal of Reproduction and Fertility 54:103‑107.

(4) Klinefelter, G.R. et al.,

(1991). The Method of Sperm Collection Significantly Influences Sperm
Motion Parameters Following Ethane Dimethanesulfonate Administration in the Rat. Reproductive
Toxicology 5:39 44

(5)

Seed, J. et al. (1996). Methods for Assessing Sperm Motility, Morphology, and Counts in the Rat,
Rabbit, and Dog: a Consensus Report. Reproductive Toxicology 10(3):237- 244.

(6) Chapin, R.E. et al., (1992).Methods for Assessing Rat Sperm Motility. Reproductive Toxicology 6:267-

273

(7) Klinefelter, G.R. et al., (1992). Direct Effects of Ethane Dimethanesulphonate on Epididymal Function

in Adult Rats: an In Vitro Demonstration. Journal of Andrology 13:409‑421.

(8)

(9)

Slott, V.L. et al.,
Toxicology 5:449‑458.

(1991). Rat Sperm Motility Analysis: Methodologic Considerations. Reproductive

Slott, V.L. and Perreault, S.D.,
Sperm Motility Using the Hamilton‑Thorn Motility Analyzer.
Academic, Orlando, Florida. pp. 319‑333.

(1993). Computer‑Assisted Sperm Analysis of Rodent Epididymal
In: Methods in Toxicology, Part A.,

(10) Toth, G.P. et al.

(1989). The Automated Analysis of Rat Sperm Motility Following Subchronic
Epichlorhydrin Administration: Methodologic and Statistical Considerations. Journal of Andrology
10: 401-415.

(11) Working, P.K. and M. Hurtt,

(1987). Computerized Videomicrographic Analysis of Rat Sperm

Motility. Journal of Andrology 8:330-337.

(12) Linder, R.E. et al., (1992). Endpoints of Spermatoxicity in the Rat After Short Duration Exposures to

Fourteen Reproductive Toxicants. Reproductive Toxicology 6:491‑505.

(13) Korenbrot, C.C. et al., (1977). Preputial Separation as an External Sign of Pubertal Development in

the Male Rat. Biological Reproduction 17:298303.

(14) Russell, L.D. et al., (1990). Histological and Histopathological Evaluation of the Testis, Cache River

Press, Clearwater, Florida.

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(15) Heindel,

J.J. and R.E. Chapin,
Toxicology, Academic, Orlando, Florida.

(eds.)

(1993). Part B. Female Reproductive Systems, Methods in

(16) Heindel, J.J. et al., (1989) Histological Assessment of Ovarian Follicle Number in Mice As a Screen of
Ovarian Toxicity. In: Growth Factors and the Ovary, A.N. Hirshfield (ed.), Plenum, New York, pp.
421‑426.

(17) Manson,

J.M. and Y.J. Kang,

for Assessing Female Reproductive and
Developmental Toxicology. In: Principles and Methods of Toxicology, A.W. Hayes (ed.), Raven, New
York.

(1989). Test Methods

(18) Smith, B.J. et al,. (1991). Comparison of Random and Serial Sections in Assessment of Ovarian

Toxicity. Reproductive Toxicology 5:379-383.

(19) Heindel, J.J. (1999). Oocyte Quantitation and Ovarian Histology. In: An Evaluation and Interpretation
of Reproductive Endpoints for Human Health Risk Assessment, G. Daston,. and C.A. Kimmel, (eds.),
ILSI Press, Washington, DC.

(20) Thomas,

J. A.

(1991). Toxic Responses of

the Reproductive System.

In: Casarett and Doull’s

Toxicology, M.O. Amdur, J. Doull, and C.D. Klaassen (eds.), Pergamon, New York.

(21) Zenick, H. and E.D. Clegg, (1989). Assessment of Male Reproductive Toxicity: A Risk Assessment
Approach. In: Principles and Methods of Toxicology, A.W. Hayes (ed.), Raven Press, New York.

(22) Palmer, A.K. (1981). In: Developmental Toxicology, Kimmel, C.A. and J. Buelke-Sam (eds.), Raven

Press, New York.

(23) Palmer, A.K. (1978). In Handbook of Teratology, Vol. 4, J.G. Wilson and F.C. Fraser (eds.), Plenum

Press, New York.’

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ANNEX 2H

‘B.42. SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY

1.

METHOD

This test method is equivalent to the OECD TG 429 (2002)

1.1

INTRODUCTION

The Local Lymph Node Assay (LLNA) has been sufficiently validated and accepted to justify its adoption as
a new Method (1)(2)(3). This is the second method for assessing skin sensitisation potential of chemicals in
animals. The other method (B.6) utilises guinea pig tests, notably the guinea pig maximisation test and the
Buehler test (4).

The LLNA provides an alternative method for identifying skin sensitising chemicals and for confirming
that chemicals lack a significant potential to cause skin sensitisation. This does not necessarily imply that
in all instances the LLNA should be used in place of guinea pig test, but rather that the assay is of equal
merit and may be employed as an alternative in which positive and negative results generally no longer
require further confirmation.

The LLNA provides certain advantages with regard to both scientific progress and animal welfare. It studies
the induction phase of skin sensitisation and provides quantitative data suitable for dose response
assessment. The details of the validation of the LLNA and a review of the associated work have been
published (5)(6)(7)(8).
it should be noted that the mild/moderate sensitisers, which are
recommended as suitable positive control substances for guinea pig test methods, are also appropriate for
use with the LLNA (6)(8)(9).

In addition,

The LLNA is an in vivo method and, as a consequence, will not eliminate the use of animals in the
assessment of contact sensitising activity. It has, however, the potential to reduce the number of animals
required for this purpose. Moreover, the LLNA offers a substantial refinement of the way in which animals
are used for contact sensitisation testing. The LLNA is based upon consideration of immunological events
stimulated by chemicals during the induction phase of sensitisation. Unlike guinea pig tests the LLNA does
not require that challenged-induced dermal hypersensitivity reactions be elicited. Furthermore, the LLNA
does not require the use of an adjuvant, as is the case for the guinea pig maximisation test. Thus, the
LLNA reduces animal distress. Despite the advantages of the LLNA over traditional guinea pig tests, it
should be recognised that there are certain limitations that may necessitate the use of traditional guinea
pigs tests (e.g., false negative findings in the LLNA with certain metals, false positive findings with certain
skin irritants)(10).

See also Introduction part B.

1.2

PRINCIPLE OF THE TEST METHOD

The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes
in the lymph node draining the site of chemical application. This proliferation is proportional to the dose
applied (and to the potency of the allergen) and provides a simple means of obtaining an objective,
quantitative measurement of sensitisation. The LLNA assesses this proliferation as a dose-response
relationship in which the proliferation in test groups is compared to that in vehicle treated controls. The
ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, is
determined, and must be at least three before a test substance can be further evaluated as a potential skin
sensitiser. The methods described here are based on the use of radioactive labelling to measure cell
proliferation. However, other endpoints for assessment of proliferation may be employed provided there is
justification and appropriate scientific support, including full citations and description of the methodology.

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1.3

DESCRIPTION OF THE TEST METHOD

1.3.1

Preparations

1.3.1.1

Housing and feeding conditions

Animals should be individually housed. The temperature of the experimental animals room should be
22ºC (±3ºC). Although the relative humidity should be at least 30 % and preferably not exceed 70 % other
than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence being
12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited
supply of drinking water.

1.3.1.2

Preparation of animals

The animals are randomly selected, marked to permit individual identification (but not by any form of ear
marking), and kept in their cages for at least 5 days prior to the start of dosing to allow for acclimatisation
to the laboratory conditions. Prior to the start of treatment all animals are examined to ensure that they
have no observable skin lesions.

1.3.2

Test Conditions

1.3.2.1

Experimental animals

The mouse is the species of choice for this test. Young adult female mice of CBA/Ca or CBA/J strain, which
are nulliparous and non-pregnant are used. At the start of the study, animals should be between 8-12
weeks old, and the weight variation of the animals should be minimal and not exceed 20 % of the mean
weight. Other strains and males may be used when sufficient data are generated to demonstrate that
significant strain and/or gender-specific differences in the LLNA response do not exist.

1.3.2.2

Reliability check

Positive controls are used to demonstrate appropriate performance of the assay and competency of the
laboratory to successfully conduct
the assay. The positive control should produce a positive LLNA
response at an exposure level expected to give an increase in the stimulation index (SI) >3 over the
negative control group. The positive control dose should be chosen such that the induction is clear but
not excessive. Preferred substances are hexyl cinnamic aldehyde (CAS No 101-86-0, EINECS No 202-983-
3) and mercaptobenzothiazole (CAS No 149-30-4, EINECS No 205-736-8). There may be circumstances
in which, given adequate justification, other control substances, meeting the above criteria, may be used.
While ordinarily a positive control group may be required in each assay, there may be situations in which
test laboratories will have available historic positive control data to show consistency of a satisfactory
response over a six-month or more extended period. In those situations, less frequent testing with positive
controls may be appropriate at intervals no greater than 6 months. Although the positive control
substance should be tested in the vehicle that is known to elicit a consistent response (e.g., acetone: olive
oil), there may be certain regulatory situations in which testing in non-standard vehicle (clinically/
chemically relevant formulation) will also be necessary. In such situation the possible interaction of a
positive control with this unconventional vehicle should be tested.

1.3.2.3

Number of animals, dose levels and vehicle selection.

A minimum of four animals is used per dose group, with a minimum of three concentrations of the test
substance, plus a negative control group treated only with the vehicle for the test substance, and, as
appropriate, a positive control. In those cases in which individual animal data are to be collected, a
minimum of
treatment with the test
substance, animals in the control groups should be handled and treated in a manner identical to that of
animals in the treatment groups.

five animals per dose group are used. Except for absence of

Dose and vehicle selection should be based on the recommendations given in reference (1). Doses are
selected from the concentration series 100 %, 50 %, 25 %, 10 %, 5 %, 2.5 %, 1 %, 0.5 % etc. Existing acute
toxicity and dermal irritation data should be considered, where available, in selecting the three consecutive
concentrations so that the highest concentration maximises exposure whilst avoiding systemic toxicity and
excessive local skin irritation (2)(11).

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The vehicle should be selected on the basis of maximising the test concentrations and solubility whilst
producing a solution/suspension suitable for application of the test substance. In order of preference,
recommended vehicles are acetone/olive oil (4:1 v/v), dimethylformamide, methyl ethyl ketone, propylene
glycol and dimethyl sulphoxide (2)(10), but others may be used if sufficient scientific rationale is provided.
In certain situations it may be necessary to use a clinically relevant solvent or the commercial formulation
in which the test substance is marketed as an additional control. Particular care should be taken to ensure
that hydrophilic materials are incorporated into a vehicle system, which wets the skin and does not
immediately run off. Thus, wholly aqueous vehicles are to be avoided.

1.3.3

Test procedure

1.3.3.1

Experimental schedule

The experimental schedule of the assay is as follows:

● Day 1:

Individually identify and record the weight of each animal. Open application of 25µl of
the
appropriate dilution of the test substance, the vehicle alone, or the positive control (as appropriate), to
the dorsum of each ear.

● Days 2 and 3:

Repeat the application procedure carried out on day 1.

● Days 4 and 5

No treatment.

● Day 6

Record the weight of each animal. Inject 250µl of phosphate-buffered saline (PBS) containing 20 µCi
(7.4e + 8 Bq) of 3H-methyl thymidine into all test and control mice via the tail vein. Alternatively
125I-iododeoxyuridine and 10-5 M
inject 250 µL PBS containing 2 µCi
fluorodeoxyuridine into all mice via the tail vein.

(7.4e + 7 Bq) of

Five hours later, the animals are killed. The draining auricular lymph nodes from each ear are excised
and pooled in PBS for each experimental group (pooled treatment group approach); alternatively pairs
of
lymph nodes from individual animals may be excised and pooled in PBS for each animal
(individual animal approach). Details and diagrams of the node identification and dissection can be
found in Annex I of reference 10.

1.3.3.2

Preparation of cell suspensions

A single cell suspension of lymph node cells (LNC) either from pooled treatment groups or bilaterally
from individual animals is prepared by gentle mechanical disaggregation through 200 µm-mesh stainless
steel gauze. Lymph node cells are washed twice with an excess of PBS and precipitated with 5 %
trichloroacetic acid (TCA) at 4 °C for 18h (2). Pellets are either re-suspended in 1 ml TCA and transferred
to scintillation vials containing 10 ml of scintillation fluid for 3H-counting, or transferred directly to
gamma counting tubes for 125I-counting.

1.3.3.3

Determination of cell proliferation (incorporated radioactivity)

Incorporation of 3H-methyl thymidine is measured by β-scintillation counting as disintegrations per
minute (DPM). Incorporation of 125I-iododeoxyuridine is measured by 125I-counting and also is expressed as
DPM. Depending on the approach used, the incorporation will be expressed as DPM/treatment group
(pooled approach) or the DPM/animal (individual approach).

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1.3.3.4

Observations

1.3.3.4.1

Clinical observations

Animals should be carefully observed once daily for any clinical signs, either of local irritation at the
application site or of systemic toxicity. All observations are systematically recorded with individual records
being maintained for each animal.

1.3.3.4.2

Body Weights

As stated in section 1.3.3.1, individual animal body weights should be measured at the start of the test and
at the scheduled kill of the animals.

1.3.4

Calculation of results

Results are expressed as the Stimulation Index (SI). When using the pooled approach, the SI is obtained by
dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled
vehicle control group; this yields a mean SI. When using the individual approach, the SI is derived by
dividing the mean DPM/animal within each test substance group and the positive control group by the
mean DPM/animal for the solvent/vehicle control group. The average SI for vehicle treated controls is then
1.

Use of the individual approach to calculate the SI will enable the performance of a statistical analysis of
the data. In choosing an appropriate method of statistical analysis the investigator should maintain an
awareness of possible inequalities of variances and other related problems that may necessitate a data
transformation or a non-parametric statistical analysis. An adequate approach for interpreting the data is
to evaluate all individual data of treated and vehicle controls, and derive from these the best fitting dose
response curve, taking confidence limits into account (8)(12)(13). However, the investigator should be alert
to possible “outlier„ responses for individual animals within a group that may necessitate the use of an
alternative measure of response (e.g., median rather than mean) or elimination of the outlier.

The decision process with regard to a positive response includes a stimulation index ≥3 together with
consideration of dose-response and, where appropriate, statistical significance (3)(6)(8)(12)(14).

If it is necessary to clarify the results obtained, consideration should be given to various properties of the
test substance, including whether it has a structural relationship to known skin sensitisers, whether it
causes excessive skin irritation and the nature of the dose response seen. These and other considerations
are discussed in detail elsewhere (7).

2.

DATA

Data should be summarised in tabular form showing the mean and individual DPM values and stimulation
indexes for each dose (including vehicle control) group.

3.

3.1

REPORTING

TEST REPORT

The test report should contain the following information:

Test substance:

— identification data (e.g., CAS number, if available; source; purity; known impurities; lotnumber);

— physical nature and physicochemical properties (e.g., volatility, stability, solubility);

— if mixture, composition and relative percentages of components.

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Vehicle:

— identification data [purity; concentration (where appropriate); volume used]

— justification for choice of vehicle.

Test animals:

— strain of mice used;

— microbiological status of the animals, when known;

— source of animals, housing conditions, diet, etc.

— number, age and sex of animals;

Test conditions:

— details of test substance preparation and application;

— justification for dose selection, including results from range finding study, if conducted; vehicle

and test substance concentrations used and the total amount of substance applied

— details of food and water quality (including diet type/source, water source).

Reliability check:

— a summary of the results of the latest reliability check including information on substance,

concentration and vehicle used.

— concurrent and/or historical positive and negative control data for testing laboratory

Results:

— individual weights of animals at the start of dosing and at scheduled kill.

— a table of mean (pooled approach) and individual (individual approach) DPM values as well as the
range of values for both approaches and the stimulation indices for each dose (including vehicle
control) group.

— statistical analysis where appropriate

— time course of onset and signs of toxicity, including dermal irritation at site of administration, if

any, for each animal.

Discussion of results:

— A brief commentary on the results, the dose-response analysis, and statistical analyses, where
appropriate, with a conclusion as to whether the test substance should be considered a skin
sensitiser.

4.

REFERENCES

(1) Kimber, I. and Basketter, D.A. (1992). The murine local lymph node assay; collaborative studies and

new directions: A commentary. Food and Chemical Toxicology 30, 165-169.

(2)

Kimber, I, Derman, R.J., Scholes E.W, and Basketter, D.A.
developments and applications. Toxicology, 93, 13-31.

(1994). The local

lymph node assay:

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(3) Kimber, I., Hilton, J., Dearman, R.J., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Lea, L., House, R.V.,
Ladies, G.S., Loveless, S.E., Hastings, K.L. (1998). Assessment of the skin sensitisation potential of
topical medicaments using the local
Journal of
Toxicology and Environmental Health, 53, 563-79.

lymph node assay: An interlaboratory exercise.

(4) Testing Method B.6.

(5) Chamberlain, M. and Basketter, D.A. (1996). The local lymph node assay: status of validation. Food

and Chemical Toxicology, 34, 999-1002.

(6)

(7)

(8)

Basketter, D.A., Gerberick, G.F., Kimber, I. and Loveless, S.E (1996). The local lymph node assay- A
viable alternative to currently accepted skin sensitisation tests. Food and Chemical Toxicology, 34,
985-997.

Basketter, D.A., Gerberick, G.F. and Kimber,
responses in predictive sensitisation tests. Food and Chemical Toxicology. 36, 327-33.

(1998). Strategies for identifying false positive

I.

Van Och, F.M.M, Slob, W., De Jong, W.H., Vandebriel, R.J., Van Loveren, H. (2000). A quantitative
method for assessing the sensitising potency of low molecular weight chemicals using a local lymph
node assay: employement of a regression method that includes determination of uncertainty margins.
Toxicology, 146, 49-59.

(9) Dearman, R.J., Hilton, J., Evans, P., Harvey, P., Basketter, D.A. and Kimber, I. (1998). Temporal stability
of local lymph node assay responses to hexyl cinnamic aldehyde. Journal of Applied Toxicology, 18,
281-4.

(10) National Institute of Environmental Health Sciences (1999). The Murine Local Lymph Node Assay: A
Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds: The
Results of an Independent Peer Review Evaluation Coordinated by the Interagency Coordinating
Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program
Center for the Evaluation of Alternative Toxicological Methods (NICETAM). NIH Publication No: 99-
4494, Research Triangle Park, N.C. (http://iccvam.niehs.nih.gov).

(11) Testing method B.4.

(12) Basketter, D.A., Selbie, E., Scholes, E.W. Lees, D. Kimber, I. and Botham, P.A. (1993) Results with
OECD recommended positive control sensitisers in the maximisation, Buehler and local lymph node
assays. Food and Chemical Toxicology, 31, 63-67.

(13) Basketter D.A., Lea L.J., Dickens A., Briggs D., Pate I., Dearman R.J., Kimber I. (1999). A comparison
of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses.
J. Appl. Toxicology, 19, 261-266.

(14) Basketter DA, Blaikie L, Derman RJ, Kimber I, Ryan CA, Gerberick GF, Harvey P, Evans P, White IR
lymph node assay for the estimation of relative contact

local

and Rycroft RTG (2000). Use of
allergenic potency. Contact Dermatitis 42, 344-48.

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B.43. NEUROTOXICITY STUDY IN RODENTS

1.

METHOD

This method is equivalent of OECD TG 424 (1997).

This Test Method has been designed to obtain the information necessary to confirm or to further
characterise the potential neurotoxicity of chemicals in adult animals. It can either be combined with
existing Test Methods for repeated dose toxicity studies or to be carried out as a separate study. It is
recommended that the OECD Guidance Document on Neurotoxicity Testing Strategies and Methods (1) be
consulted to assist in the design of studies based on this Test Method. This is particularly important when
modifications of the observations and test procedures as recommended for routine use of this Method are
considered. The Guidance Document has been prepared to facilitate the selection of other test procedures
for use in specific circumstances.The assessment of developmental neurotoxicity is not the subject of this
Method.

1.1

INTRODUCTION

In the assessment and evaluation of the toxic characteristics of chemicals, it is important to consider the
potential for neurotoxic effects. Already the Test Method for repeated dose systemic toxicity includes
observations that screen for potential neurotoxicity. This Test Method can be used to design a study to
obtain further information on, or to confirm, the neurotoxic effects observed in the repeated dose
systemic toxicity studies. However, consideration of
the potential neurotoxicity of certain classes of
chemicals may suggest that they may be more appropriately evaluated using this Method without prior
indications of the potential neurotoxicity from repeated dose systemic toxicity studies. Such considerations
include, for example:

● observation of neurological signs or neuropathological lesions in toxicity studies other than repeated

dose systemic toxicity studies, or

● structural relationship or other information linking them to known neurotoxicants.

In addition there may be other instances when use of this Test Method is appropriate; for further details
see (1).

This Method has been developed so that it can be tailored to meet particular needs to confirm the specific
histopathological and behavioural neurotoxicity of a chemical as well as provide a characterization and
quantification of the neurotoxic responses.

In the past, neurotoxicity was equated with neuropathy involving neuropathological lesions or neurological
dysfunctions, such as seizure, paralysis or tremor. Although neuropathy is an important manifestation of
neurotoxicity, it is now clear that there are many other signs of nervous system toxicity (e.g. loss of motor
co-ordination, sensory deficits,
that may not be reflected in
learning and memory dysfunctions)
neuropathy or other types of studies.

This neurotoxicity Test Method is designed to detect major neurobehavioural and neuropathological effects
in adult rodents. While behavioural effects, even in the absence of morphological changes, can reflect an
adverse impact on the organism, not all behavioural changes are specific to the nervous system. Therefore,
evaluated in conjunction with correlative histopathological,
any changes observed should be
haematological or biochemical data as well as data on other types of systemic toxicity. The testing called
for in this Method to provide a characterization and quantification of the neurotoxic responses includes
specific
by
electrophysiological and/or biochemical investigations (1)(2)(3)(4).

histopathological

behavioural

procedures

that may

supported

further

and

be

Neurotoxicants may act on a number of
targets within the nervous system and by a variety of
mechanisms. Since no single array of tests is capable of thoroughly assessing the neurotoxic potential of all
substances, it may be necessary to utilize other in vivo or in vitro tests specific to the type of neurotoxicity
observed or anticipated.

This Test Method can also be used, in conjunction with the guidance set out in the OECD Guidance
Document on Neurotoxicity Testing Strategies and Methods (1) to design studies intended to further
characterize or increase the sensitivity of the dose-response quantification in order or better estimate a no-
observed-adverse effect level or to substantiate known or suspected hazards of the chemical. For example,
studies may be designed to identify and evaluate the neurotoxic mechanism(s) or supplement the data

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already available from the use of basic neurobehavioural and neuropathological observation procedures.
Such studies need not replicate data that would be generated from the use of the standard procedures
recommended in this Method, if such data are already available and are not considered necessary for the
interpretation of the results of the study.

This neurotoxicity study, when used alone or in combination, provides information that can:

— identify whether the nervous system is permanently or reversibly affected by the chemical tested;

— contribute to the characterization of the nervous system alterations associated with exposure to the

chemical, and to understanding the underlying mechanism.

— determine dose-and time-response relationships in order to estimate a no-observed-adverse-effect level

(which can be used to establish safety criteria for the chemical).

This Test Method uses oral administration of the test substance. Other routes of administration (e.g.
dermal or inhalation) may be more appropriate, and may require modification of
the procedures
recommended. Considerations of the choice of the route of administration depend on the human exposure
profile and available toxicological or kinetic information.

1.2

DEFINITIONS

Adverse effect: is any treatment-related alteration from baseline that diminishes an organism's ability to
survive, reproduce or adapt to the environment.

Dose: is the amount of test substance administered. Dose is expressed as weight (g, mg) or as weight of
test substance per unit weight of the test animal (e.g. mg/Kg), or as constant dietary concentrations (ppm).

Dosage: is a general term comprising of dose, its frequency and the duration of dosing.

Neurotoxicity: is an adverse change in the structure or function of the nervous system that results from
exposure to a chemical, biological or physical agent.

Neurotoxicant: is any chemical, biological or physical agent having the potential to cause neurotoxicity.

NOAEL: is the abbreviation for no-observed-adverse effect level and is the highest dose level where no
adverse treatment-related findings are observed.

1.3

PRINCIPLE OF THE TEST METHOD

The test chemical is administered by the oral route across a range of doses to several groups of laboratory
rodents. Repeated doses are normally required, and the dosing regimen may be 28 days, subchronic (90
days) or chronic (1 year or longer). The procedures set out in this Test Method may also be used for an
acute neurotoxicity study. The animals are tested to allow the detection or the characterization of
behavioural and/or neurological abnormalities. A range of behaviours that could be affected by
neurotoxicants is assessed during each observation period. At the end of the test, a subset of animals of
each sex from each group are perfused in situ and sections of the brain, spinal cord, and peripheral nerves
are prepared and examined.

be

can

used

When the study is conducted as a stand-alone study to screen for neurotoxicity or to characterize
neurotoxic effects, the animals in each group not used for perfusion and subsequent histopathology (see
Table 1)
or
electrophysiological procedures that may supplement the data obtained from the standard examinations
required by this Method (1). These supplemental procedures can be particularly useful when empirical
observations or anticipated effects indicate a specific type or target of a chemical's neurotoxicity.
Alternatively, the remaining animals can be used for evaluations such as those called for in Test Methods
for repeated dose toxicity studies in rodents.

neuropathological,

neurobehavioural,

neurochemical

specific

for

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When the procedures of this Test Method are combined with those of other Test Methods, a sufficient
number of animals is needed to satisfy the requirements for the observations of both studies.

1.4

DESCRIPTION OF THE TEST METHOD

1.4.1

Selection of animal species

The preferred rodent species is the rat, although other rodent species, with justification, may be used.
Commonly used laboratory strains of young adult healthy animals should be employed. The females
should be nulliparous and non-pregnant. Dosing should normally begin as soon as possible after weaning,
preferably not later than when animals are six weeks, and, in any case, before the animals are nine weeks
age. However, when this study is combined with other studies this age requirement may need adjustment.
At the commencement of the study the weight variation of animals used should not exceed ± 20 % of the
mean weight of each sex. Where a repeated dose study of short duration is conducted as a preliminary to
a long term study, animals from the same strain and source should be used in both studies.

1.4.2

Housing and feeding conditions

The temperature in the experimental animal room should be 22 oC (± 3 oC). Although the relative
humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning, the
aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark.
Loud intermittent noise should be kept to a minimum. For feeding, conventional laboratory diets may be
used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to
ensure a suitable admixture of a test substance when administered by this method. Animals may be
housed individually, or be caged in small groups of the same sex.

1.4.3

Preparation of animals

Healthy young animals are randomly assigned to the treatment and control groups. Cages should be
arranged in such a way that possible effects due to cage placement are minimized. The animals are
identified uniquely and kept in their cages for at least (5) five days prior the start of the study to allow for
acclimatization to the laboratory conditions.

1.4.4

Route of administration and preparation of doses

This Test Method specifically addresses the oral administration of the test substance. Oral administration
may be by gavage, in the diet, in drinking water or by capsules. Other routes of administration (e.g. dermal
or inhalation) can be used but may require modification of the procedures recommended. Considerations
of
the route of administration depend on the human exposure profile and available
toxicological or kinetic information. The rationale for choosing the route of administration as well as
resulting modifications to the procedures of this Test Method should be indicated.

the choice of

the use of an aqueous

the test substance may be dissolved or suspended in a suitable vehicle.

is
Where necessary,
recommended that
followed by
consideration of a solution/suspension in oil (e.g., corn oil) and then by possible solution/suspension in
other vehicle. The toxic characteristics of the vehicle must be known. In addition, consideration should be
given to the following characteristics of the vehicle: effects of the vehicle on absorption, distribution,
metabolism, or retention of the test substance which may alter its toxic characteristics; and effects on the
food or water consumption or the nutritional status of the animals.

solution/suspension be considered first,

It

1.5

PROCEDURES

1.5.1

Number and sex animals

When the study is conducted as a separate study, at least 20 animals (10 females and 10 males) should be
used in each dose and control group for the evaluation of detailed clinical and functional observations. At
least five males and five females, selected from these 10 males and 10 females, should be perfused in situ
and used for detailed neurohistopathology at the end of the study. In cases where only a limited number

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of animals in a given dose group are observed for signs of neurotoxic effects, consideration should be
given to the inclusion of these animals in those selected for perfusion. When the study is conducted in
combination with a repeated dose toxicity study, adequate numbers of animals should be used to meet the
objectives of both studies. The minimum numbers of animals per group for various combinations of
studies are given in Table 1. If interim kills or recovery groups for observation of reversibility, persistence
or delayed occurrence of toxic effects post treatment are planned or when supplemental observations are
considered, then the number of animals should be increased to ensure that the number of animals
required for observation and histopathology are available.

1.5.2

Treatment and control group

At least three dose groups and a control group should generally be used, but if from the assessment of
other data, no effects would be expected at a repeated dose of 1 000 mg/kg body weight/day, a limit test
may be performed. If there are no suitable data available, a range finding study may be performed to aid
in the determination of the doses to be used. Except for treatment with the test substance, animals in the
control group should be handled in an identical manner to the test group subjects. If a vehicle is used in
administering the test substance, the control group should receive the vehicle at the highest volume used.

1.5.3

Reliability check

The laboratory performing the study should present data demonstrating its capability to carry out the
study and the sensitivity of the procedures used. Such data should provide evidence of the ability to detect
and quantify, as appropriate, changes in the different end points recommended for observation, such as
autonomic signs, sensory reactivity, limb grip strength and motor activity. Information on chemicals that
cause different types of neurotoxic responses and could be used as positive control substances can be
found in references 2 to 9. Historical data may be used if the essential aspects of the experimental
procedures remain the same. Periodic updating of historical data is recommended. New data that
demonstrate the continuing sensitivity of
the procedures should be developed when some essential
element of the conduct of the test or procedures has been changed by the performing laboratory.

1.5.4

Dose selection

Dose levels should be selected by taking into account any previously observed toxicity and kinetic data
available for the test compound or related materials. The highest dose level should be chosen with the aim
of inducing neurotoxic effects or clear systemic toxic effects. Thereafter, a descending sequence of dose
levels should be selected with a view to demonstrating any dose-related response and no-observed-adverse
effect (NOAEL) at the lowest dose level. In principle, dose levels should be set so that primary toxic effects
on the nervous system can be distinguished from effects related to systemic toxicity. Two to three intervals
are frequently optimum and addition of a fourth test group is often preferable to using very large intervals
(e.g., more than a factor of 10) between dosages. Where there is a reasonable estimation of human
exposure this should also be taken into account.

1.5.5

Limit test

If a study at one dose level of at least 1 000 mg/kg body weight/day, using the procedures described,
produces no observable neurotoxic effects and if toxicity would not be expected based upon data from
structurally related compounds, then a full study using three dose levels may not be considered necessary.
Expected human exposure may indicate the need for a higher oral dose level to be used in the limit test.
the physical chemical
For other types of administration, such as inhalation or dermal application,
properties of the test substance often may dictate the maximum attainable level of exposure. For the
conduct of an oral acute study, the dose for a limit test should be at least 2 000 mg/kg.

1.5.6

Administration of doses

The animals are dosed with the test substance daily, seven days each week, for a period at least 28 days;
use of a five-day dosing regime or a shorter exposure period needs to be justified. When the test substance
is administered by gavage, this should be done in a single dose using a stomach tube or a suitable

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intubation cannula. The maximum volume of a liquid that can be administered at one time depends on
the size of the test animals. The volume should not exceed 1 ml/100 g body weight. However in the case
of aqueous solutions, the use of up to 2 ml/100 g body weight can be considered. Except for irritating or
corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability
in test volume should be minimized by adjusting the concentration to ensure a constant volume at all
dose levels.

For substances administered via the diet or drinking water, it is important to ensure that the quantities of
the test substance involved do not interfere with normal nutrition or water balance. When the test
substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level
in terms of the animals' body weight may be used; the alternative used must be specified. For a substance
administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to
maintain a constant dose level in terms of animal body weight. Where a repeat dose study is used as a
preliminary to a long term study, a similar diet should be used in both studies. For acute studies, if a single
dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours.

1.6

OBSERVATION

1.6.1

Frequency of observations and tests

In repeated dose studies, the observation period should cover the dosage period. In acute studies, 14-day
post-treatment period should be observed. For animals in satellite groups which are kept without exposure
during a post-treatment period, observations should cover this period as well.

Observations should be made with sufficient frequency to maximize the probability of detection of any
behavioural and/or neurological abnormalities. Observations should be made preferably at the same times
each day with consideration given to the peak period of anticipated effects after dosing. The frequency of
clinical observations and functional tests is summarized in Table 2. If kinetic or other data generated from
previous studies indicates the need to use different time points for observations, tests or post-observation
periods, an alternative schedule should be adopted in order to achieve maximum information. The
rationale for changes to the schedule should be provided.

1.6.1.1

Observations of general health condition and mortality/morbidity

All animals should be carefully observed at least once daily with respect to their health condition as well
as at least twice daily for morbidity and mortality.

1.6.1.2

Detailed clinical observations

Detailed clinical observations should be made on all animals selected for this purpose (see Table 1) once
before the first exposure (to allow for within-subject comparisons) and at different intervals thereafter,
dependant on the duration of the study (see Table 2). Detailed clinical observations on satellite recovery
groups should be made at the end of the recovery period. Detailed clinical observations should be made
outside the home cage in a standard arena. They should be carefully recorded using scoring systems that
include criteria or scoring scales for each measurement in the observations. The criteria or scales used
should be explicitly defined by the testing laboratory. Effort should be made to ensure that variations in
(not systematically related to treatment) and that observations are
the test conditions are minimal
conducted by trained observers unaware of the actual treatment.

It is recommended that the observations be carried out in a structured fashion in which well-defined
criteria (including the definition of the normal “range”) are systematically applied to each animal at each
observation time. The “normal range” should be adequately documented. All observed signs should be
recorded. Whenever feasible, the magnitude of the observed signs should also be recorded. Clinical
observations should include, but not be limited to, changes in skin, fur, eyes, mucous membranes,
occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size,
unusual respiratory pattern and/or mouth breathing, any unusual signs of urination or defecation, and
discoloured urine).

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to body position, activity level

increased
Any unusual responses with respect
exploration of the standard arena) and co-ordination of movement should also be noted. Changes in gait
(e.g., waddling, ataxia), posture (e.g., hunched-back) and reactivity to handling, placing or other
environmental stimuli, as well as the presence of clonic or tonic movements, convulsions or tremors,
stereotypes (e.g., excessive grooming, unusual head movements, repetitive circling) or bizarre behaviour
(e.g., biting or excessive licking, self mutilation, walking backwards, vocalization) or aggression should be
recorded.

(e.g., decreased or

1.6.1.3

Functional tests

Similar to the detailed clinical observations, functional tests should also be conducted once prior to
exposure and frequently thereafter in all animals selected for this purpose (see Table 1). The frequency of
functional testing is also dependent on the study duration (see Table 2). In addition to the observation
periods as set out in Table 2, functional observations on satellite recovery groups should also be made as
close as possible to the terminal kill. Functional tests should include sensory reactivity to stimuli of
different modalities [e.g., auditory, visual and proprioceptive stimuli (5)(6)(7)], assessment of limb grip
strength (8) and assessment of motor activity (9). Motor activity should be measured with an automated
device capable of detecting both decreases and increases in activity. If another defined system is used it
should be quantitative and its sensitivity and reliability should be demonstrated. Each device should be
tested to ensure reliability across time and consistency between devices. Further details of the procedures
that can be followed are given in the respective references. If there are no data (e.g. structure-activity,
epidemiological data, other toxicology studies) to indicate the potential neurotoxic effects, the inclusion of
more specialized tests of sensory and motor function or learning and memory to examine these possible
effects in greater details should be considered. More information on more specialized tests and their use is
provided in (1).

Exceptionally, animals that reveal signs of toxicity to an extent that would significantly interfere with the
functional test may be omitted from that test. Justification for the elimination of animals from a functional
test should be provided.

1.6.2

Body weight and food/water consumption

For studies up to 90 days duration, all animals should be weighed at least once a week and measurements
should be made of food consumption (water consumption, when the test substance is administered by that
medium) at least weekly. For long term studies, all animals should be weighed at least once at week for the
first 13 weeks and at least once every 4 weeks thereafter. Measurements should be made of food
consumption (water consumption, when the test substance is administered by that medium) at least
weekly for the first 13 weeks and then at approximately three-month intervals unless the health status or
body weight changes dictate otherwise.

1.6.3

Ophthalmology

For studies longer than 28 days duration, ophthalmologic examination, using an ophthalmoscope or an
equivalent suitable instrument, should be made prior to the administration of the test substance and at the
termination of the study, preferably on all animals, but at least on animals in the high dose and control
groups. If changes in the eyes are detected or, if clinical signs indicate the need, all animals should be
examined. For long term studies, an ophthalmologic examination should also be carried out at 13 weeks.
Ophthalmologic examinations need not to be conducted if this data is already available from others
studies of similar duration and at similar dose levels.

1.6.4

Haematology and clinical biochemistry

When the neurotoxicity study is carried out in combination with a repeated dose systemic toxicity study,
haematological examinations and clinical biochemistry determinations should be carried out as set out in
the respective Method of the systemic toxicity study. Collection of samples should be carried out in such a
way that any potential effects on neurobehaviour are minimized.

1.6.5

Histopathology

The neuropathological examination should be designed to complement and extend the observations made
during the in vivo phase of the study. Tissues from at least 5 animals/sex/group (see Table 1 and next
paragraph) should be fixed in situ, using generally recognized perfusion and fixation techniques (see

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the remainder of

the animals may be used either

reference 3, chapter 5 and reference 4, chapter 50). Any observable gross changes should be recorded.
When the study is conducted as a stand-alone study screen for neurotoxicity or to characterize neurotoxic
effects,
(10)(11),
neuropathological (10)(11)(12)(13), neurochemical (10)(11)(14)(15) or electrophysiological (10)(11)(16)(17)
procedures that may supplement the procedures and examinations described here, or to increase the
number of subjects examined for histophatology. These supplementary procedures are of particular use
when empirical observations or anticipated effects indicate a specific type or target of neurotoxicity (2)(3).
Alternatively, the remainder of the animals can also be used for routine pathological evaluations as
described in Method for repeated dose studies.

for specific neurobehavioural

A general staining procedure, such as haematoxylin and eosin (H&E), should be performed on all tissue
specimens embedded in paraffin and microscopic examination should be carried out. If signs of peripheral
neuropathy are observed or suspected, plastic-embedded samples of peripheral nerve tissue should be
examined. Clinical signs may also suggest additional sites for examination or the use of special staining
procedures. Guidance on additional sites to be examined can be found in (3)(4). Appropriate special stains
to demonstrate specific types of pathological change may also be helpful (18).

Representative sections of the central and peripheral nervous system should be examined histologically
(see reference 3, chapter 5 and reference 4, chapter 50). The areas examined should normally include: the
forebrain, the centre of the cerebrum, including a section through the hippocampus, the midbrain, the
cerebellum, the pons, the medulla oblongata, the eye with optic nerve and retina, the spinal cord at the
cervical and lumbar swellings, the dorsal root ganglia, the dorsal and ventral root fibres, the proximal
sciatic nerve, the proximal tibial nerve (at the knee) and the tibial nerve calf muscle branches. The spinal
cord and peripheral nerve sections should include both cross or transverse and longitudinal sections.
Attention should be given to the vasculature of
the nervous system. A sample of skeletal muscle,
particularly calf muscle, should also be examined. Special attention should be paid to sites with cellular
and fibre structure and pattern in the CNS and PNS known to be particularly affected by neurotoxicants.

Guidance on neurophatological alterations that typically result from toxicant exposure can be found in the
references (3)(4). A stepwise examination of tissue samples is recommended in which sections from the
high dose group are first compared with those of the control group. If no neurophatological alterations
are observed in the samples from these groups, subsequent analysis is not required. If neuropathological
alterations are observed in the high dose group, sample from each of the potentially affected tissues from
the intermediate and low dose groups should then be coded and examined sequentially.

If any evidence of neuropathological alterations is found in the qualitative examination, then a second
examination should be performed on all regions of the nervous system showing these alterations. Sections
from all dose groups from each of the potentially affected regions should be coded and examined at
random without knowledge of the code. The frequency and severity of each lesion should be recorded.
After all regions from all dose groups have been rated, the code can be broken and statistical analysis
performed to evaluate dose-response relationships. Examples of different degrees of severity of each lesion
should be described.

The neuropathological
findings should be evaluated in the context of behavioural observations and
measurements, as well as other data from preceding and concurrent systemic toxicity studies of the test
substance.

2.

DATA

2.1

TREATMENT OF RESULTS

Individual data should be provided. Additionally, all data should be summarized in tabular form showing
for each test or control group the number of animals at the start of the test, the number of animals found
dead during the test or killed for humane reasons and the time of any death or humane kill, the number
showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration,
type and severity of any toxic effects, the number of animals showing lesions, including the type and
severity of the lesion(s).

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2.2

EVALUATION AND INTERPRETATION OF RESULTS

The findings of the study should be evaluated in terms of the incidence, severity and correlation of
neurobehavioural and neuropathological effects (neurochemical or electrophysiological effects as well if
supplementary examinations are included) and any other adverse effects observed. When possible,
numerical results should be evaluated by an appropriate and generally acceptable statistical method. The
statistical methods should be selected during the design of the study.

3.

3.1

REPORTING

TEST REPORT

The test report must include the following information:

Test substance:

— physical nature (including isomerism, purity and physicochemical properties);

— identification data.

Vehicle (if appropriate):

— justification for choice of vehicle.

Test animals:

— species/strain used;

— number, age and sex of animals;

— source, housing conditions, acclimatization, diet, etc;

— individual weights of animals at the start of the test.

Test conditions:

— details of

test substance formulation/diet preparation, achieved concentration, stability and

homogeneity of the preparation:

— specification of the doses administered, including details of the vehicle, volume and physical form

of the material administered;

— details of the administration of the test substance;

— rationale for dose levels selected;

— rationale for the route and duration of the exposure;

— conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg

body weight/day), if applicable;

— details of the food and water quality.

Observation and Test Procedures:

— details of the assignment of animals in each group to the perfusion subgroups;

— details of scoring systems,

including criteria and scoring scales for each measurement in the

detailed clinical observations;

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— details on the functional tests for sensory reactivity to stimuli of different modalities (e.g.,
auditory, visual and proprioceptive); for assessment of
limb grip strength; for motor activity
assessment (including details of automated devices for detecting activity); and other procedures
used;

— details of ophthalmologic examinations and,

if appropriate, haematological examinations and

clinical biochemistry tests with relevant base-line values;

— details for specific neurobehavioural, neuropathological, neurochemical or electrophysiological

procedures.

Results:

— body weight/body weight changes including body weight at kill;

— food consumption and water consumption, as appropriate;

— toxic response data by sex and dose level, including signs of toxicity or mortality;

— nature, severity and duration (time of onset and subsequent course) of the detailed clinical

observations (whether reversible or not);

— a detailed description of all functional test results;

— necropsy findings;

— a detailed description of all neurobehavioural, neuropathological, and neurochemical or

electrophysiological findings, if available;

— absorption and metabolism data, if available;

— statistical treatment of results, where appropriate.

Discussion of results;

— dose response information;

— relationship of any other toxic effects to a conclusion about the neurotoxic potential of the test

chemical;

— no-observed-adverse effect level.

Conclusions:

— a specific statement of the overall neurotoxicity of the test chemical is encouraged.

4.

REFERENCES

(1) OECD Giudance Document on Neurotoxicity Testing Strategies and Test Methods. OECD, Paris, In

Preparation.

(2)

Test Guideline for a Developmental Neurotoxicity Study, OECD Guidelines for the Testing of
Chemicals. In preparation.

(3) World Health Organization (WHO) (1986). Environmental Health Criteria document 60: Principles

and Methods for the Assessment of Neurotoxicity associated with Exposure to Chemicals.

(4)

(5)

Spencer, P.S. and Schaumburg, H.H. (1980). Experimental and Clinical Neurotoxicology. Eds. Spencer,
P.S. and Schaumburg, H.H. eds. Williams and Wilkins, Baltimore/ London.

Tupper, D.E. and Wallace, R.B.
Neurobiol. Exp., 40, 999-1003.

(1980). Utility of

the Neurological Examination in Rats. Acta

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(6) Gad, S.C. (1982). A Neuromuscular Screen for Use in Industrial Toxicology. J. Toxicol. Environ.

Health, 9, 691-704.

(7) Moser, V.C., McDaniel, K.M. and Phillips, P.M. (1991). Rat Strain and Stock Comparisons Using a
Functional Observational Battery: Baseline Values and Effects of amitraz. Toxic. Appl. Pharmacol.,
108, 267-283.

(8) Meyer, O.A., Tilson, H.A., Byrd, W.C. and Riley, M.T. (1979). A Method for the Routine Assessment of

Fore- and Hind- limb Grip Strength of Rats and Mice. Neurobehav. Toxicol., 1, 233-236.

(9) Crofton, K.M., Haward, J.L., Moser, V.C., Gill, M.W., Reirer, L.W., Tilson, H.A. and MacPhail, R.C.
for

(1991)
Comparison
Neurotoxicological Assessments. Neurotoxicol. Teratol., 13, 599-609.

of Motor Activity

Interlaboratory

Experiments:

Implication

(10) Tilson, H.A., and Mitchell, C.L. eds. (1992). Neurotoxicology Target Organ Toxicology Series. Raven

Press, New York.

(11) Chang, L.W., ed. (1995). Principles of Neurotoxicology. Marcel Dekker, New York.

(12) Broxup, B. (1991). Neuopathology as a screen for Neurotoxicity Assessment. J. Amer. Coll. Toxicol.,

10, 689-695.

(13) Moser, V.C., Anthony, D.C., Sette, W.F. and MacPhail, R.C.

(1992). Comparison of Subchronic
Neurotoxicity of 2-Hydroxyethyl Acrylate and Acrylamide in Rats. Fund. Appl.Toxicol., 18, 343-352.

(14) O'Callaghan, J.P. (1988). Neurotypic and Gliotypic Proteins as Biochemical Markers of Neurotoxicity.

Eurotoxicol. Teratol., 10, 445-452.

(15) O'Callaghan J.P. and Miller, D.B. (1988). Acute Exposure of the Neonatal Rat to Triethyltin Results in
Persistent Changes in Neurotypic and Gliotypic Proteins. J. Pharmacol. Exp. Ther., 244, 368-378.

(16)

(17)

Fox. D.A., Lowndes, H.E. and Birkamper, G.G.
in
Neurotoxicology. In: Nervous System Toxicology. Mitchell, C.L. ed. Raven Press, New York, pp 299-
335.

(1982). Electrophysiological Techniques

Johnson, B.L. (1980). Electrophysiological Methods in neurotoxicity Testing. In: Experimental and
Clinical Neurotoxicology. Spencer, P.S. and Schaumburg, H.H. eds., Williams and Wilkins Co.,
Baltimore/London, pp. 726-742.

(18) Bancroft, J.D. and Steven A. (1990). Theory and Pratice of Histological Techniques. Chapter 17,

Neuropathological Techniques. Lowe, James and Cox, Gordon eds. Churchill Livingstone.

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Minimum numbers of animals needed per group when the neurotoxicity study is conducted separately or in combination whit studies

Table 1

NEUROTOXICITY STUDY CONDUCTED AS:

Separate study

Combined study with the
28-day study

Combined study with the
90-day study

Combined study with the
chronic toxicity study

Total number of animals per group

10 males and 10 females 10 males and 10 females

15 males and 15 females

25 males and 25 females

Number of animals selected for
functional testing including detailed
clinical observations

Number of animals selected per
perfusion in situ and
neurohistopathology

Number of animals selected for
repeated dose/subchronic/chronic
toxicity observations, haematology,
clinical biochemistry,
histopathology, etc. as indicate in
the respective Guidelines

10 males and 10 females 10 males and 10 females

10 males and 10 females

10 males and 10 females

5 males and 5 females

5 males and 5 females

5 males and 5 females

5 males and 5 females

5 males and 5 females

10 males †
10 females

and
†

20 males †
20 females

and
†

Supplemental observations, as
appropriate

5 males and 5 females

† Includes five animals selected for functional testing and detailed clinical observations as part of the neurotoxicity study

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Table 2

Frequency of clinical observation and functional tests

Type of observations

Study duration

Acute

28-day

90-day

Chronic

In all animals

General health
condition

daily

daily

daily

daily

Mortality/morbidity

Twice daily

Twice daily

Twice daily

Twice daily

In animals selected for
functional observations

Detailed clinical
observations

— prior to first
exposure
— once weekly
thereafter

— prior to first
exposure

— within 8 hours of
dosing at estimate
time of peak effect

— at day 7 and 14
after dosing

— prior to first
exposure

— prior to first
exposure

— once during the
first or second
week of exposure
— monthly thereafter

— once at the end of
the first month of
exposure

— every three months

thereafter

Functional tests

— prior to first
exposure

— prior to first
exposure

— prior to first
exposure

— prior to first
exposure

— within 8 hours of
dosing at estimate
time of peak effect

— at day 7 and 14
after dosing

— during the fourth
week of treatment
as close as possible
to the end of the
exposure period

— once during the
first or second
week of exposure
— monthly thereafter

— once at the end of
the first month of
exposure

— every three months

thereafter’

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ANNEX 2I

‘C.21. SOIL MICROORGANISMS: NITROGEN TRANSFORMATION TEST

1.

METHOD

This test method is a replicate of OECD TG 216 (2000).

1.1

INTRODUCTION

This Testing method describes a laboratory method designed to investigate the long-term effects of
chemicals, after a single exposure, on nitrogen transformation activity of soil microorganisms. The test is
principally based on the recommendations of
the European and Mediterranean Plant Protection
Organization (1). However, other guideline, including those of the German Biologische Bundesanstalt (2),
the US Environmental Protection Agency (3) SETAC (4) and the International Organization for
Standardization (5), were also taken into account. An OECD Workshop on soil/sediment Selection held at
Belgirate, Italy, in 1995 (6) agreed on the number and type of soils for use in this test. Recommendations
for collection, handling and storage of soil sample are based on an ISO Guidance Document (7) and
recommendations from the Belgirate Workshop. In the assessment and evaluation of toxic characteristics
of test substances, determination of effects on soil microbial activity may be required, e.g. when data on
the potential side effects of crop protection products on soil microflora are required or when exposure of
than crop protection products is expected. The nitrogen
soil microorganisms to chemicals other
transformation test is carried out to determine the effects of such chemicals on soil microflora.
If
forestry chemicals) are tested, both nitrogen
agrochemicals (e.g. crop protection products,
If non agrochemicals are tested, the
transformation and carbon transformation tests are conducted.
nitrogen transformation test is sufficient. However, if EC50 values of the nitrogen transformation test for
such chemicals fall within the range found for commercially available nitrification inhibitors (e.g.
nitrapyrin), a carbon transformation test can be conducted to gain further information.

fertilisers,

Soils consist of living and non-living components which exist in complex and heterogeneous mixtures.
Microorganisms play an important role in break-down and transformation of organic matter in fertile soils
with many species contributing to different aspects of soil fertility. Any long-term interference with these
biochemical processes could potentially interfere with nutrient cycling and this could alter soil fertility.
Transformation of carbon and nitrogen occurs in all fertile soils. Although the microbial communities
responsible for these processes differ from soil to soil, the pathways of transformation are essentially the
same.

This Testing method described is designed to detect long-term adverse effects of a substance on the
process of nitrogen transformation in aerobic surface soils. The test method also allows estimation of the
effects of substances on carbon transformation by the soil microflora. Nitrate formation takes place
subsequent to the degradation of carbon-nitrogen bonds. Therefore, if equal rates of nitrate production are
found in treated and control soils, it is highly probable that the major carbon degradation pathways are
intact and functional. The substrate chosen for the test (powdered lucerne meal) has a favourable carbon
to nitrogen ratio (usually between 12/1 and 16/1). Because of this, carbon starvation is reduced during the
test and if microbial communities are damaged by a chemical, they might recover within 100 days.

The tests from which this Testing Method was developed were primarily designed for substances for which
the amount reaching the soil can be anticipated. This is the case, for example, for crop protection products
for which the application rate in the field is known. For agrochemicals, testing of two doses relevant to the
anticipated or predicted application rate is sufficient. Agrochemicals can be tested as active ingredients (a.i.)
or as formulated products. However, the test is not limited to agrochemicals. By changing both the
amounts of test substance applied to the soil, and the way in which the data are evaluated, the test can
also be used for chemicals for which the amount expected to reach the soil is not known. Thus, with
chemicals other than agrochemicals, the effects of a series of concentrations on nitrogen transformation
are determined. The data from these tests are used to prepare a dose-response curve and calculate ECx
values, where x is defined % effect.

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1.2

DEFINITIONS

Nitrogen transformation: is the ultimate degradation by microorganisms of nitrogen-containing organic
matter, via the process of ammonification and nitrification, to the respective inorganic end-product nitrate.

ECx (Effective Concentration):
percent inhibition of nitrogen transformation to nitrate.

is the concentration of the test substance in soil that results in a x

EC50 (Median Effective Concentration): is the concentration of the test substance in soil that results in a
50 percent (50 %) inhibition of nitrogen transformation to nitrate.

1.3

REFERENCE SUBSTANCES

None.

1.4

Principle of the test method

is amended with powdered plant meal and either treated with the test substance or left
Sieved soil
untreated (control). If agrochemicals are tested, a minimum of two test concentrations are recommended
and these should be chosen in relation to the highest concentration anticipated in the field. After 0, 7, 14
days and 28 days of incubation, samples of treated and control soils are extracted with an appropriate
solvent, and the quantities of nitrate in the extracts are determined. The rate of nitrate formation in treated
samples is compared with the rate in the controls, and the percent deviation of the treated from the
control is calculated. All tests run for at least 28 days. If, on the 28th day, differences between treated and
untreated soils are equal to or greater than 25 %, measurements are continued to a maximum of 100 days.
If non agrochemicals are tested, a series of concentrations of the test substance are added to samples of
the soil, and the quantities of nitrate formed in treated and control samples are measured after 28 days of
incubation. Results from tests with multiple concentrations are analysed using a regression model, and the
ECx values are calculated (i.e. EC50, EC25 and/or EC10). See definitions.

1.5

VALIDITY OF THE TEST

Evaluations of test results with agrochemicals are based on relatively small differences (i.e. average value
±25 %) between nitrate concentrations in control and treated soil samples, so large variations in the
controls can lead to false results. Therefore, the variation between replicate control samples should be less
than ±15 %.

1.6

DESCRIPTION OF THE TEST METHOD

1.6.1

Apparatus

Test containers made of chemically inert material are used. They should be of a suitable capacity in
compliance with the procedure used for incubation of soils,
incubation in bulk or as a series of
individual soil samples (see section 1.7.1.2). Care should be taken both to minimise water loss and to allow
gas exchange during the test (e.g. the test containers may be covered with perforated polyethylene foil).
When volatile substances are tested, sealable and gas-tight containers should be used. These should be of a
size such that approximately one quarter of their volume is filled with the soil sample.

i.e.

Standard laboratory equipment including the following is used:

— agitation device: mechanical shaker or equivalent equipment;

— centrifuge (3 000 g) or filtration device (using nitrate-free filter paper);

— instrument of adequate sensitivity and reproducibility for nitrate analysis.

1.6.2

Selection and number of soils

One single soil is used. The recommended soil characteristics are as follows:

— sand content: not less than 50 % and not greater than 75 %;

— pH: 5,5-7,5;

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— organic carbon content: 0.5 - 1.5 %;

— the microbial biomass should be measured (8)(9) and its carbon content should be at least 1 % of the

total soil organic carbon.

In most cases, a soil with these characteristics represents a worst case situation, since adsorption of the test
chemical is minimum and its availability to the microflora is maximum. Consequently, tests with other
soils are generally unnecessary. However, in certain circumstances, e.g. where the anticipated major use of
the test substance is in particular soils such as acidic forest soils, or for electrostatically charged chemicals,
it may be necessary to use an additional soil.

1.6.3

Collection and storage of soil samples

1.6.3.1

Collection

is collected should be
Detailed information on the history of the field site from where the test soil
available. Details include exact
treatments with crop protection
location, vegetation cover, dates of
products, treatments with organic and inorganic fertilisers, additions of biological materials or accidental
contaminations. The site chosen for soil collection should be one which allows long-term use. Permanent
pastures, fields with annual cereal crops (except maize) or densely sown green manures are suitable. The
selected sampling site should not have been treated with crop protection products for a minimum of one
year before sampling. Also, no organic fertiliser should have been applied for at least six months. The use
of mineral fertiliser is only acceptable when in accordance with the requirements of the crop and soil
samples should not be taken until at least three months after fertiliser application. The use of soil treated
with fertilisers with known biocidal effects (e.g. calcium cyanamide) should be avoided.

Sampling should be avoided during or immediately following long periods (greater than 30 days) of
drought or water logging. For ploughed soils, samples should be taken from a depth of 0 down to 20 cm.
For grassland (pasture) or other soils where ploughing does not occur over longer periods (at least one
growing season), the maximum depth of sampling may be slightly more than 20 cm (e.g. to 25 cm).

Soil samples should be transported using containers and under temperature conditions which guarantee
that the initial soil properties are not significantly altered.

1.6.3.2

Storage

The use of soils freshly collected from the field is preferred. If storage in the laboratory cannot be avoided,
soils may be stored in the dark at 4±2°C for a maximum of three months. During the storage of soils,
aerobic conditions must be ensured. If soils are collected from areas where they are frozen for at least
three months per year, storage for six months at minus 18°C to minus 22°C can be considered. The
microbial biomass of stored soils is measured prior to each experiment and the carbon in the biomass
should be at least 1 % of the total soil organic carbon content (see section 1.6.2).

1.6.4

Handling and preparation of soil for the test

1.6.4.1

Pre-incubation

If the soil was stored (see section 1.6.3.2), pre-incubation is recommended for a period between 2 and 28
days. The temperature and moisture content of the soil during pre-incubation should be similar to that
used in the test (see sections 1.6.4.2 and 1.7.1.3).

1.6.4.2

Physical-chemical characteristics

The soil
is manually cleared of large objects (e.g. stones, parts of plants, etc.) and then moist sieved
without excess drying to a particle size less than or equal to 2 mm. The moisture content of the soil
sample should be adjusted with distilled or deionised water to a value between 40 % and 60 % of the
maximum water holding capacity.

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1.6.4.3

Amendment with organic substrate

The soil should be amended with a suitable organic substrate, e.g. powdered lucerne-grass-green meal
(main component: Medicago sativa) with a C/N ratio between 12/1 and 16/1. The recommended lucerne-
soil ratio is 5 g of lucerne per kilogram of soil (dry weight).

1.6.5

Preparation of the test substance for the application to soil

The test substance is normally applied using a carrier. The carrier can be water (for water soluble
substances) or an inert solid such as fine quartz sand (particle size: 0.1-0.5mm). Liquid carriers other than
water (e.g. organic solvents such as acetone, chloroform) should be avoided since they can damage the
microflora. If sand is used as a carrier, it can be coated with the test substance dissolved or suspended in
an appropriate solvent. In such cases, the solvent should be removed by evaporation before mixing with
the soil. For an optimum distribution of the test substance in soil, a ratio of 10 g of sand per kilogram of
soil (dry weight) is recommended. Control samples are treated with an equivalent amount of water and/or
quartz sand only.

losses during treatment should be avoided as far as possible and an
When testing volatile chemicals,
attempt should be made to ensure homogeneous distribution in the soil (e.g. the test substance should be
injected into the soil at several places).

1.6.6

Test concentrations

the lower concentration. The concentrations of

If agrochemicals are tested, at least two concentrations should be used. The lower concentration should
reflect at least the maximum amount expected to reach the soil under practical conditions whereas the
test
higher concentration should be a multiple of
substance added to soil are calculated assuming uniform incorporation to a depth of 5 cm and a soil bulk
density of 1.5. For agrochemicals that are applied directly to soil, or for chemicals for which the quantity
reaching the soil can be predicted, the test concentrations recommended are the maximum Predicted
Environmental Concentration (PEC) and five times that concentration. Substances that are expected to be
applied to soils several times in one season should be tested at concentrations derived from multiplying
the PEC by the maximum anticipated number of applications. The upper concentration tested, however,
should not exceed ten times the maximum single application rate. If non-agrochemicals are tested, a
geometric series of at least five concentrations is used. The concentrations tested should cover the range
needed to determine the ECx values.

1.7

PERFORMANCE OF THE TEST

1.7.1

Conditions of exposure

1.7.1.1

Treatment and control

If agrochemicals are tested, the soil is divided into three portions of equal weight. Two portions are mixed
with the carrier containing the product, and the other is mixed with the carrier without the product
(control). A minimum of three replicates for both treated and untreated soils is recommended. If non-
agrochemicals are tested, the soil is divided into six portions of equal weight. Five of the samples are
mixed with the carrier containing the test substance, and the sixth sample is mixed with the carrier
without the chemical. Three replicates for both treatments and control are recommended. Care should be
taken to ensure homogeneous distribution of the test substance in the treated soil samples. During mixing,
compacting or balling of the soil should be avoided.

1.7.1.2

Incubation of soil samples

Incubation of soil samples can be performed in two ways: as bulk samples of each treated and untreated
soil or as a series of individual and equally sized subsamples of each treated and untreated soil. However,
individual
when volatile substances are tested, the test should only be performed with a series of
subsamples. When soils are incubated in bulk,
large quantities of each treated and untreated soils are
prepared and subsamples to be analysed are taken as needed during the test. The amount initially prepared
for each treatment and control depends on the size of the subsamples, the number of replicates used for
analysis and the anticipated maximum number of sampling times. Soils incubated in bulk should be
thoroughly mixed before subsampling. When soils are incubated as a series of individual soil samples, each
treated and untreated bulk soil is divided into the required number of subsamples, and these are utilised as

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needed. In the experiments where more than two sampling times can be anticipated, enough subsamples
should be prepared to account for all replicates and all sampling times. At least three replicate samples of
the test soil should be incubated under aerobic conditions (see section 1.7.1.1). During all tests, appropriate
containers with sufficient headspace should be used to avoid development of anaerobic conditions. When
volatile substances are tested, the test should only be performed with a series of individual subsamples.

1.7.1.3

Test conditions and duration

The test is carried out in the dark at room temperature of 20±2°C. The moisture content of soil samples
should be maintained during the test between 40 % and 60 % of the maximum water holding capacity of
the soil (see section 1.6.4.2) with a range of ±5 %. Distilled, deionized water can be added as needed.

The minimum duration of tests is 28 days. If agrochemicals are tested, the rates of nitrate formation in
treated and control samples are compared. If these differ by more than 25 % on day 28, the test is
continued until a difference equal to or less than 25 % is obtained, or for a maximum of 100 days,
whichever is shorter. For non-agrochemicals, the test is terminated after 28 days. On day 28, the quantities
of nitrate in treated and control soil samples are determined and the ECx values are calculated.

1.7.2

Sampling and analysis of soils

1.7.2.1

Soil sampling schedule

If agrochemicals are tested, soil samples are analysed for nitrate on days 0, 7, 14 and 28. If a prolonged
test is required, further measurements should be made at 14 days intervals after day 28.

If non-agrochemicals are tested, at least five test concentrations are used and soil samples are analysed for
nitrate at the beginning (day 0) and at the end of the exposure period (28 days). An intermediate
measurement, e.g. at day 7, may be added if deemed necessary. The data obtained on day 28 are used to
determine ECx value for the chemical. If desired, data from day 0 control samples can be used to report
the initial quantity of nitrate in the soil.

1.7.2.2

Analysis of soil samples

The amount of nitrate formed in each treated and control replicate is determined at each sampling time.
Nitrate is extracted from soil by shaking samples with a suitable extraction solvent, e.g. a 0.1 M potassium
chloride solution. A ratio of 5 ml of KCl solution per gram dry weight equivalent of soil is recommended.
To optimise extraction, containers holding soil and extraction solution should not be more than half full.
The mixtures are shaken at 150 rpm for 60 minutes. The mixtures are centrifuged or filtered and the
liquid phases are analysed for nitrate. Particle-free liquid extracts can be stored prior to analysis at minus
20±5 °C for up to six months.

2.

2.1

DATA

TREATMENT OF RESULTS

If tests are conducted with agrochemicals, the quantity of nitrate formed in each replicate soil sample
should be recorded, and the mean values of all replicates should be provided in tabular form. Nitrogen
transformation rates should be evaluated by appropriate and generally acceptable statistical methods (e.g.
F-test, 5 % significance level). The quantities of nitrate formed are expressed in mg nitrate/kg dry weight
soil/day. The nitrate formation rate in each treatment is compared with that in the control, and the percent
deviation from the control is calculated.

tests are conducted with non-agrochemicals,

If
the quantity of nitrate formed in each replicate is
determined, and a dose-response curve is prepared for estimation of the ECx values. The quantities of
nitrate (i.e. mg nitrate/kg dry weight soil) found in the treated samples after 28 days are compared to that
found in the control. From these data, the % inhibition values for each test concentration are calculated.

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These percentages are plotted against concentration, and statistical procedures are then used to calculate
the ECx values. Confidence limits (p = 0.95) for the calculated ECx are also determined using standard
procedures (10)(11)(12).

Test substances that contain high quantities of nitrogen may contribute to the quantities of nitrate formed
during the test. If these substances are tested at a high concentration (e.g. chemicals which are expected to
be used in repeated applications) appropriate controls must be included in the test (i.e. soil plus test
substance but without plant meal). Data from these controls must be accounted for in the ECx calculations.

2.2

INTERPRETATION OF RESULTS

When results from tests with agrochemicals are evaluated, and the difference in the rates of nitrate
formation between the lower treatment (i.e. the maximum predicted concentration) and control is equal to
or less than 25 % at any sampling time after day 28, the product can be evaluated as having no long-term
influence on nitrogen transformation in soils. When results from tests with chemicals other than
agrochemicals are evaluated, the EC50, EC25 and/or EC10 values are used.

3.

REPORTING

The test report must include the following information:

Complete identification of the soil used including:

— geographical reference of the site (latitude, longitude);

— information on the history of the site (i.e. vegetation cover, treatments with crop protection

products, treatments with fertilisers, accidental contamination, etc.);

— use pattern (e.g. agricultural soil, forest, etc.);

— depth of sampling (cm);

— sand/silt/clay content (% dry weight);

— pH (in water);

— organic carbon content (% dry weight);

— nitrogen content (% dry weight);

— initial nitrate concentration (mg nitrate/kg dry weight);

— cation exchange capacity (mmol/kg);

— microbial biomass in terms of percentage of the total organic carbon;

— reference of the methods used for the determination of each parameter;

— all information relating to the collection and storage of soil samples;

— details of pre-incubation of soil if any.

Test substance:

— physical nature and, where relevant, physical-chemical properties;

— chemical

identification data, where relevant,

including structural formula, purity (i.e. for crop

protection products the percentage of active ingredient), nitrogen content.

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Substrate:

— source of substrate;

— composition (i.e. lucerne meal, lucerne-grass-green meal);

— carbon, nitrogen content (% dry weight);

— sieve size (mm).

Test conditions:

— details of the amendment of soil with organic substrate;

— number of concentrations of test chemical used and, where appropriate,

justification of the

selected concentrations;

— details of the application of test substance to soil;

— incubation temperature;

— soil moisture content at the beginning and during the test;

— method of soil incubation used (i.e. as bulk or as a series of individual subsamples);

— number of replicates;

— sampling times;

— method used for extraction of nitrate from soil;

Results:

— analytical procedure and equipment used to analyse nitrate;

— tabulated data including individual and mean values for nitrate measurements;

— variation between the replicates in treated and control samples;

— explanations of corrections made in the calculations, if relevant;

— the percent variation in nitrate formation rates at each sampling time or, if appropriate, the EC50
value with 95 per cent confidence limit, other ECx (i.e. EC25 or EC10) with confidence intervals,
and a graph of the dose-response curve;

— statistical treatment of results;

— all information and observations helpful for the interpretation of the results.

4.

REFERENCES

(1)

EPPO (1994). Decision-Making Scheme for the Environmental Risk Assessment of Plant Protection
Chemicals. Chapter 7: Soil Microflora. EPPO Bulletin 24: 1-16, 1994.

(2) BBA (1990). Effects on the Activity of the Soil Microflora. BBA Guidelines for the Official Testing of

Plant Protection Products, VI, 1-1 (2nd eds., 1990).

(3)

(4)

EPA (1987). Soil Microbial Community Toxicity Test. EPA 40 CFR Part 797.3700. Toxic Substances
Control Act Test Guidelines; Proposed rule. September 28, 1987.

SETAC-Europe (1995). Procedures for assessing the environmental fate and ecotoxicity of pesticides,
Ed. M.R. Lynch, Pub. SETAC-Europe, Bruxelles.

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(5)

ISO/DIS 14238 (1995). Soil Quality - Determination of Nitrogen Mineralisation and Nitrification in
Soils and the Influence of Chemicals on these Processes. Technical Committee ISO/TC 190/SC 4: Soil
Quality - Biological Methods.

(6) OECD (1995). Final Report of the OECD Workshop on Selection of Soils/Sediments, Belgirate, Italie,

18-20 janvier 1995.

(7)

(8)

(9)

ISO 10381-6 (1993). Soil quality - Sampling. Guidance on the collection, handling and storage of soil
for the assessment of aerobic microbial processes in the laboratory.

ISO 14240-1 (1997). Soil quality - Determination of soil microbial biomass - Part 1: Substrate-
induced respiration method.

ISO 14240-2 (1997). Soil quality - Determination of soil microbial biomass - Part 2: Fumigation-
extraction method.

(10) Litchfield, J.T. and Wilcoxon F. (1949). A simplified method of evaluating dose-effect experiments.

Jour. Pharmacol. and Exper. Ther., 96, 99-113.

(11) Finney, D.J. (1971). Probit Analysis. 3rd ed., Cambridge, London and New-York.

(12) Finney, D.J. (1978). Statistical Methods in biological Assay. Griffin, Weycombe, UK.

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C.22. SOIL MICROORGANISMS: CARBON TRANSFORMATION TEST

1.

METHOD

This method is a replicate of OECD TG 217 (2000).

1.1

INTRODUCTION

This Testing method describes a laboratory method designed to investigate long term potential effects of a
single exposure of crop protection products and possibly other chemicals on carbon transformation
activity of soil microorganisms. The test is principally based on the recommendations of the European and
Mediterranean Plant Protection Organization (1). However, other guideline, including those of the German
Biologische Bundesanstalt (2), the US Environmental Protection Agency (3) and SETAC (4), were also taken
into account. An OECD Workshop on Soil/Sediment Selection held at Belgirate, Italy, in 1995 (5) agreed
on the number and type of soils for use in this test. Recommendations for collection, handling and storage
of soil sample are based on an ISO Guidance Document (6) and recommendations from the Belgirate
Workshop.

In the assessment and evaluation of toxic characteristics of test substances, determination of effects on soil
microbial activity may be required, e.g. when data on the potential side effects of crop protection products
on soil microflora are required or when exposure of soil microorganisms to chemicals other than crop
protection products is expected. The carbon transformation test is carried out to determine the effects of
such chemicals on soil microflora. If agrochemicals (e.g. crop protection products, fertilisers, forestry
chemicals) are tested, both carbon transformation and nitrogen transformation tests are conducted. If non-
agrochemicals are tested, the nitrogen transformation test is sufficient. However,
if EC50 values of the
nitrogen transformation test for such chemicals fall within the range found for commercially available
nitrification inhibitors (e.g. nitrapyrin), a carbon transformation test can be conducted to gain further
information.

Soils consist of living and non-living components which exist in complex and heterogeneous mixtures.
Microorganisms play an important role in breakdown and transformation of organic matter in fertile soils
with many species contributing to different aspects of soil fertility. Any long-term interference with these
biochemical processes could potentially interfere with nutrient cycling and this could alter the soil fertility.
Transformation of carbon and nitrogen occurs in all fertile soils. Although the microbial communities
responsible for these processes differ from soil to soil, the pathways of transformation are essentially the
same.

This Testing Method is designed to detect long-term adverse effects of a substance on the process of
carbon transformation in aerobic surface soils. The test is sensitive to changes in size and activity of
microbial communities responsible for carbon transformation since it subjects these communities to both
chemical stress and carbon starvation. A sandy soil low in organic matter is used. This soil is treated with
the test substance and incubated under conditions that allow rapid microbial metabolism. Under these
conditions, sources of readily available carbon in the soil are rapidly depleted. This causes carbon
starvation which both kills microbial cells and induces dormancy and/or sporulation. If the test runs for
these reactions can be measured in (untreated soil) controls as a
more than 28 days, the sum of
progressive loss of metabolically active microbial biomass (7). If the biomass in carbon-stressed soil, under
the conditions of the test, is affected by the presence of a chemical, it may not return to the same level as
the control. Hence, disturbances caused by the test substance at any time during the test will often last
until the end of the test.

The tests from which this Testing Method was developed were primarily designed for substances for which
the amount reaching the soil can be anticipated. This is the case, for example, for crop protection products
for which the application rate in the field is known. For agrochemicals, testing of two doses relevant to the
anticipated or predicted application rate is sufficient. Agrochemicals can be tested as active ingredients (a.i.)
or as formulated products. However, the test is not limited to chemicals with predictable environmental
concentrations. By changing both the amounts of test substance applied to the soil, and the way in which
the data are evaluated, the test can also be used for chemicals for which the amount expected to reach the
soil
is not known. Thus, with non-agrochemicals, the effects of a series of concentrations on carbon
transformation are determined. The data from these tests are used to prepare a dose-response curve and
calculate ECx values, where x is defined % effect.

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1.2

DEFINITIONS

Carbon transformation: is the degradation by microorganisms of organic matter to form inorganic end-
product carbon dioxide.

ECx (Effective Concentration):
percent inhibition of carbon transformation in carbon dioxide.

is the concentration of the test substance in soil that results in a x

EC50 (Median Effective Concentration): is the concentration of test substance in soil that results in a 50
per cent inhibition of carbon transformation in carbon dioxide.

1.3

REFERENCE SUBSTANCES

None.

1.4

PRINCIPLE OF THE TEST METHOD

Sieved soil is either treated with the test substance or left untreated (control). If agrochemicals are tested, a
minimum of two test concentrations are recommended and these should be chosen in relation to the
highest concentration anticipated in the field. After 0, 7, 14 and 28 days incubation, samples of treated
and control soils are mixed with glucose, and glucose-induced respiration rates are measured for 12
consecutive hours. Respiration rates are expressed as carbon dioxide released (mg carbon dioxide/kg dry
soil/h) or oxygen consumed (mg oxygen/kg soil/h). The mean respiration rate in the treated soil samples is
compared with that in control and the percent deviation of the treated from the control is calculated. All
tests run for at least 28 days. If, on the 28th day, differences between treated and untreated soils are equal
to or greater than 25 % measurements are continued in 14 day intervals for a maximum of 100 days. If
chemicals other than agrochemicals are tested, a series of concentrations of the test substance are added to
samples of the soil, and glucose induced respiration rates (i.e. the mean of the quantities of carbon dioxide
formed or oxygen consumed) are measured after 28 days. Results from tests with a series of
concentrations are analysed using a regression model, and the ECx values are calculated (i.e. EC50, EC25 and/
or EC10). See definitions.

1.5

VALIDITY OF THE TEST

Evaluations of test results with agrochemicals are based on relatively small differences (i.e. average value
±25 %) between the carbon dioxide released or the oxygen consumed in (or by) control and treated soil
samples, so large variations in the controls can lead to false results. Therefore, the variation between
replicate control samples should be less than ±15 %.

1.6

DESCRIPTION OF THE TEST METHOD

1.6.1

Apparatus

Test containers made of chemically inert material are used. They should be of a suitable capacity in
compliance with the procedure used for incubation of soils,
incubation in bulk or as a series of
individual soil samples (see section 1.7.1.2). Care should be taken both to minimise water loss and to allow
gas exchange during the test (e.g. the test containers may be covered with perforated polyethylene foil).
When volatile substances are tested, sealable and gas-tight containers should be used. These should be of a
size such that approximately one quarter of their volume is filled with the soil sample.

i.e.

For determination of glucose-induced respiration, incubation systems and instruments for measurement of
carbon dioxide production or oxygen consumption are required. Examples of such systems and
instruments are found in the literature (8) (9) (10) (11).

1.6.2

Selection and number of soils

One single soil is used. The recommended soil characteristics are as follows:

— sand content: not less than 50 % and not greater than 75 %;

— pH: 5.5-7.5;

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— organic carbon content: 0.5-1.5 %;

— the microbial biomass should be measured (12)(13) and its carbon content should be at least 1 % of

the total soil organic carbon.

In most cases, a soil with these characteristics represents a worst case situation, since adsorption of the test
chemical is minimised and its availability to the microflora is maximum. Consequently, tests with other
soils are generally unnecessary. However, in certain circumstances, e.g. where the anticipated major use of
the test substance is in particular soils such as acidic forest soils, or for electrostatically charged chemicals,
it may be necessary to substitute an additional soil.

1.6.3

Collection and storage of soil samples

1.6.3.1

Collection

is collected should be
Detailed information on the history of the field site from where the test soil
available. Details include exact
treatments with crop protection
location, vegetation cover, dates of
products, treatments with organic and inorganic fertilisers, additions of biological materials or accidental
contaminations. The site chosen for soil collection should be one which allows long-term use. Permanent
pastures, fields with annual cereal crops (except maize) or densely sown green manures are suitable. The
selected sampling site should not have been treated with crop protection products for a minimum of one
year before sampling. Also, no organic fertiliser should have been applied for at least six months. The use
of mineral fertiliser is only acceptable when in accordance with the requirements of the crop and soil
samples should not be taken until at least three months after fertiliser application. The use of soil treated
with fertilisers with known biocidal effects (e.g. calcium cyanamide) should be avoided.

Sampling should be avoided during or immediately following long periods (greater than 30 days) of
drought or water logging. For ploughed soils, samples should be taken from a depth of 0 down to 20 cm.
For grassland (pasture) or other soils where ploughing does not occur over longer periods (at least one
growing season), the maximum depth of sampling may be slightly more than 20 cm (e.g. to 25 cm). Soil
samples should be transported using containers and under temperature conditions which guarantee that
the initial soil properties are not significantly altered.

1.6.3.2

Storage

The use of soils freshly collected from the field is preferred. If storage in the laboratory cannot be avoided,
soils may be stored in the dark at 4 ± 2 °C for a maximum of three months. During the storage of soils,
aerobic conditions must be ensured. If soils are collected from areas where they are frozen for at least
three months per year, storage for six months at minus 18 °C can be considered. The microbial biomass of
stored soils is measured prior to each experiment and the carbon in the biomass should be at least 1 % of
the total soil organic carbon content (see section 1.6.2).

1.6.4

Handling and preparation of soil for the test

1.6.4.1

Pre-incubation

If the soil was stored (see sections 1.6.4.2 and 1.7.1.3), pre-incubation is recommended for a period
between 2 and 28 days. The temperature and moisture content of the soil during pre-incubation should be
similar to that used in the test (see sections 1.6.4.2 and 1.7.1.3).

1.6.4.2

Physical-chemical characteristics

The soil
is manually cleared of large objects (e.g. stones, parts of plants, etc.) and then moist sieved
without excess drying to a particle size less than or equal to 2 mm. The moisture content of the soil
sample should be adjusted with distilled or deionised water to a value between 40 % and 60 % of the
maximum water holding capacity.

1.6.5

Preparation of the test substance for the application to soil

The test substance is normally applied using a carrier. The carrier can be water (for water soluble
substances) or an inert solid such as fine quartz sand (particle size: 0.1-0.5 mm). Liquid carriers other than
water (e.g. organic solvents such as acetone, chloroform) should be avoided since they can damage the

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microflora. If sand is used as a carrier, it can be coated with the test substance dissolved or suspended in
an appropriate solvent. In such cases, the solvent should be removed by evaporation before mixing with
the soil. For an optimum distribution of the test substance in soil, a ratio of 10 g of sand per kilogram of
soil (dry weight) is recommended. Control samples are treated with the equivalent amount of water and/or
quartz sand only.

When testing volatile chemicals, losses during treatment should be avoided and an attempt should be
made to ensure homogeneous distribution in the soil (e.g. the test substance should be injected into the
soil at several places).

1.6.6

Test concentrations

If crop protection products or other chemicals with predictable environmental concentrations are tested, at
least two concentrations should be used. The lower concentration should reflect at least the maximum
amount expected to reach the soil under practical conditions whereas the higher concentration should be a
multiple of the lower concentration. The concentrations of test substance added to soil are calculated
assuming uniform incorporation to a depth of 5 cm and a soil bulk density of 1.5. For agrochemicals that
are applied directly to soil, or for chemicals for which the quantity reaching the soil can be predicted, the
test concentrations recommended are the Predictable Environmental Concentration (PEC) and five times
that concentration. Substances that are expected to be applied to soils several times in one season should
be tested at concentrations derived from multiplying the PEC by the maximum anticipated number of
applications. The upper concentration tested, however, should not exceed ten times the maximum single
application rate.

If non-agrochemicals are tested, a geometric series of at
concentrations tested should cover the range needed to determine the ECx values.

least

five concentrations is used. The

1.7

PERFORMANCE OF THE TEST

1.7.1

Conditions of exposure

1.7.1.1

Treatment and control

If agrochemicals are tested, the soil is divided into three portions of equal weight. Two portions are mixed
with the carrier containing the product, and the other is mixed with the carrier without the product
(control). A minimum of three replicates for both treated and untreated soils is recommended. If non-
agrochemicals are tested, the soil is divided into six portions of equal weight. Five of the samples are
mixed with the carrier containing the test substance, and the sixth sample is mixed with the carrier
without the chemical. Three replicates for both treatments and control are recommended. Care should be
taken to ensure homogeneous distribution of the test substance in the treated soil samples. During mixing,
compacting or balling of the soil should be avoided.

1.7.1.2

Incubation of soil samples

Incubation of soil samples can be performed in two ways: as bulk samples of each treated and untreated
soil or as a series of individual and equally sized subsamples of each treated and untreated soil. However,
individual
when volatile substances are tested, the test should only be performed with a series of
subsamples. When soils are incubated in bulk,
large quantities of each treated and untreated soils are
prepared and subsamples to be analysed are taken as needed during the test. The amount initially prepared
for each treatment and control depends on the size of the subsamples, the number of replicates used for
analysis and the anticipated maximum number of sampling times. Soils incubated in bulk should be
thoroughly mixed before subsampling. When soils are incubated as a series of individual soil samples, each
treated and untreated bulk soil is divided into the required number of subsamples, and these are utilised as
needed. In the experiments where more than two sampling times can be anticipated, enough subsamples
should be prepared to account for all replicates and all sampling times. At least three replicate samples of
the test soil should be incubated under aerobic conditions (see section 1.7.1.1). During all tests, appropriate
containers with sufficient headspace should be used to avoid development of anaerobic conditions. When
volatile substances are tested, the test should only be performed with a series of individual subsamples.

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1.7.1.3

Test conditions and duration

The test is carried out in the dark at room temperature of 20±2°C. The moisture content of soil samples
should be maintained during the test between 40 % and 60 % of the maximum water holding capacity of
the soil (see section 1.6.4.2) with a range of ±5 %. Distilled, deionised water can be added as needed.

The minimum duration of tests is 28 days. If agrochemicals are tested, the quantities of carbon dioxide
released or oxygen consumed in treated and control samples are compared. If these differ by more than
25 % on day 28, the test is continued until a difference equal to or less than 25 % is obtained, or for a
maximum of 100 days, whichever is shorter. If non-agrochemicals are tested, the test is terminated after
28 days. On day 28, the quantities of carbon dioxide released or oxygen consumed in treated and control
soil samples are determined and the ECx values are calculated.

1.7.2

Sampling and analysis of soils

1.7.2.1

Soil sampling schedule

If agrochemicals are tested, soil samples are analysed for glucose-induced respiration rates on days 0, 7, 14
and 28. If a prolonged test is required, further measurements should be made at 14 days intervals after day
28.

If non-agrochemicals are tested, at least five test concentrations are used and soil samples are analysed for
glucose-induced respiration at the beginning (day 0) and at the end of the exposure period (28 days). An
intermediate measurement, e.g. at day 7, may be added if deemed necessary. The data obtained on day 28
are used to determine ECx value for the chemical. If desired, data from day 0 control samples can be used
to estimate the initial quantities of metabolically active microbial biomass in the soil (12).

1.7.2.2

Measurement of glucose-induced respiration rates

The glucose-induced respiration rate in each treated and control replicate is determined at each sampling
time. The soil samples are mixed with a sufficient amount of glucose to elicit an immediate maximum
respiratory response. The amount of glucose needed to elicit a maximum respiratory response from a
given soil can be determined in a preliminary test using a series of concentrations of glucose (14).
However, for sandy soils with 0.5-1.5 % organic carbon, 2 000 mg to 2 000 mg glucose per kg dry
weight soil is usually sufficient. The glucose can be ground to a powder with clean quartz sand (10 g sand/
kg dry weight soil) and homogeneously mixed with the soil.

The glucose amended soil samples are incubated in a suitable apparatus for measurement of respiration
rates either continuously, every hour, or every two hours (see section 1.6.1) at 20 ± 2 °C. The carbon
dioxide released or the oxygen consumed is measured for 12 consecutive hours and measurements should
start as soon as possible, i.e. within 1 to 2 hours after glucose supplement. The total quantities of carbon
dioxide released or oxygen consumed during the 12 hours are measured and mean respiration rates are
determined.

2.

2.1

DATA

TREATMENT OF RESULTS

If agrochemicals are tested, the carbon dioxide released from, or oxygen consumed by each replicate soil
sample should be recorded, and the mean values of all replicates should be provided in tabular form.
Results should be evaluated by appropriate and generally acceptable statistical methods (e.g. F-test, 5 %
significance level). Glucose-induced respiration rates are expressed in mg carbon dioxide/kg dry weight
soil/h or mg oxygen/dry weight soil/h. The mean carbon dioxide formation rate or mean oxygen
consumption rate in each treatment is compared with that in control, and the percent deviation from the
control is calculated.

tests are conducted with non-agrochemicals, the quantities of carbon dioxide released or oxygen
If
consumed by each replicate is determined, and a dose-response curve is prepared for estimation of the ECx

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values. The glucose-induced respiration rates (i.e. mg carbon dioxide/kg dry weight soil/h or mg oxygen/
dry weight soil/h) found in the treated samples after 28 days are compared to that found in control. From
these data, the % inhibition values for each test concentration are calculated. These percentages are plotted
against concentration, and statistical procedures are used to calculate the ECx values. Confidence limits (p =
0.95) for the calculated ECx are also determined using standard procedures (15)(16)(17).

2.2

INTERPRETATION OF RESULTS

When results from tests with agrochemicals are evaluated, and the difference in respiration rates between
the lower treatment (i.e. the maximum predicted concentration) and control is equal to or less than 25 %
at any sampling time after day 28, the product can be evaluated as having no long-term influence on
carbon transformation in soils. When results from tests with chemicals other than agrochemicals are
evaluated, the EC50, EC25 and/or EC10 values are used.

3.

3.1

REPORTING

TEST REPORT

The test report must include the following information:

Complete identification of the soil used including:

— geographical reference of the site (latitude, longitude);

— information on the history of the site (i.e. vegetation cover, treatments with crop protection

products, treatments with fertilisers, accidental contamination, etc.)

— use pattern (e.g. agricultural soil, forest, etc.);

— depth of sampling (cm);

— sand/silt/clay content (% dry weight);

— pH (in water);

— organic carbon content (% dry weight);

— nitrogen content (% dry weight);

— cation exchange capacity (mmol/kg);

— initial microbial biomass in terms of percentage of the total organic carbon;

— reference of the methods used for the determination of each parameter;

— all information relating to the collection and storage of soil samples;

— details of pre-incubation of soil if any.

Test substance:

— physical nature and, where relevant, physical-chemical properties;

— chemical

identification data, where relevant,

including structural formula, purity (i.e. for crop

protection products the percentage of active ingredient), nitrogen content.

Test conditions:

— details of the amendment of soil with organic substrate;

— number of concentrations of test chemical used and, where appropriate,

justification of the

selected concentrations;

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— details of the application of test substance to soil;

— incubation temperature;

— soil moisture content at the beginning and during the test;

— method of soil incubation used (i.e. as bulk or as a series of individual subsamples);

— number of replicates;

— sampling times.

Results:

— method and equipment used for measurement of respiration rates;

— tabulated data including individual and mean values for quantities of carbon dioxide or oxygen;

— variation between the replicates in treated and control samples;

— explanations of corrections made in the calculations, if relevant;

— the percent variation of glucose-induced respiration rates at each sampling time or, if appropriate,
the EC50 with 95 per cent confidence limit, other ECx (i.e. EC25 or EC10) with confidence intervals,
and a graph of the dose-response curve;

— statistical treatment of results, where appropriate;

— all information and observations helpful for the interpretation of the results.

4.

REFERENCES

(1)

EPPO (1994). Decision-Making Scheme for the Environmental Risk Assessment of Plant Protection
Chemicals. Chapter 7: Soil Microflora. EPPO Bulletin 24: 1-16, 1994.

(2) BBA (1990). Effects on the Activity of the Soil Microflora. BBA Guidelines for the Official Testing of

Plant Protection Products, VI, 1-1 (2nd eds., 1990).

(3)

(4)

EPA (1987). Soil Microbial Community Toxicity Test. EPA 40 CFR Part 797.3700. Toxic Substances
Control Act Test Guidelines; Proposed rule. September 28, 1987.

SETAC-Europe (1995). Procedures for assessing the environmental fate and ecotoxicity of pesticides,
Ed. M.R. Lynch, Pub. SETAC-Europe, Brussels.

(5) OECD (1995). Final Report of the OECD Workshop on Selection of Soils/Sediments, Belgirate, Italy,

18-20 January 1995.

(6)

ISO 10381-6 (1993). Soil quality - Sampling. Guidance on the collection, handling and storage of soil
for the assessment of aerobic microbial processes in the laboratory.

(7) Anderson, J.P.E. (1987). Handling and Storage of Soils for Pesticide Experiments, in “Pesticide Effects

on Soil Microflora„. Eds. L. Somerville and M.P. Greaves, Chap. 3: 45-60.

(8) Anderson,

J.P.E.

in “Methods of Soil Analysis - Part 2: Chemical and
Microbiological Properties„. Agronomy Monograph No 9. Eds. A.L. Page, R.H. Miller and D.R.
Keeney. 41: 831- 871.

(1982). Soil Respiration,

(9)

ISO 11266-1. (1993). Soil Quality - Guidance on Laboratory Tests for Biodegradation in Soil: Part 1.
Aerobic Conditions.

(10) ISO 14239 (1997E). Soil Quality - Laboratory incubation systems for measuring the mineralization of

organic chemicals in soil under aerobic conditions.

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(11) Heinemeye,r O.,

(1989). Soil microbial biomass and
respiration measurements; an automated technique based on infrared gas analyses. Plant and Soil,
116: 77-81.

Insam, H., Kaiser, E.A, and Walenzik, G.

(12)

ISO 14240-1 (1997). Soil quality - Determination of soil microbial biomass - Part 1: Substrate-
induced respiration method.

(13)

ISO 14240-2 (1997). Soil quality - Determination of soil microbial biomass - Part 2: Fumigation-
extraction method.

(14) Malkomes, H.-P. (1986). Einfluß von Glukosemenge auf die Reaktion der Kurzzeit-Atmung im Boden
Gegenüber Pflanzenschutzmitteln, Dargestellt am Beispiel eines Herbizide. (Influence of the Amount
of Glucose Added to the Soil on the Effect of Pesticides in Short-Term Respiration, using a Herbicide
as an Example). Nachrichtenbl. Deut. Pflanzenschutzd., Braunschweig, 38: 113-120.

(15) Litchfield, J.T. and Wilcoxon, F. (1949). A simplified method of evaluating dose-effect experiments.

Jour. Pharmacol. and Exper. Ther., 96, 99-113.

(16) Finney, D.J. (1971). Probit Analysis. 3rd ed., Cambridge, London and New-York.

(17) Finney D.J. (1978). Statistical Methods in biological Assay. Griffin, Weycombe, UK.

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C.23. AEROBIC AND ANAEROBIC TRANSFORMATION IN SOIL

1.

METHOD

This Test Method is a replicate of the OECD TG 307 (2002)

1.1

INTRODUCTION

This Test Method is based on existing guidelines (1)(2)(3)(4)(5)(6)(7)(8)(9). The method described in this
Test Method is designed for evaluating aerobic and anaerobic transformation of chemicals in soil. The
experiments are performed to determine (i) the rate of transformation of the test substance, and (ii) the
nature and rates of formation and decline of transformation products to which plants and soil organisms
may be exposed. Such studies are required for chemicals which are directly applied to soil or which are
likely to reach the soil environment. The results of such laboratory studies can also be used to develop
sampling and analysis protocols for related field studies.

Aerobic and anaerobic studies with one soil
the evaluation of
transformation pathways (8)(10)(11). Rates of transformation should be determined in at least three
additional soils (8)(10).

type are generally sufficient

for

An OECD Workshop on soil and sediment selection, held at Belgirate, Italy in 1995 (10) agreed,
in
particular, on the number and types of soils for use in this test. The types of soils tested should be
representative of the environmental conditions where use or release will occur. For example, chemicals
that may be released in subtropical to tropical climates should be tested with Ferrasols or Nitosols (FAO
system). The Workshop also made recommendations relating to collection, handling and storage of soil
samples, based on the ISO Guidance (15). The use of paddy (rice) soils is also considered in this method.

1.2

DEFINITIONS

Test substance: any substance, whether the parent compound or relevant transformation products.

Transformation products: all substances resulting from biotic or abiotic transformation reactions of the
test substance including CO2 and products that are in bound residues.

Bound residues: “Bound residues” represent compounds in soil, plant or animal, which persist in the
matrix in the form of the parent substance or its metabolite(s)/transformation products after extraction.
The extraction method must not substantially change the compounds themselves or the structure of the
matrix. The nature of the bond can be clarified in part by matrix-altering extraction methods and
sophisticated analytical techniques. To date, for example, covalent ionic and sorptive bonds, as well as
entrapments, have been identified in this way. In general, the formation of bound residues reduces the
bioaccessibility and the bioavailability significantly (12) [modified from IUPAC 1984 (13)].

Aerobic transformation: reactions occurring in the presence of molecular oxygen (14).

Anaerobic transformation: reactions occurring under exclusion of molecular oxygen (14).

Soil: is a mixture of mineral and organic chemical constituents, the latter containing compounds of high
carbon and nitrogen content and of high molecular weights, animated by small (mostly micro-) organisms.
Soil may be handled in two states:

(a)

undisturbed, as it has developed with time, in characteristic layers of a variety of soil types;

(b)

disturbed, as it is usually found in arable fields or as occurs when samples are taken by digging and
used in this test method (14).

Mineralisation: is the complete degradation of an organic compound to CO2 and H2O under aerobic
conditions, and CH4, CO2 and H2O under anaerobic conditions. In the context of this test method,

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when 14C-labelled compound is used, mineralisation means extensive degradation during which a labelled
carbon atom is oxidised with release of the appropriate amount of 14CO2 (14).

Half-life: t0.5, is the time taken for 50 % transformation of a test substance when the transformation can
be described by first-order kinetics; it is independent of the concentration.

DT50 (Disappearance Time 50):
is the time within which the concentration of the test substance is
reduced by 50 %; it is different from the half-life t0.5 when transformation does not follow first order
kinetics.

DT75 (Disappearance Time 75):
reduced by 75 %.

DT90 (Disappearance Time 90):
reduced by 90 %.

is the time within which the concentration of the test substance is

is the time within which the concentration of the test substance is

1.3

REFERENCE SUBSTANCES

Reference substances should be used for the characterisation and/or identification of transformation
products by spectroscopic and chromatographic methods.

1.4

APPLICABILITY OF THE TEST

The method is applicable to all chemical substances (non-labelled or radiolabelled) for which an analytical
method with sufficient accuracy and sensitivity is available. It is applicable to slightly volatile, non-volatile,
water-soluble or water-insoluble compounds. The test should not be applied to chemicals which are highly
volatile from soil (e.g. fumigants, organic solvents) and thus cannot be kept in soil under the experimental
conditions of this test.

1.5

INFORMATION ON THE TEST SUBSTANCE

Non-labelled or labelled test substance can be used to measure the rate of transformation. Labelled
material is required for studying the pathway of transformation and for establishing a mass balance. 14C-
labelling is recommended but the use of other isotopes, such as 13C, 15N, 3H, 32P, may also be useful. As far
as possible, the label should be positioned in the most stable part(s) of the molecule (1). The purity of the
test substance should be at least 95 %.

Before carrying out a test on aerobic and anaerobic transformation in soil, the following information on
the test substance should be available:

(a)

solubility in water (Method A.6)

(b)

solubility in organic solvents;

(c)

vapour pressure (Method A.4) and Henry’s law constant;

(d)

n-octanol/water partition coefficient (Method A.8);

(e)

chemical stability in dark (hydrolysis) (Method C.7);

(f)

pKa if a molecule is liable to protonation or deprotonation [OECD Guideline 112 ] (16).

Other useful
[Testing Methods C.21 and C.22] (16).

information may include data on toxicity of the test substance to soil micro-organisms

Analytical methods (including extraction and clean-up methods) for quantification and identification of the
test substance and its transformation products should be available.

(1)

For example, if the test substance contains one ring, labelling on this ring is required; if the test substance contains two or more rings,
separate studies may be needed to evaluate the fate of each labelled ring and to obtain suitable information on formation of
transformation products.

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1.6

PRINCIPLE OF THE TEST METHOD

Soil samples are treated with the test substance and incubated in the dark in biometer-type flasks or in
flow-through systems under controlled laboratory conditions (at constant temperature and soil moisture).
After appropriate time intervals, soil samples are extracted and analysed for the parent substance and for
transformation products. Volatile products are also collected for analysis using appropriate absorption
devices. Using 14C-labelled material, the various mineralisation rates of the test substance can be measured
by trapping evolved 14CO2 and a mass balance, including the formation of soil bound residues, can be
established.

1.7

QUALITY CRITERIA

1.7.1

Recovery

Extraction and analysis of, at least, duplicate soil samples immediately after the addition of the test
substance gives a first indication of the repeatability of the analytical method and of the uniformity of the
application procedure for the test substance. Recoveries for later stages of the experiments are given by the
respective mass balances. Recoveries should range from 90 % to 110 % for labelled chemicals (8) and from
70 % to 110 % for non-labelled chemicals (3).

1.7.2

Repeatability and sensitivity of analytical method

Repeatability of the analytical method (excluding the initial extraction efficiency) to quantify test substance
and transformation products can be checked by duplicate analysis of
the soil,
incubated long enough for formation of transformation products.

the same extract of

The limit of detection (LOD) of the analytical method for the test substance and for the transformation
products should be at least 0.01 mg⋅kg-1 soil (as test substance) or 1 % of applied dose whichever is lower.
The limit of quantification (LOQ) should also be specified.

1.7.3

Accuracy of transformation data

Regression analysis of the concentrations of the test substance as a function of time gives the appropriate
information on the reliability of the transformation curve and allows the calculation of the confidence
limits for half-lives (in the case of pseudo first order kinetics) or DT50 values and, if appropriate, DT75 and
DT90 values.

1.8

DESCRIPTION OF THE TEST METHOD

1.8.1

Equipment and chemical reagents

Incubation systems consist of static closed systems or suitable flow-through systems (7)(17). Examples of
suitable flow-through soil incubation apparatus and biometer-type flask are shown in Figures 1 and 2,
respectively. Both types of incubation systems have advantages and limitations (7)(17).

Standard laboratory equipment is required and especially the following:

— Analytical

instruments such as GLC, HPLC, TLC-equipment,

including the appropriate detection

systems for analysing radiolabelled or non-labelled substances or inverse isotopes dilution method;

— Instruments for identification purposes (e.g. MS, GC-MS, HPLC-MS, NMR, etc.);

— Liquid scintillation counter;

— Oxidiser for combustion of radioactive material;

— Centrifuge;

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— Extraction apparatus (for example, centrifuge tubes for cold extraction and Soxhlet apparatus for

continuous extraction under reflux);

— Instrumentation for concentrating solutions and extracts (e.g. rotating evaporator);

— Water bath;

— Mechanical mixing device (e.g. kneading machine, rotating mixer).

Chemical reagents used include, for example:

— NaOH, analytical grade, 2 mol ⋅ dm-3, or other appropriate base (e.g. KOH, ethanolamine);

— H2SO4, analytical grade, 0.05 mol ⋅ dm-3;

— Ethylene glycol, analytical grade;

— Solid absorption materials such as soda lime and polyurethane plugs;

— Organic solvents, analytical grade, such as acetone, methanol, etc.;

— Scintillation liquid.

1.8.2

Test substance application

For addition to and distribution in soil, the test substance can be dissolved in water (deionised or distilled)
or, when necessary, in minimum amounts of acetone or other organic solvents (6) in which the test
substance is sufficiently soluble and stable. However, the amount of solvent selected should not have a
significant influence on soil microbial activity (see sections 1.5 and 1.9.2-1.9.3 The use of solvents which
inhibit microbial activity, such as chloroform, dichloromethane and other halogenated solvents, should be
avoided.

The test substance can also be added as a solid, e.g. mixed in quartz sand (6) or in a small sub-sample of
the test soil which has been air-dried and sterilised. If the test substance is added using a solvent the
solvent should be allowed to evaporate before the spiked sub-sample is added to the original non-sterile
soil sample.

For general chemicals, whose major route of entry into soil is through sewage sludge/farming application,
the test substance should be first added to sludge which is then introduced into the soil sample. (see
sections 1.9.2 and 1.9.3)

The use of formulated products is not routinely recommended. However, e.g. for poorly soluble test
substances, the use of formulated material may be an appropriate alternative.

1.8.3

Soils

1.8.3.1

Soil selection

To determine the transformation pathway, a representative soil can be used; a sandy loam or silty loam or
loam or loamy sand [according to FAO and USDA classification (18)] with a pH of 5.5-8.0, an organic
carbon content of 0.5-2.5 % and a microbial biomass of at
total organic carbon is
recommended (10).

least 1 % of

For transformation rate studies at least three additional soils should be used representing a range of
relevant soils. The soils should vary in their organic carbon content, pH, clay content and microbial
biomass (10).

All soils should be characterised, at least, for texture (% sand, % silt, % clay) [according to FAO and USDA
classification (18)], pH, cation exchange capacity, organic carbon, bulk density, water
retention
characteristic (2) and microbial biomass (for aerobic studies only). Additional
information on soil
in interpreting the results. For determination of the soil characteristics the
properties may be useful

(2) Water retention characteristic of a soil can be measured as field capacity, as water holding capacity or as water suction tension (pF).
For explanations see Annex 1. It should be reported in the test report whether water retention characteristics and bulk density of soils
were determined in undisturbed field samples or in disturbed (processed) samples.

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methods recommended in references (19)(20)(21)(22)(23) can be used. Microbial biomass should be
determined by using the substrate-induced respiration (SIR) method (25)(26) or alternative methods (20).

1.8.3.2

Collection, handling, and storage of soils

Detailed information on the history of the field site from where the test soil
is collected should be
available. Details include exact location, vegetation cover, treatments with chemicals, treatments with
organic and inorganic fertilisers, additions of biological materials or other contamination. If soils have
been treated with the test substance or its structural analogues within the previous four years, these should
not be used for transformation studies (10)(15).

The soil should be freshly collected from the field (from the A horizon or top 20 cm layer) with a soil
water content which facilitates sieving. For soils other than those from paddy fields, sampling should be
avoided during or immediately following long periods (> 30 days) of drought, freezing or flooding (14).
Samples should be transported in a manner which minimises changes in soil water content and should be
kept in the dark with free access of air, as much as possible. A loosely-tied polyethylene bag is generally
adequate for this purpose.

The soil should be processed as soon as possible after sampling. Vegetation, larger soil fauna and stones
should be removed prior to passing the soil through a 2 mm sieve which removes small stones, fauna and
plant debris. Extensive drying and crushing of the soil before sieving should be avoided (15).

When sampling in the field is difficult in winter (soil frozen or covered by layers of snow), it may be taken
from a batch of soil stored in the greenhouse under plant cover (e.g. grass or grass-clover mixtures).
Studies with soils freshly collected from the field are strongly preferred, but if the collected and processed
soil has to be stored prior to the start of the study storage conditions must be adequate and for a limited
time only (4 ± 2 °C for a maximum of three months) to maintain microbial activity (3). Detailed
instructions on collection, handling and storage of soils to be used for biotransformation experiments can
be found in (8)(10)(15)(26)(27).

Before the processed soil is used for this test, it should be pre-incubated to allow germination and removal
of seeds, and to re-establish equilibrium of microbial metabolism following the change from sampling or
storage conditions
to incubation conditions. A pre-incubation period between 2 and 28 days
approximating the temperature and moisture conditions of the actual test is generally adequate (15).
Storage and pre-incubation time together should not exceed three months.

1.9

PERFORMANCE OF THE TEST

1.9.1

Test conditions

1.9.1.1

Test temperature

During the whole test period, the soils should be incubated in the dark at a constant temperature
representative of the climatic conditions where use or release will occur. A temperature of 20 ± 2 °C is
recommended for all test substances which may reach the soil in temperate climates. The temperature
should be monitored.

For chemicals applied or released in colder climates (e.g. in northern countries, during autumn/winter
periods), additional soil samples should be incubated but at a lower temperature (e.g. 10 ± 2 °C).

(3) Recent research results indicate that soils from temperate zones can also be stored at –20 °C for more than three months (28)(29)

without significant losses of microbial activity.

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1.9.1.2

Moisture content

For transformation tests under aerobic conditions, the soil moisture content (4) should be adjusted to and
maintained at a pF between 2.0 and 2.5 (3). The soil moisture content is expressed as mass of water per
mass of dry soil and should be regularly controlled (e.g. in 2 week intervals) by weighing of the incubation
flasks and water losses compensated by adding water (preferably sterile-filtered tap water). Care should be
given to prevent or minimise losses of test substance and/or transformation products by volatilisation and/
or photodegradation (if any) during moisture addition.

For transformation tests under anaerobic and paddy conditions, the soil is water-saturated by flooding.

1.9.1.3

Aerobic incubation conditions

In the flow-through systems, aerobic conditions will be maintained by intermittent
flushing or by
continuously ventilating with humidified air. In the biometer flasks, exchange of air is maintained by
diffusion.

1.9.1.4

Sterile aerobic conditions

To obtain information on the relevance of abiotic transformation of a test substance, soil samples may be
sterilised (for sterilisation methods see references 16 and 29), treated with sterile test substance (e.g.
addition of solution through a sterile filter) and aerated with humidified sterile air as described in section
1.9.1.3. For paddy soils, soil and water should be sterilised and the incubation should be carried out as
described in section 1.9.1.6.

1.9.1.5

Anaerobic incubation conditions

To establish and maintain anaerobic conditions, the soil treated with the test substance and incubated
under aerobic conditions for 30 days or one half-life or DT50 (whichever is shorter) is then water-logged
(1-3 cm water layer) and the incubation system flushed with an inert gas (e.g. nitrogen or argon) (5). The
test system must allow for measurements such as pH, oxygen concentration and redox potential and
include trapping devices for volatile products. The biometer-type system must be closed to avoid entrance
of air by diffusion.

1.9.1.6

Paddy incubation conditions

To study transformation in paddy rice soils, the soil is flooded with a water layer of about 1-5 cm and the
test substance applied to the water phase (9). A soil depth of at least 5 cm is recommended. The system is
ventilated with air as under aerobic conditions. pH, oxygen concentration and redox potential of the
aqueous layer should be monitored and reported. A pre-incubation period of at least two weeks is
necessary before commencing transformation studies (see section 1.8.3.2).

1.9.1.7

Test duration

The rate and pathway studies should normally not exceed 120 days (6) (3)(6)(8), because thereafter a
decrease of the soil microbial activity with time would be expected in an artificial
laboratory system
isolated from natural replenishment. Where necessary to characterise the decline of the test substance and
the formation and decline of major transformation products, studies can be continued for longer periods
(e.g. 6 or 12 months)
(8). Longer incubation periods should be justified in the test report and
accompanied by biomass measurements during and at the end of these periods.

(4)

The soil should neither be too wet nor too dry to maintain adequate aeration and nutrition of soil microflora. Moisture contents
recommended for optimal microbial growth range from 40-60 % water holding capacity (WHC) and from 0.1-0.33 bar (6). The latter
range is equivalent to a pF-range of 2.0 – 2.5. Typical moisture contents of various soil types are given in Annex 2.

(5) Aerobic conditions are dominant in surface soils and even in sub-surface soils as shown in an EU sponsored research project [K.
Takagi et al. (1992). Microbial diversity and activity in subsoils: Methods, field site, seasonal variation in subsoil temperatures and
oxygen contents. Proc. Internat. Symp. Environm. Aspects Pesticides Microbiol., 270-277, 17-21 August 1992, Sigtuna, Sweden].
Anaerobic conditions may only occur occasionally during flooding of soils after heavy rainfalls or when paddy conditions are
established in rice fields.

(6) Aerobic studies might be terminated much before 120 days provided that ultimate transformation pathway and ultimate
mineralisation are clearly reached at that time. Termination of the test is possible after 120 days, or when at least 90 % of the test
substance is transformed, but only if at least 5 % CO2 is formed.

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1.9.2

Performance of the test

About 50 to 200 g of soil (dry weight basis) are placed into each incubation flask (see Figures 1 and 2 in
Annex 3) and the soil treated with the test substance by one of the methods described in section 1.8.2.
When organic solvents are used for the application of the test substance, they should be removed from
soil by evaporation. Then the soil is thoroughly mixed with a spatula and/or by shaking of the flask. If the
study is conducted under paddy field conditions, soil and water should be thoroughly mixed after
application of the test substance. Small aliquots (e.g. 1 g) of the treated soils should be analysed for the
test substance to check for uniform distribution. For alternative method, see below.

The treatment rate should correspond to the highest application rate of a crop protection product
recommended in the use instructions and uniform incorporation to an appropriate depth in the field (e.g.
top 10 cm layer (7) of soil). For example, for chemicals foliarly or soil applied without incorporation, the
appropriate depth for computing how much chemical should be added to each flask is 2.5 cm. For soil
incorporated chemicals, the appropriate depth is the incorporation depth specified in the use instructions.
For general chemicals, the application rate should be estimated based on the most relevant route of entry;
for example, when the major route of entry in soil is through sewage sludge, the chemical should be dosed
into the sludge at a concentration that reflects the expected sludge concentration and the amount of
sludge added to the soil should reflect normal sludge loading to agricultural soils. If this concentration is
not high enough to identify major transformation products, incubation of separate soil samples containing
higher rates may be helpful, but excessive rates influencing soil microbial functions should be avoided (see
sections 1.5 and 1.8.2).

Alternatively, a larger batch (i.e. 1 to 2 kg) of soil can be treated with the test substance, carefully mixed in
an appropriate mixing machine and then transferred in small portions of 50 to 200 g into the incubation
flasks (for example with the use of sample splitters). Small aliquots (e.g. 1 g) of the treated soil batch
should be analysed for the test substance to check for uniform distribution. Such a procedure is preferred
since it allows for more uniform distribution of the test substance into the soil.

Also untreated soil samples are incubated under the same conditions (aerobic) as the samples treated with
the test substance. These samples are used for biomass measurements during and at the end of the studies.

When the test substance is applied to the soil dissolved in organic solvent(s), soil samples treated with the
same amount of solvent(s) are incubated under the same conditions (aerobic) as the samples treated with
the test substance. These samples are used for biomass measurements initially, during and at the end of the
studies to check for effects of the solvent(s) on microbial biomass.

The flasks containing the treated soil are either attached to the flow-through system described in Figure 1
or closed with the absorption column shown in Figure 2 (see Annex 3).

1.9.3

Sampling and measurement

Duplicate incubation flasks are removed at appropriate time intervals and the soil samples extracted with
appropriate solvents of different polarity and analysed for the test substance and/or transformation
products. A well-designed study includes sufficient flasks so that two flasks are sacrificed at each sampling
event. Also, absorption solutions or solid absorption materials are removed at various time intervals (7-
day intervals during the first month and after one month in 17-day intervals) during and at the end of
incubation of each soil sample and analysed for volatile products. Besides a soil sample taken directly after

(7) Calculation of the initial concentration on an area basis using the following equation:

Csoil = Initial concentration in soil [mg⋅kg-1]
A = Application rate [kg⋅ha-1]; 1 = thickness of field soil layer [m]; d = dry bulk density of soil [kg⋅m-3].
As a rule of thumb, an application rate of 1 kg⋅ha-1 results in a soil concentration of approximately 1 mg⋅kg-1 in a 10 cm layer
(assuming a bulk density of 1 g ⋅ cm-3).

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application (0-day sample) at least 5 additional sampling points should be included. Time intervals should
be chosen in such a way that pattern of decline of the test substance and patterns of formation and
decline of transformation products can be established (e.g. 0, 1, 3, 7 days; 2, 3 weeks; 1, 2, 3 months,
etc.).

When using 14C-labelled test substance, non-extractable radioactivity will be quantified by combustion and
a mass balance will be calculated for each sampling interval.

In the case of anaerobic and paddy incubation, the soil and water phases are analysed together for test
substance and transformation products or separated by filtration or centrifugation before extraction and
analysis.

1.9.4

Optional tests

Aerobic, non-sterile studies at additional temperatures and soil moistures may be useful for the estimation
of the influence of temperature and soil moisture on the rates of transformation of a test substance and/or
its transformation products in soil.

A further characterisation of non-extractable radioactivity can be attempted using,
supercritical fluid extraction.

for example,

2.

2.1

DATA

TREATMENT OF RESULTS

The amounts of
test substance, transformation products, volatile substances (in % only), and non-
extractable should be given as % of applied initial concentration and, where appropriate, as mg⋅kg-1 soil
(based on soil dry weight) for each sampling interval. A mass balance should be given in percentage of the
applied initial concentration for each sampling interval. A graphical presentation of the test substance
concentrations against
its transformation half-life or DT50. Major
transformation products should be identified and their concentrations should also be plotted against time
to show their rates of formation and decline. A major transformation product is any product representing
≥ 10 % of applied dose at any time during the study.

time will allow an estimation of

The volatile products trapped give some indication of the volatility potential of a test substance and its
transformation products from soil.

More accurate determinations of half-lives or DT50 values and, if appropriate, DT75 and DT90 values should
be obtained by applying appropriate kinetic model calculations. The half-life and DT50 values should be
reported together with the description of the model used, the order of kinetics and the determination
coefficient (r2). First order kinetics is favoured unless r2 < 0.7. If appropriate, the calculations should also
be applied to the major transformation products. Examples of appropriate models are described in
references 31 to 35.

In the case of rate studies carried out at various temperatures, the transformation rates should be described
as a function of temperature within the experimental temperature range using the Arrhenius relationship
of the form:

where ln A and B are regression constants from the intercept and slope, respectively, of a best fit line
generated from linearly regressing ln k against 1/T, k is the rate constant at temperature T and T is the
temperature in Kelvin. Care should be given to the limited temperature range in which the Arrehenius
relationship will be valid in case transformation is governed by microbial action.

2.2

EVALUATION AND INTERPRETATION OF RESULTS

Although the studies are carried out in an artificial laboratory system, the results will allow estimation of
formation and decline of
the rate of
transformation products under field conditions (36)(37).

the test substance and also of

transformation of

rate of

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A study of the transformation pathway of a test substance provides information on the way in which the
applied substance is structurally changed in the soil by chemical and microbial reactions.

3.

3.1

REPORTING

TEST REPORT

The test report must include:

Test substance:

— common name, chemical name, CAS number, structural formula (indicating position of label(s)
when radiolabelled material is used) and relevant physical-chemical properties (see section 1.5);

— purity (impurities) of test substance;

— radiochemical purity of labelled chemical and specific activity (where appropriate);

Reference substances:

— chemical name and structure of reference substances used for the characterisation and/or

identification of transformation product;

Test soils:

— details of collection site;

— date and procedure of soil sampling;

— properties of soils, such as pH, organic carbon content, texture (% sand, % silt, % clay), cation

exchange capacity, bulk density, water retention characteristic, and microbial biomass;

— length of soil storage and storage conditions (if stored);

Test conditions:

— dates of the performance of the studies;

— amount of test substance applied;

— solvents used and method of application for the test substance;

— weight of soil treated initially and sampled at each interval for analysis;

— description of the incubation system used;

— air flow rates (for flow-through systems only);

— temperature of experimental set-up;

— soil moisture content during incubation;

— microbial biomass initially, during and at the end of the aerobic studies;

— pH, oxygen concentration and redox potential initially, during and at the end of the anaerobic

and paddy studies;

— method(s) of extraction;

— methods for quantification and identification of the test substance and major transformation

products in soil and absorption materials;

— number of replicates and number of controls.

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Results:

— result of microbial activity determination;

— repeatability and sensitivity of the analytical methods used;

— rates of recovery (% values for a valid study are given in section 1.7.1);

— tables of results expressed as % of applied initial dose and, where appropriate, as mg⋅kg-1 soil (on

a dry weight basis);

— mass balance during and at the end of the studies;

— characterisation of non-extractable (bound) radioactivity or residues in soil;

— quantification of released CO2 and other volatile compounds;

— plots of soil concentrations versus time for the test substance and, where appropriate, for major

transformation products;

— half-life or DT50, DT75 and DT90 for the test substance and, where appropriate,

for major

transformation products including confidence limits;

— estimation of abiotic degradation rate under sterile conditions;

— an assessment of transformation kinetics for the test substance and, where appropriate, for major

transformation products;

— proposed pathways of transformation, where appropriate;

— discussion and interpretation of results;

— raw data (i.e. sample chromatograms, sample calculations of transformation rates and means used

to identify transformation products).

4.

REFERENCES

(1) US- Environmental Protection Agency (1982). Pesticide Assessment Guidelines, Subdivision N.

Chemistry: Environmental Fate.

(2)

(3)

Agriculture Canada (1987). Environmental Chemistry and Fate. Guidelines for registration of
pesticides in Canada.

European Union (EU) (1995). Commission Directive 95/36/EC of 14 July 1995 amending Council
Directive 91/414/EEC concerning the placing of plant protection products on the market. Annex II,
Part A and Annex III, Part A: Fate and Behaviour in the Environment.

(4) Dutch Commission for Registration of Pesticides (1995). Application for registration of a pesticide.

Section G: Behaviour of the product and its metabolites in soil, water and air.

(5) BBA (1986). Richtlinie für die amtliche Prüfung von Pflanzenschutzmitteln, Teil IV, 4-1. Verbleib von

Pflanzenschutzmitteln im Boden - Abbau, Umwandlung und Metabolismus.

(6)

(7)

(8)

ISO/DIS 11266-1 (1994). Soil Quality -Guidance on laboratory tests for biodegradation of organic
chemicals in soil - Part 1 : Aerobic conditions.

ISO 14239 (1997). Soil Quality – Laboratory incubation systems for measuring the mineralization of
organic chemicals in soil under aerobic conditions.

SETAC (1995). Procedures for Assessing the Environmental Fate and Ecotoxicity of Pesticides. Mark R.
Lynch, Ed.

(9) MAFF - Japan 2000 - Draft Guidelines for transformation studies of pesticides in soil - Aerobic

metabolism study in soil under paddy field conditions (flooded).

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(10) OECD (1995). Final Report of the OECD Workshop on Selection of Soils/Sediments. Belgirate, Italy,

18-20 January 1995.

(11) Guth, J.A. (1980). The study of transformations. In Interactions between Herbicides and the Soil (R.J.

Hance, Ed.), Academic Press, 123-157.

(12) DFG: Pesticide Bound Residues in Soil. Wiley – VCH (1998).

(13) T.R. Roberts: Non-extractable pesticide residue in soils and plants. Pure Appl. Chem. 56, 945-956

(IUPAC 1984).

(14) OECD Test Guideline 304 A: Inherent Biodegradability in Soil (adopted 12 May 1981).

(15)

ISO 10381-6 (1993). Soil Quality - Sampling - Part 6: Guidance on the collection, handling and
storage of soil for the assessment of aerobic microbial processes in the laboratory.

(16) Annex V to Dir. 67/548/EEC.

(17) Guth, J.A. (1981). Experimental approaches to studying the fate of pesticides in soil. In Progress in

Pesticide Biochemistry. D.H. Hutson, T.R. Roberts, Eds. J. Wiley & Sons. Vol 1, 85-114.

(18) Soil Texture Classification (US and FAO systems): Weed Science, 33, Suppl. 1 (1985) and Soil Sci.

Soc. Amer. Proc. 26:305 (1962).

(19) Methods of Soil Analysis (1986). Part 1, Physical and Mineralogical Methods. A. Klute, Ed.)

Agronomy Series No 9, 2nd Edition.

(20) Methods of Soil Analysis (1982). Part 2, Chemical and Microbiological Properties. A.L. Page, R.H.

Miller and D.R. Kelney, Eds. Agronomy Series No 9, 2nd Edition.

(21)

ISO Standard Compendium Environment (1994). Soil Quality - General aspects; chemical and
physical methods of analysis; biological methods of analysis. First Edition.

(22) Mückenhausen, E.

(1975). Die Bodenkunde und ihre geologischen, geomorphologischen,

mineralogischen und petrologischen Grundlagen. DLG-Verlag, Frankfurt, Main.

(23) Scheffer, F., Schachtschabel, P. (1975). Lehrbuch der Bodenkunde. F. Enke Verlag, Stuttgart.

(24) Anderson, J.P.E., Domsch, K.H. (1978) A physiological method for the quantitative measurement of

microbial biomass in soils. Soil Biol. Biochem. 10, 215-221.

(25)

ISO 14240-1 and 2 (1997). Soil Quality - Determination of soil microbial biomass - Part 1:
Substrate-induced respiration method. Part 2: fumigation-extraction method.

(26) Anderson, J.P.E. (1987). Handling and storage of soils for pesticide experiments. In Pesticide Effects on

Soil Microflora. L. Somerville, M.P. Greaves, Eds. Taylor & Francis, 45-60.

(27) Kato, Yasuhiro. (1998). Mechanism of pesticide transformation in the environment: Aerobic and bio-
the 16th Symposium on

transformation of pesticides in aqueous environment. Proceedings of
Environmental Science of Pesticide, 105-120.

(28) Keuken O., Anderson J.P.E.

(1996).
Pesticides, Soil Microbiology and Soil Quality, 59-63 (SETAC-Europe).

Influence of storage on biochemical processes in soil.

In

(29) Stenberg B., Johansson M., Pell M., Sjödahl-Svensson K., Stenström J., Torstensson L. (1996). Effect of
freeze and cold storage of soil on microbial activities and biomass. In Pesticides, Soil Microbiology
and Soil Quality, 68-69 (SETAC-Europe).

(30) Gennari, M., Negre, M., Ambrosoli, R. (1987). Effects of ethylene oxide on soil microbial content and

some chemical characteristics. Plant and Soil 102, 197-200.

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(31) Anderson,

J.P.E.

(1975). Einfluss von Temperatur und Feuchte auf Verdampfung, Abbau und

Festlegung von Diallat im Boden. Z. PflKrankh Pflschutz, Sonderheft VII, 141-146.

(32) Hamaker,

J.W.

(1976). The application of mathematical modelling to the soil persistence and
accumulation of pesticides. Proc. BCPC Symposium: Persistence of Insecticides and Herbicides, 181-
199.

(33) Goring, C.A.I., Laskowski, D.A., Hamaker,

(1975). Principles of pesticide
degradation in soil. In “Environmental Dynamics of Pesticides”. R. Haque and V.H. Freed, Eds., 135-
172.

J.W., Meikle, R.W.

(34) Timme, G., Frehse, H., Laska, V. (1986). Statistical interpretation and graphic representation of the
degradational behaviour of pesticide residues. II. Pflanzenschutz - Nachrichten Bayer 39, 188-204.

(35) Timme, G., Frehse, H.

interpretation and graphic representation of
degradational behaviour of pesticide residues. I. Pflanzenschutz - Nachrichten Bayer 33, 47-60.

(1980). Statistical

the

(36) Gustafson D.I., Holden L.R. (1990). Non-linear pesticide dissipation in soil; a new model based on

spatial variability. Environm. Sci. Technol. 24, 1032-1041.

(37) Hurle K., Walker A. (1980). Persistence and its prediction. In Interactions between Herbicides and the

Soil (R.J. Hance, Ed.), Academic Press, 83-122.

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WATER TENSION, FIELD CAPACITY (FC) AND WATER HOLDING CAPACITY (WHC) (1)

ANNEX 1

Height of Water
Column [cm]

pF (a)

bar (b)

Remarks

107

1,6 · 104

104

103

6 · 102

3,3 · 102

102

60

33

10

1

7

4,2

4

3

2,8

2,5

2

1,8

1,5

1

0

104

16

10

1

0,6

0,33 (c)

0,1

0,06

0,033

0,01

0,001

9
>>>>=
>>>>;

Dry Soil

Wilting point

Range of Field capacity (d)

WHC (approximation)

Water saturated soil

(a) pF = log of cm water column.
(b) 1 bar = 105 Pa.
(c) Corresponds to an approximate water content of 10 % in sand, 35 % in loam and 45 % in clay.
(d) Field capacity is not constant but varies with soil type between pF 1.5 and 2.5.

Water tension is measured in cm water column or in bar . Due to the large range of suction tension it is expressed simply
as pF value which is equivalent to the logarithm of cm water column.

Field capacity is defined as the amount of water which can be stored against gravity by a natural soil 2 days after a longer
raining period or after sufficient irrigation. It is determined in undisturbed soil in situ in the field. The measurement is
thus not applicable to disturbed laboratory soil samples. FC values determined in disturbed soils may show great
systematic variances.

Water holding capacity (WHC) is determined in the laboratory with undisturbed and disturbed soil by saturating a soil
column with water by capillary transport. It is particularly useful for disturbed soils and can be up to 30 % greater than
field capacity (1). It is also experimentally easier to determine than reliable FC-values.

(1) Mückenhausen, E.

(1975). Die Bodenkunde und ihre geologischen, geomorphologischen, mineralogischen und petrologischen

Grundlagen. DLG-Verlag, Frankfurt, Main.

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SOIL MOISTURE CONTENTS (g water per 100 g dry soil) OF VARIOUS SOIL TYPES FROM VARIOUS
COUNTRIES

ANNEX 2

Soil Moisture

Content at

Soil Type Country

WHC (1)

pF = 1,8

pF = 2,5

Sand

Loamy sand

Loamy sand

Silt loam

Clay loam

Clay loam

Sandy loam

Silt loam

Sandy loam

(1) Water Holding Capacity

Germany

Germany

Switzerland

Switzerland

Brazil

Japan

Japan

USA

USA

28,7

50,4

44,0

72,8

69,7

74,4

82,4

47,2

40,4

8,8

17,9

35,3

56,6

38,4

57,8

59,2

33,2

25,2

3,9

12,1

9,2

28,4

27,3

31,4

36,0

18,8

13,3

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ANNEX 3

Figure 1

Example of a flow-through apparatus to study transformation of chemicals in soil (1)(2)

1:
2:
3:
4:
5:
6:
7, 8:
9:

needle valve
gas washing bottle containing water
ultramembrane (sterile conditions only), pore size 0.2 μm
soil metabolism flask (water-logged only for anaerobic and paddy conditions;)
ethylene glycol trap for organic volatile compounds
sulphuric acid trap for alkaline volatile compounds
sodium hydroxide trap for CO2 & other acidic volatiles
flow meter.

Example of a biometer-type flask for studying the transformation of chemicals in soil (3)

Figure 2

(1) Guth, J.A. (1980). The study of transformations. In Interactions between Herbicides and the Soil (R.J. Hance, Ed.),

Academic Press, 123-157.

(2) Guth,

J.A.

(1981). Experimental approaches to studying the fate of pesticides in soil.

In Progress in Pesticide

Biochemistry. D.H. Hutson, T.R. Roberts, Eds. J. Wiley & Sons. Vol 1, 85-114.

(3) Anderson, J.P.E. (1975). Einfluss von Temperatur und Feuchte auf Verdampfung, Abbau und Festlegung von Diallat im

Boden. Z. PflKrankh Pflschutz, Sonderheft VII, 141-146.

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C.24. AEROBIC AND ANAEROBIC TRANSFORMATION IN AQUATIC SEDIMENT SYSTEMS

1.

METHOD

This test method is a replicate of the OECD TG 308 (2002).

1.1

INTRODUCTION

Chemicals can enter shallow or deep surface waters by such routes as direct application, spray drift, run-
off, drainage, waste disposal, industrial, domestic or agricultural effluent and atmospheric deposition. This
Testing Method describes a laboratory method to assess aerobic and anaerobic transformation of organic
chemicals in aquatic sediment systems. It is based on existing Guidelines (1)(2)(3)(4)(5)(6). An OECD
Workshop on Soil/Sediment Selection, held in Belgirate, Italy in 1995 (7) agreed, in particular, on the
number and type of sediments for use in this test. It also made recommendations relating to collection,
handling and storage of sediment samples, based on the ISO Guidance (8). Such studies are required for
chemicals which are directly applied to water or which are likely to reach the aqueous environment by the
routes described above.

The conditions in natural aquatic sediment systems are often aerobic in the upper water phase. The surface
layer of sediment can be either aerobic or anaerobic, whereas the deeper sediment is usually anaerobic. To
encompass all of these possibilities both aerobic and anaerobic tests are described in this document. The
aerobic test simulates an aerobic water column over an aerobic sediment layer that is underlain with an
If
anaerobic gradient. The anaerobic test simulates a completely anaerobic water-sediment system.
circumstances indicate that it is necessary to deviate significantly from these recommendations,
for
example by using intact sediment cores or sediments that may have been exposed to the test substance,
other methods are available for this purpose (9).

1.2

DEFINITIONS

Standard International (SI) units should be used in any case.

Test substance: any substance, whether the parent or relevant transformation products.

Transformation products: all substances resulting from biotic and abiotic transformation reactions of the
test substance including CO2 and bound residues.

Bound residues: “Bound residues” represent compounds in soil, plant or animal that persist in the matrix
in the form of the parent substance or its metabolite(s) after extractions. The extraction method must not
substantially change the compounds themselves or the structure of the matrix. The nature of the bond can
be clarified in part by matrix-altering extraction methods and sophisticated analytical techniques. To date,
for example, covalent ionic and sorptive bonds, as well as entrapments, have been identified in this way. In
general, the formation of bound residues reduces the bioaccessibility and the bioavailability significantly
(10) [modified from IUPAC 1984 (11)].

Aerobic transformation: (oxidising): reactions occurring in the presence of molecular oxygen (12).

Anaerobic transformation: (reducing): reactions occurring under exclusion of molecular oxygen (12).

Natural waters: are surface waters obtained from ponds, rivers, streams, etc.

Sediment: is a mixture of mineral and organic chemical constituents, the latter containing compounds of
high carbon and nitrogen content and of high molecular masses. It is deposited by natural water and
forms an interface with that water.

Mineralisation:
is the complete degradation of an organic compound to CO2, H2O under aerobic
conditions, and CH4, CO2 and H2O under anaerobic conditions. In the context of this test method, when
radiolabelled compound is used, mineralisation means extensive degradation of a molecule during which a
labelled carbon atom is oxidised or reduced quantitatively with release of the appropriate amount of 14CO2
or 14CH4, respectively.

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Half-life, t0.5, is the time taken for 50 % transformation of a test substance when the transformation can
be described by first-order kinetics; it is independent of the initial concentration.

DT50 (Disappearance Time 50): is the time within which the initial concentration of the test substance is
reduced by 50 %.

DT75 (Disappearance Time 75): is the time within which the initial concentration of the test substance is
reduced by 75 %.

DT90 (Disappearance Time 90): is the time within which the initial concentration of the test substance is
reduced by 90 %.

1.3

REFERENCE SUBSTANCES

Reference substances should be used for the identification and quantification of transformation products
by spectroscopic and chromatographic methods.

1.4

INFORMATION ON THE TEST SUBSTANCE

Non-labelled or isotope-labelled test substance can be used to measure the rate of transformation although
labelled material is preferred. Labelled material is required for studying the pathway of transformation
and for establishing a mass balance. 14C-labelling is recommended, but the use of other isotopes, such
as 13C, 15N, 3H, 32P, may also be useful. As far as possible, the label should be positioned in the most stable
part(s) of the molecule (1). The chemical and/or radiochemical purity of the test substance should be at
least 95 %.

Before carrying out a test, the following information about the test substance should be available:

(a)

solubility in water (Method A.6);

(b)

solubility in organic solvents;

(c)

vapour pressure (Method A.4) and Henry’s Law constant;

(d)

n-octanol/water partition coefficient (Method A.8);

(e)

adsorption coefficient (Kd, Kf or Koc, where appropriate) (Method C.18);

(f)

hydrolysis (Method C.7);

(g)

dissociation constant (pKa) [OECD Guideline 112] (13);

(h)

chemical structure of the test substance and position of the isotope-label(s), if applicable.

Note: The temperature at which these measurements were made should be reported.

Other useful information may include data on toxicity of the test substance to microorganisms, data on
ready and/or inherent biodegradability, and data on aerobic and anaerobic transformation in soil.

Analytical methods (including extraction and clean-up methods) for identification and quantification of the
test substance and its transformation products in water and in sediment should be available (see section
1.7.2).

1.5

PRINCIPLE OF THE TEST METHOD

The method described in this test employs an aerobic and an anaerobic aquatic sediment (see Annex 1)
system which allows:

(i)

the measurement of the transformation rate of the test substance in a water-sediment system,

(ii)

the measurement of the transformation rate of the test substance in the sediment,

(1)

For example, if the substance contains one ring, labelling on this ring is required; if the test substance contains two or more rings,
separate studies may be needed to evaluate the fate of each labelled ring and to obtain suitable information on formation of
transformation products.

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(iii)

the measurement of the mineralisation rate of the test substance and /or its transformation products
(when 14C-labelled test substance is used),

(iv)

(v)

the identification and quantification of
including mass balance (when labelled test substance is used),

transformation products in water and sediment phases

the measurement of the distribution of the test substance and its transformation products between
the two phases during a period of incubation in the dark (to avoid, for example, algal blooms) at
constant temperature. Half-lives, DT50, DT75 and DT90 values are determined where the data warrant,
but should not be extrapolated far past the experimental period (see section 1.2).

At least two sediments and their associated waters are required for both the aerobic and the anaerobic
studies respectively (7). However, there may be cases where more than two aquatic sediments should be
used, for example, for a chemical that may be present in freshwater and/or marine environments.

1.6

APPLICABILITY OF THE TEST

The method is generally applicable to chemical substances (unlabelled or labelled) for which an analytical
method with sufficient accuracy and sensitivity is available. It is applicable to slightly volatile, non-volatile,
water-soluble or poorly water-soluble compounds. The test should not be applied to chemicals which are
highly volatile from water (e.g. fumigants, organic solvents) and thus cannot be kept in water and/or
sediment under the experimental conditions of this test.

The method has been applied so far to study the transformation of chemicals in fresh waters and
sediments, but in principle can also be applied to estuarine/marine systems. It is not suitable to simulate
conditions in flowing water (e.g. rivers) or the open sea.

1.7

QUALITY CRITERIA

1.7.1

Recovery

Extraction and analysis of, at least, duplicate water and sediment samples immediately after the addition of
the
the test substance gives a first indication of
uniformity of
the
experiments are given by the respective mass balances (when labelled material is used). Recoveries should
range from 90 % to 110 % for labelled chemicals (6) and from 70 % to 110 % for non-labelled chemicals.

the analytical method and of
the repeatability of
the application procedure for the test substance. Recoveries for later stages of

1.7.2

Repeatability and sensitivity of analytical method

Repeatability of the analytical method (excluding the initial extraction efficiency) to quantify test substance
and transformation products can be checked by duplicate analysis of the same extract of the water or the
sediment samples which were incubated sufficiently long enough for formation of
transformation
products.

The limit of detection (LOD) of the analytical method for the test substance and for the transformation
products should be at least 0.01 mg⋅kg-1 in water or sediment (as test substance) or 1 % of the initial
amount applied to a test system whichever is lower. The limit of quantification (LOQ) should also be
specified.

1.7.3

Accuracy of transformation data

Regression analysis of the concentrations of the test substance as a function of time gives the appropriate
information on the accuracy of the transformation curve and allows the calculation of the confidence
limits for half-lives (if pseudo first-order kinetics apply) or DT50 values and, if appropriate, DT75 and DT90
values.

1.8

DESCRIPTION OF THE METHOD

1.8.1

Test system and apparatus

The study should be performed in glass containers (e.g. bottles, centrifuge tubes), unless preliminary
indicates that the test
information (such as n-octanol-water partition coefficient, sorption data, etc.)

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substance may adhere to glass, in which case an alternative material (such as Teflon) may have to be
considered. Where the test substance is known to adhere to glass, it may be possible to alleviate this
problem using one or more of the following methods:

— determine the mass of test substance and transformation products sorbed to glass;

— ensure a solvent wash of all glassware at the end of the test;

— use of formulated products (see also section 1.9.2);

— use an increased amount of co-solvent for addition of test substance to the system; if a co-solvent is

used it should be a co-solvent that does not solvolyse the test substance.

Examples of typical test apparatus, i.e. gas flow-through and biometer-type systems, are shown in Annexes
2 and 3, respectively (14). Other useful incubation systems are described in reference 15. The design of the
experimental apparatus should permit the exchange of air or nitrogen and the trapping of volatile
products. The dimensions of the apparatus must be such that the requirements of the test are complied
with (see section 1.9.1). Ventilation may be provided by either gentle bubbling or by passing air or
nitrogen over the water surface. In the latter case gentle stirring of the water from above may be advisable
for better distribution of the oxygen or nitrogen in the water. CO2-free air should not be used as this can
result in increases in the pH of the water. In either case, disturbance of the sediment is undesirable and
should be avoided as far as possible. Slightly volatile chemicals should be tested in a biometer-type system
with gentle stirring of the water surface. Closed vessels with a headspace of either atmospheric air or
nitrogen and internal vials for the trapping of volatile products can also be used (16). Regular exchange of
the headspace gas is required in the aerobic test in order to compensate for the oxygen consumption by
the biomass.

Suitable traps for collecting volatile transformation products include but are not restricted to 1 mol⋅dm-3
solutions of potassium hydroxide or sodium hydroxide for carbon dioxide (2) and ethylene glycol,
ethanolamine or 2 % paraffin in xylene for organic compounds. Volatiles formed under anaerobic
conditions, such as methane, can be collected, for example, by molecular sieves. Such volatiles can be
combusted, for example, to CO2 by passing the gas through a quartz tube filled with CuO at a temperature
of 900 °C and trapping the CO2 formed in an absorber with alkali (17).

Laboratory instrumentation for chemical analysis of test substance and transformation products is required
(e.g. gas liquid chromatography (GLC), high performance liquid chromatography (HPLC),
thin-layer
chromatography (TLC), mass spectroscopy (MS), gas chromatography-mass spectroscopy (GC-MS), liquid
chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), etc.), including detection
systems for radiolabelled or non-labelled chemicals as appropriate. When radiolabelled material is used a
liquid scintillation counter and combustion oxidiser (for the combustion of sediment samples prior to
analysis of radioactivity) will also be required.

Other standard laboratory equipment for physical-chemical and biological determinations (see section
Table 1, section 1.8.2.2), glassware, chemicals and reagents are required as appropriate.

1.8.2

Selection and number of aquatic sediments

The sampling sites should be selected in accordance with the purpose of the test in any given situation. In
selecting sampling sites, the history of possible agricultural, industrial or domestic inputs to the catchment
they have been
and the waters upstream must be considered. Sediments should not be used if
contaminated with the test substance or its structural analogues within the previous 4 years.

(2) As these alkaline absorption solutions also absorb the carbon dioxide from the ventilation air and that formed by respiration in
aerobic experiments, they have to be exchanged in regular intervals to avoid their saturation and thus loss of their absorption
capacity.

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1.8.2.1

Sediment selection

Two sediments are normally used for the aerobic studies (7). The two sediments selected should differ with
respect to organic carbon content and texture. One sediment should have a high organic carbon content
(2.5-7.5 %) and a fine texture, the other sediment should have a low organic carbon content (0.5-2.5 %)
and a coarse texture. The difference between the organic carbon contents should normally be at least 2 %.
‘Fine texture’ is defined as a [clay + silt] (3) content of > 50 % and ‘coarse texture’ is defined as a [clay +
silt] content of < 50 %. The difference in [clay + silt] content for the two sediments should normally be at
least 20 %. In cases, where a chemical may also reach marine waters, at least one of the water-sediment
systems should be of marine origin.

For the strictly anaerobic study, two sediments (including their associated waters) should be sampled from
the anaerobic zones of surface water bodies (7). Both the sediment and the water phases should be
handled and transported carefully under exclusion of oxygen.

Other parameters may be important in the selection of sediments and should be considered on a case-by-
case basis. For example, the pH range of sediments would be important for testing chemicals for which
transformation and/or sorption may be pH-dependent. pH-dependency of sorption might be reflected by
the pKa of the test substance.

1.8.2.2

Characterisation of water-sediment samples

Key parameters that must be measured and reported (with reference to the method used) for both water
and sediment, and the stage of the test at which those parameters are to be determined are summarised in
the Table hereafter. For information, methods for determination of these parameters are given in references
(18)(19)(20)(21).

In addition, other parameters may need to be measured and reported on a case by case basis (e.g. for
freshwater: particles, alkalinity, hardness, conductivity, NO3/PO4 (ratio and individual values); for sediments:
cation exchange capacity, water holding capacity, carbonate, total nitrogen and phosphorus; and for
marine systems: salinity). Analysis of sediments and water for nitrate, sulfate, bioavailable iron, and
possibly other electron acceptors may be also useful in assessing redox conditions, especially in relation to
anaerobic transformation.

Measurement of parameters for characterisation of water-sediment samples (7)(22)(23)

Parameter

Water

Origin/source

Temperature

pH

TOC

O2 concentration*

Redox Potential*

Sediment

Origin/source

Depth of layer

pH

Particle size distribution

Stage of test procedure

field
sampling

post-
handling

start of
acclimation

start of test

during test

end of test

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

(3)

[Clay + silt] is the mineral fraction of the sediment with particle size of < 50 μm

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Parameter

TOC

Microbial biomass **

Redox potential *

Stage of test procedure

field
sampling

post-
handling

start of
acclimation

start of test

during test

end of test

x

x

x

x

x

x

x

x

x

x

x

Observa-
tion (col-
our/smell)

*

Recent research results have shown that measurements of water oxygen concentrations and of redox
potentials have neither a mechanistic nor a predictive value as far as growth and development of
microbial populations in surface waters are concerned (24)(25). Determination of the biochemical
oxygen demand (BOD, at field sampling, start and end of test) and of concentrations of micro/macro
nutrients Ca, Mg and Mn (at start and end of test) in water and the measurement of total N and total
P in sediments (at field sampling and end of test) may be better tools to interpret and evaluate
aerobic biotransformation rates and routes.

** Microbial respiration rate method (26), fumigation method (27) or plate count measurements (e.g.
for aerobic studies; methanogenesis rate for

fungi and total colonies)

bacteria, actinomycetes,
anaerobic studies.

1.8.3

Collection, Handling and Storage

1.8.3.1

Collection

The draft ISO guidance on sampling of bottom sediment (8) should be used for sampling of sediment.
Sediment samples should be taken from the entire 5 to 10 cm upper layer of the sediment. Associated
water should be collected from the same site or location and at the same time as the sediment. For the
anaerobic study, sediment and associated water should be sampled and transported under exclusion of
oxygen (28) (see section 1.8.2.1). Some sampling devices are described in the literature (8)(23).

1.8.3.2

Handling

The sediment is separated from the water by filtration and the sediment wet-sieved to a 2 mm-sieve using
excess location water that is then discarded. Then known amounts of sediments and water are mixed at
the desired ratio (see section 1.9.1) in incubation flasks and prepared for the acclimation period (see
section 1.8.4). For the anaerobic study, all handling steps have to be done under exclusion of oxygen
(29)(30)(31)(32)(33).

1.8.3.3

Storage

Use of freshly sampled sediment and water is strongly recommended, but if storage is necessary, sediment
and water should be sieved as described above and stored together, water-logged (6-10 cm water layer), in
the dark, at 4 ± 2 °C4 for a maximum of 4 weeks (7)(8)(23). Samples to be used for aerobic studies should
be stored with free access of air (e.g.
in open containers), whereas those for anaerobic studies under
exclusion of oxygen. Freezing of sediment and water and drying-out of the sediment must not occur
during transportation and storage.

1.8.4

Preparation of the sediment/water samples for the test

A period of acclimation should take place prior to adding the test substance, with each sediment/water
sample being placed in the incubation vessel to be used in the main test, and the acclimation to be carried
out under exactly the same conditions as the test incubation (see section 1.9.1). The acclimation period is
the time needed to reach reasonable stability of the system, as reflected by pH, oxygen concentration in
water, redox potential of the sediment and water, and macroscopic separation of phases. The period of
acclimation should normally last between one week and two weeks and should not exceed four weeks.
Results of determinations performed during this period should be reported.

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1.9

PERFORMANCE OF THE TEST

1.9.1

Test conditions

The test should be performed in the incubation apparatus (see section 1.8.1) with a water sediment
volume ratio between 3:1 and 4:1, and a sediment layer of 2.5 cm (± 0.5 cm) (4). A minimum amount of
50 g of sediment (dry weight basis) per incubation vessel is recommended.

The test should be performed in the dark at a constant temperature in the range of 10 to 30 °C. A
temperature of (20 ± 2) °C is appropriate. Where appropriate, an additional lower temperature (e.g. 10 °C)
may be considered on a case-by-case basis, depending on the information required from the test.
Incubation temperature should be monitored and reported.

1.9.2

Treatment and application of test substance

One test concentration of chemical is used (5). For crop protection chemicals applied directly to water
bodies, the maximum dosage on the label should be taken as, the maximum application rate calculated on
the basis of the surface area of the water in the test vessel. In all other cases, the concentration to be used
should be based on predictions from environmental emissions. Care must be taken to ensure that an
adequate concentration of test substance is applied in order to characterise the route of transformation
and the formation and decline of transformation products. It may be necessary to apply higher doses (e.g.
10 times) in situations where test substance concentrations are close to limits of detection at the start of
the study and/or where major transformation products could not readily be detected when present at 10 %
of the test substance application rate. However, if higher test concentrations are used they should not have
a significant adverse effect on the microbial activity of the water-sediment system. In order to achieve a
constant concentration of test substance in vessels of differing dimensions an adjustment to the quantity
of the material applied may be considered appropriate, based on the depth of the water column in the
vessel in relation to the depth of water in the field (which is assumed to be 100 cm, but other depths can
be used). See Annex 4 for an example calculation.

Ideally the test substance should be applied as an aqueous solution into the water phase of the test system.
If unavoidable, the use of low amounts of water miscible solvents (such as acetone, ethanol) is permitted
for application and distribution of the test substance, but this should not exceed 1 % v/v and should not
have adverse effects on microbial activity of the test system. Care should be exercised in generating the
aqueous solution of the test substance - use of generator columns and pre-mixing may be appropriate to
ensure complete homogeneity. Following addition of the aqueous solution to the test system, gentle
mixing of the water phase is recommended, disturbing the sediment as little as possible.

The use of formulated products is not routinely recommended as the formulation ingredients may affect
the distribution of the test substance and/or transformation products between water and sediment phases.
However, for poorly water-soluble test substances, the use of formulated material may be an appropriate
alternative.

The number of incubation vessels depends on the number of sampling times (see section 1.9.3). A
sufficient number of test systems should be included so that two systems may be sacrificed at each
sampling time. Where control units of each aquatic sediment system are employed, they should not be
treated with the test substance. The control units can be used to determine the microbial biomass of the
sediment and the total organic carbon of the water and sediment at the termination of the study. Two of
the control units (i.e. one control unit of each aquatic sediment) can be used to monitor the required
parameters in the sediment and water during the acclimation period (see Table in section 1.8.2.2). Two
additional control units have to be included in case the test substance is applied by means of a solvent to
measure adverse effects on the microbial activity of the test system.

1.9.3

Test duration and sampling

The duration of the experiment should normally not exceed 100 days (6), and should continue until the
degradation pathway and water/sediment distribution pattern are established or when 90 % of the test
substance has dissipated by transformation and/or volatilisation. The number of sampling times should be

(4) Recent studies have shown that storage at 4 °C can lead to a decrease of the organic carbon content of the sediment which may

(5)

possibly result in a decrease of microbial activity (34).
Test with a second concentration can be useful for chemicals that reach surface waters by different entry routes resulting in
significantly different concentrations, as long as the lower concentration can be analysed with sufficient accuracy.

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at least six (including zero time), with an optional preliminary study (see section 1.9.4) being used to
establish an appropriate sampling regime and the duration of the test, unless sufficient data is available on
the test substance from previous studies. For hydrophobic test substances, additional sampling points
during the initial period of the study may be necessary in order to determine the rate of distribution
between water and sediment phases.

At appropriate sampling times, whole incubation vessels (in replicate) are removed for analysis. Sediment
and overlying water are analysed separately (6). The surface water should be carefully removed with
minimum disturbance of the sediment. The extraction and characterisation of the test substance and
transformation products should follow appropriate analytical procedures. Care should be taken to remove
material that may have adsorbed to the incubation vessel or to interconnecting tubing used to trap
volatiles.

1.9.4

Optional preliminary test

If duration and sampling regime cannot be estimated from other relevant studies on the test substance, an
optional preliminary test may be considered appropriate, which should be performed using the same test
conditions proposed for the definitive study. Relevant experimental conditions and results from the
preliminary test, if performed, should be briefly reported.

1.9.5

Measurements and analysis

Concentration of the test substance and the transformation products at every sampling time in water and
sediment should be measured and reported (as a concentration and as percentage of applied). In general,
transformation products detected at ≥ 10 % of the applied radioactivity in the total water-sediment system
at any sampling time should be identified unless reasonably justified otherwise. Transformation products
for which concentrations are continuously increasing during the study should also be considered for
identification, even if their concentrations do not exceed the limits given above, as this may indicate
persistence. The latter should be considered on a case by case basis, with justifications being provided in
the report.

Results from gases/volatiles trapping systems (CO2 and others, i.e. volatile organic compounds) should be
reported at each sampling time. Mineralisation rates should be reported. Non-extractable (bound) residues
in sediment are to be reported at each sampling point.

2.

2.1

DATA

TREATMENT OF RESULTS

Total mass balance or recovery (see section 1.7.1) of added radioactivity is to be calculated at every
sampling time. Results should be reported as a percentage of added radioactivity. Distribution of
radioactivity between water and sediment should be reported as concentrations and percentages, at every
sampling time.

Half-life, DT50 and, if appropriate, DT75 and DT90 of the test substance should be calculated along with
their confidence limits (see section 1.7.3). Information on the rate of dissipation of the test substance in
the water and sediment can be obtained through the use of appropriate evaluation tools. These can range
from application of pseudo-first order kinetics, empirical curve-fitting techniques which apply graphical or
numerical solutions and more complex assessments using, for example, single- or multi-compartment
models. Further details can be obtained from the relevant published literature (35)(36)(37).

All approaches have their strengths and weaknesses and vary considerably in complexity. An assumption
of first-order kinetics may be an oversimplification of the degradation and distribution processes, but
when possible gives a term (the rate constant or half-life) which is easily understood and of value in
simulation modelling and calculations of predicted environmental concentrations. Empirical approaches or
linear transformations can result in better fits of curves to data and therefore allow better estimation of
half-lives, DT50 and, if appropriate, DT75 and DT90 values., The use of the derived constants, however, is
limited. Compartment models can generate a number of useful constants of value in risk assessment that

(6)

In cases where rapid re-oxidation of anaerobic transformation products may readily occur, anaerobic conditions should be maintained
during sampling and analysis.

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describe the rate of degradation in different compartments and the distribution of the chemical. They
should also be used for estimation of rate constants for the formation and degradation of major
transformation products. In all cases, the method chosen must be justified and the experimenter should
demonstrate graphically and/or statistically the goodness of fit.

3.

3.1

REPORTING

TEST REPORT

The report must include the following information:

Test substance:

— common name, chemical name, CAS number, structural formula (indicating position of the

label(s) when radiolabelled material is used) and relevant physical-chemical properties;

— purity (impurities) of test substance;

— radiochemical purity of labelled chemical and molar activity (where appropriate).

Reference substances:

— chemical name and structure of reference substances used for the characterisation and/or

identification of transformation products

Test sediments and waters:

— location and description of aquatic sediment sampling site(s) including, if possible, contamination

history;

— all

information relating to the collection, storage (if any) and acclimation of water-sediment

systems;

— characteristics of the water-sediment samples as listed in Table in section 1.8.2.2.

Test conditions:

— test system used (e.g. flow-through, biometer, way of ventilation, method of stirring, water
volume, mass of sediment, thickness of both water and sediment layer, dimension of test vessels,
etc.)

— application of test substance to test system: test concentration used, number of replicates and

controls mode of application of test substance (e.g. use of solvent if any), etc.

— incubation temperature;

— sampling times;

— extraction methods and efficiencies as well as analytical methods and detection limits;

— methods for characterisation/identification of transformation products;

— deviations from the test protocol or test conditions during the study.

Results:

— raw data figures of representative analyses (all raw data have to be stored in the GLP-archive);

— repeatability and sensitivity of the analytical methods used;

— rates of recovery (% values for a valid study are given in section 1.7.1);

— tables of results expressed as % of the applied dose and in mg⋅kg-1 in water, sediment and total
system (% only) for the test substance and, if appropriate, for transformation products and non-
extractable radioactivity;

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— mass balance during and at the end of the studies;

— a graphical representation of the transformation in the water and sediment fractions and in total

system (including mineralisation);

— mineralisation rates;

— half-life, DT50 and,

if appropriate, DT75 and DT90 values for the test substance and, where
appropriate, for major transformation products including confidence limits in water, sediment
and in total system;

— an assessment of the transformation kinetics of the test substance and, where appropriate, the

major transformation products;

— a proposed pathway of transformation, where appropriate;

— discussion of results.

4

REFERENCES

(1)

BBA-Guidelines for the examination of plant protectors in the registration process. (1990). Part IV,
Section 5-1: Degradability and fate of plant protectors in the water/sediment system. Germany.

(2) Commission for registration of pesticides: Application for registration of a pesticide. (1991). Part G.
the product and its metabolites in soil, water and air, Section G.2.1 (a). The

Behaviour of
Netherlands.

(3) MAFF Pesticides Safety Directorate. (1992). Preliminary guideline for the conduct of biodegradability

tests on pesticides in natural sediment/water systems. Ref No SC 9046. United-Kingdom.

(4)

Agriculture Canada: Environmental chemistry and fate.
pesticides in Canada. Aquatic (Laboratory) - Anaerobic and aerobic. Canada. pp 35-37.

(1987). Guidelines for

registration of

(5) US-EPA: Pesticide assessment guidelines, Subdivision N. Chemistry: Environmental

fate (1982).

Section 162-3, Anaerobic aquatic metabolism.

(6)

SETAC-Europe publication. (1995). Procedures for assessing the environmental fate and ecotoxicity of
pesticides. Ed. Dr Mark R. Lynch. SETAC-Europe, Brussels.

(7) OECD Test Guidelines Programme. (1995). Final Report of the OECD Workshop on Selection of

Soils/sediments, Belgirate, Italy, 18-20 January 1995.

(8)

ISO/DIS 5667-12. (1994). Water quality - Sampling - Part 12: Guidance on sampling of bottom
sediments.

(9) US-EPA (1998a). Sediment/water microcosm biodegradation test. Harmonised Test Guidelines (OPPTS

835.3180). EPA 712-C-98-080.

(10) DFG: Pesticide Bound Residues in Soil. Wiley-VCH (1998).

(11) T.R. Roberts: Non-extractable pesticide residues in soils and plants. Pure Appl. Chem. 56, 945-956

(IUPAC 1984).

(12) OECD Test Guideline 304A: Inherent Biodegradability in Soil (adopted 12 May 1981).

(13) OECD (1993): Guidelines for Testing of Chemicals. Paris. OECD (1994-2000): Addenda 6-11 to

Guidelines for the Testing of Chemicals.

(14) Scholz, K., Fritz R., Anderson C. and Spiteller M. (1988) Degradation of pesticides in an aquatic

model ecosystem. BCPC - Pests and Diseases, 3B-4, 149-158.

(15) Guth, J.A. (1981). Experimental approaches to studying the fate of pesticides in soil. In Progress in

Pesticide Biochemistry (D.H. Hutson, T.R. Roberts, Eds.), Vol. 1, 85-114. J. Wiley & Sons.

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(16) Madsen, T., Kristensen, P.

(1997). Effects of bacterial

inoculation and non-ionic surfactants on

degradation of polycyclic aromatic hydrocarbons in soil. Environ. Toxicol. Chem. 16, 631-637.

(17) Steber, J., Wierich, P. (1987). The anaerobic degradation of detergent range fatty alcohol ethoxylates.

Studies with 14C-labelled model surfactants. Water Research 21, 661-667.

(18) Black, C.A. (1965). Methods of Soil Analysis. Agronomy Monograph No. 9. American Society of

Agronomy, Madison.

(19) APHA (1989). Standard Methods for Examination of Water and Wastewater (17th edition). American
Public Health Association, American Water Works Association and Water Pollution Control
Federation, Washington D.C.

(20) Rowell, D.L. (1994). Soil Science Methods and Applications. Longman.

(21) Light, T.S. (1972). Standard solution for redox potential measurements. Anal. Chemistry 44, 1038-

1039.

(22) SETAC-Europe publication (1991). Guidance document on testing procedures for pesticides in
freshwater mesocosms. From the Workshop ‘A Meeting of Experts on Guidelines for Static Field
Mesocosms Tests’, 3-4 July 1991.

(23) SETAC-Europe publication. (1993). Guidance document on sediment toxicity tests and bioassays for
freshwater and marine environments. From the Workshop On Sediment Toxicity Assessment
(WOSTA), 8-10 November 1993. Eds.: I.R. Hill, P. Matthiessen and F. Heimbach.

(24) Vink, J.P.M., van der Zee, S.E.A.T.M. (1997). Pesticide biotransformation in surface waters: multivariate

analyses of environmental factors at field sites. Water Research 31, 2858-2868.

(25) Vink, J.P.M., Schraa, G., van der Zee, S.E.A.T.M. (1999). Nutrient effects on microbial transformation

of pesticides in nitrifying waters. Environ. Toxicol, 329-338.

(26) Anderson, T.H., Domsch, K.H. (1985). Maintenance carbon requirements of actively-metabolising

microbial populations under in-situ conditions. Soil Biol. Biochem. 17, 197-203.

(27)

ISO-14240-2. (1997). Soil quality - Determination of soil microbial biomass - Part 2: Fumigation-
extraction method.

(28) Beelen, P. Van and F. Van Keulen. (1990), The Kinetics of the Degradation of Chloroform and

Benzene in Anaerobic Sediment from the River Rhine. Hydrobiol. Bull. 24 (1), 13-21.

(29) Shelton, D.R. and Tiedje, J.M. (1984). General method for determining anaerobic biodegradation

potential. App. Environ. Microbiol. 47, 850-857.

(30) Birch, R.R., Biver, C., Campagna, R., Gledhill, W.E., Pagga, U., Steber, J., Reust, H. and Bontinck, W.J.

(1989). Screening of chemicals for anaerobic biodegradation. Chemosphere 19, 1527-1550.

(31) Pagga, U. and Beimborn, D.B.

(1993). Anaerobic biodegradation tests for organic compounds.

Chemoshpere 27, 1499-1509.

(32) Nuck, B.A. and Federle, T.W. (1986). A batch test for assessing the mineralisation of 14C-radiolabelled

compounds under realistic anaerobic conditions. Environ. Sci. Technol. 30, 3597-3603.

(33) US-EPA (1998b). Anaerobic biodegradability of organic chemicals. Harmonised Test Guidelines

(OPPTS 835.3400). EPA 712-C-98-090.

(34) Sijm, Haller and Schrap (1997). Influence of storage on sediment characteristics and drying sediment
on sorption coefficients of organic contaminants. Bulletin Environ. Contam. Toxicol. 58, 961-968.

(35) Timme, G., Frehse H. and Laska V. (1986) Statistical interpretation and graphic representation of the
degradational behaviour of pesticide residues II. Pflanzenschutz - Nachrichten Bayer, 39, 187-203.

(36) Timme, G., Frehse, H. (1980) Statistical interpretation and graphic representation of the degradational

behaviour of pesticide residues I. Pflanzenschutz - Nachrichten Bayer, 33, 47-60.

(37) Carlton, R.R. and Allen, R. (1994). The use of a compartment model for evaluating the fate of
pesticides in sediment/water systems. Brighton Crop Protection Conference - Pest and Diseases, pp
1349-1354.

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ANNEX 1

GUIDANCE ON THE AEROBIC AND THE ANAEROBIC TEST SYSTEMS

Aerobic test system

The aerobic test system described in this test method consists of an aerobic water layer (typical oxygen concentrations
range from 7 to 10 mg⋅l-1) and a sediment layer, aerobic at the surface and anaerobic below the surface (typical average
redox potentials (Eh) in the anaerobic zone of the sediment range from –80 to –190 mV). Moistened air is passed over the
surface of the water in each incubation unit to maintain sufficient oxigen in the head space.

Anaerobic test system

For the anaerobic test system, the test procedure is essentially the same as that outlined for the aerobic system with the
exception that moistened nitrogen is passed above the surface of the water in each incubation unit to maintain a head
space of nitrogen. The sediment and water are regarded as anaerobic once the redox potential (Eh) is lower than –100 mV.

In the anaerobic test, assessment of mineralisation includes measurement of evolved carbon dioxide and methane.

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ANNEX 2

EXAMPLE OF A GAS FLOW-THROUGH APPARATUS

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ANNEX 3

EXAMPLE OF A BIOMETER APPARATUS

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ANNEX 4

EXAMPLE CALCULATION FOR APPLICATION DOSE TO TEST VESSELS

Cylinder internal diameter:

Water column depth not including sediment:

Surface area: 3,142 × 42

Application rate: 500 g test substance/ha corresponds to 5 µg/cm2

Total µg: 5 × 50,3

Adjust quantity in relation to a depth of 100 cm: 12 × 251,5 ÷ 100

Volume of water column: 50,3 × 12

Concentration in water: 30,18 ÷ 603

= 8 cm

= 12 cm

= 50,3 cm2

= 251,5 µg

= 30,18 µg

= 603 ml

= 0,050 µg/ml or 50 µg/l ’