Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Limited data is available for Toluene-4-sulphonohydrazide (T). The conclusion is therefore also based on read across to OBSH (S1).

Toluene-4-sulphonohydrazide was mutagenic in two reverse mutation assays (OECD 471) with Salmonella typhimurium and Escherichia coli with and wihtout S9 and in mouse lymphoma mutagenicity assay, but not did not induce chromosomal aberrations.

In summary, OBSH was shown to be mutagenic in two reliable reverse mutation assays (OECD 471) with Salmonella typhimurium and Escherichia coli with and wihtout S9.

Further a reliable a mammalian chromosome aberation test (OECD 473) was positive with and without S9 and also a hepatocyte DNA repair test was positive in rat as well as mouse heaptocytes.

In an in vivo OECD 474 OBSH did not induce micronuclei and chromosome aberations in mouse erythrocytes. Although no structural anomalies were found in this vivo test, OBSH with its reactive hydrazide structure most still be highly suspected to be a mutagen due to the clear positive in vitro tests showing direct mutagenic response, clastogenic repsonse and impact on DNA repair.

Based on this it is considered most appropriate to assign a classification with Muta 2 H341 to OBSH, as further more targeted in vivo testing on the direct mutagenic properties is considered superfluous as positive response is considered to be the most probable outcome.

Based on read-across to these data Toluene-4-sulphonohydrazide (T) should also be classified with Muta 2 H341, as well. 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read-across from chromosome aberration study on one closely structural and physicochemical related substance: OBSH (4,4'-Oxybis (benzenesulfonyl hydrazide)).
For further read-across justification se document attached in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Species / strain:
other: CHL/IU cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
-S9: at 1360 µg/mL and above. +S9: at 696µg/mL and above
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Conclusions:
No data is available for Toluene-4-sulphonohydrazide (T).
The genetic toxicity of OBSH was evaluated in an in vitro Mammalian chromosome aberration test with chinese hamster lung (CHL) cells. The study was performed in accordance with the OECD guideline 473. OBSH exhibited clastogenic activity on cultured Chinese Hamster Lung cells.
Due to the close chemical similarity to Toluene-4-sulphonohydrazide the clastogenic potential may be considered relevant for this substances as well.
Executive summary:

No data is available for Toluene-4-sulphonohydrazide (T).

The genetic toxicity of OBSH was evaluated in an in vitro Mammalian chromosome aberration test with chinese hamster lung (CHL) cells. The study was performed in accordance with the OECD guideline 473. OBSH exhibited clastogenic activity on cultured Chinese Hamster Lung cells.

Due to the close chemical similarity to Toluene-4-sulphonohydrazide theclastogenicpotential may be considered relevant for this substances as well.

See justification for read-across attached in section 13

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read-across from Ames test studies on one closely structural and physicochemical related substance: OBSH (4,4'-Oxybis (benzenesulfonyl hydrazide)).
For further read-across justification se document attached in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Remarks on result:
other: Study II
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Remarks on result:
other: Study II
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Remarks on result:
other: Study II
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
at 3000µg/plate
Remarks on result:
other: Study I
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Remarks on result:
other: Study I
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
at 3000µg/plate
Remarks on result:
other: Study I
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Remarks on result:
other: Study I
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
at 3000µg/plate
Remarks on result:
other: Study I
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Remarks on result:
other: Study I
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
300µg/plate
Remarks on result:
other: Study I
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Remarks on result:
other: Study I
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Remarks on result:
other: Study I
Conclusions:
No data is available for Toluene-4-sulphonohydrazide (T). The source substance OBSH demonstrated a mutagenic potential in both studies and therefore considered mutagenic in bacteria.
Due to the close chemical similarity to Toluene-4-sulphonohydrazide the mutagenic potential may be considered relevant for this substances as well.
Executive summary:

No data is available for Toluene-4-sulphonohydrazide (T).

The genetic toxicity in vitro of OBSH was evaluation in two studies using bacterial reverse mutation assay. The studies were performed in accordance with the OECD guideline 471. In the first study OBSH showed mutagenic effects in Salmonella typhimurium TA 98, TA 100, and TA 1535 without metabolic activation and in Salmonella typhimurium TA 100 and TA 1535 with metabolic activation system. In the second study, OBSH showed mutagenic effects in Salmonella typhimurium TA 100, TA 1535, and WP2 uvrA without and with metabolic activation system.

Conclusively OBSH demonstrated a mutagenic potential in both studies and therefore considered mutagenic in bacteria.

Due to the close chemical similarity to Toluene-4-sulphonohydrazide the mutagenic potential may be considered relevant for this substances as well.

See justification for read-across attached in section 13

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read-across from DNA damage/repair study on one closely structural and physicochemical related substance: OBSH (4,4'-Oxybis (benzenesulfonyl hydrazide)).
For further read-across justification se document attached in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Species / strain:
hepatocytes:
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: heaptocytes
Conclusions:
No data is available for Toluene-4-sulphonohydrazide (T).

The genetic toxicity of OBSH was evaluated in using a protocol similar with the OECD guideline 482. In the DNA repair test with rat hepatocytes OBSH elicited positive DNA repair synthesis and the nuclear grain counts were roughly related to the doses.
OBSH was positive in the DNA repair test with mouse hepatocytes.
The test substance was positive in the DNA repair test with hepatocytes of rat and mouse.
Due to the close chemical similarity to Toluene-4-sulphonohydrazide it may a have a similar effects on DNA repair as well.
Executive summary:

No data is available for Toluene-4-sulphonohydrazide (T).

The genetic toxicity of OBSH was evaluated in using a protocol similar with the OECD guideline 482. In the DNA repair test with rat hepatocytes OBSH elicited positive DNA repair synthesis and the nuclear grain counts were roughly related to the doses.

OBSH was positive in the DNA repair test with mouse hepatocytes.

The test substance was positive in the DNA repair test with hepatocytes of rat and mouse.

Due to the close chemical similarity to Toluene-4-sulphonohydrazide it may a have a similar effects on DNA repair as well.

See justification for read-across attached in section 13

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was performed in accordance with the OECD guideline 471 and the GLP standard.
Reason / purpose:
reference to same study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Principles of method if other than guideline:
NA
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
no data
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell lines (if applicable):
NA
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Male rat, liver, S9 mix induced with Aroclor 1254
Test concentrations with justification for top dose:
11.1, 33.3, 100, 300, 1,000, and 3,000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Negative controls:
yes
Remarks:
solvent
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 4-nitroquinoline and 2-aminoanthracene
Details on test system and conditions:
Description of follow up repeat study: Dose range finding experiment was carried out using dose levels with 5-fold intervals of 1.6, 8, 40, 200, 1,000, and 5,000 µg/plate both in the absence and in the presence of metabolic activation system.
Evaluation criteria:
The number of revertant colonies increased significantly in at least one strain at one or more concentrations or the data set showed a dose related correlation.
Statistics:
no data
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
at 3000 µg/plate
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
at 3000 µg/plate
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
at 3000 µg/plate
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
above 300 µg/plate
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Additional information on results:
none

Table.Result of bacterial reverse mutation assay with4,4’-oxybis(benzenesulfonyl hydrazide)

Tester strain

Chemical treated

Dose (mg/plate)

Colonies/plate (mean±SD)

without S9 mix

with S9 mix

TA 98

Test item

    0

31±1

32±3

      11.1

20±6

29±3

     33.3

24±5

35±2

  100

34±3

40±6

  300

101±2

30±10

1,000

305±741

44±4

3,000

0±04

41±9

TA 100

Test item

    0

100±16

113±0

      11.1

189±83

169±16

     33.3

219±7

269±12

  100

315±13

439±18

  300

349±1

438±68

1,000

302±12

318±8

3,000

0±04

349±8

TA 1535

Test item

    0

10±0

8±4

      11.1

73±8

120±10

     33.3

118±3

295±13

  100

243±2

437±61

  300

187±20

320±61

1,000

12±32

259±15

3,000

0±04

449±47

TA 1537

Test item

    0

14±3

16±1

      11.1

24±2

13±4

     33.3

18±1

17±1

  100

12±6

17±0

  300

-   3

18±4

1,000

-   3

24±1

3,000

0±04

16±4

E. coli

WP2uvrA

Test item

    0

15±1

15±2

      11.1

12±1

21±1

     33.3

13±6

15±6

  100

22±4

13±1

  300

19±1

15±3

1,000

22±6

15±3

3,000

13±2

22±2

Positive control

TA 98

2-NF

    1.0

209±1

 

2-AA

    2.0

 

550±88

TA 100

SA

    0.5

502±62

 

2-AA

    2.0

 

699±13

TA 1535

SA

    0.5

251±24

 

2-AA

    5.0

 

131±9

TA 1537

9-AA

  50.0

355±8

 

2-AA

    5.0

 

259±39

WP2uvrA

4-NQ

    2.0

926±86

 

2-AA

10

 

297±115

1;Colony counting and slight thin lawn, some very slight toxicity                                                                   

2;Colony counting and thin lawn but microcolonies still evident

3;No colony count and thin lawn and probably macroscopic colonies grown from microcolonies

4;Complete killing

2-NF; 2-Nitrofluorene, 2-AA; 2-Aminoanthracene, SA; Sodium azide, 9-AA; 9-Aminoacridine, 4-NQ; 4-Nitroquinoline

Conclusions:
Interpretation of results (migrated information):
positive

OBSH showed mutagenic effects in Salmonella typhimurium TA 98, TA 100, and TA 1535 without metabolic activation and in Salmonella typhimurium TA 100 and TA 1535 with metabolic activation system.
Executive summary:

The genetic toxicity in vitro of OBSH was evaluation in a bacterial reverse mutation assay. The study was performed in accordance with the OECD guideline 471. OBSH showed mutagenic effects in Salmonella typhimurium TA 98, TA 100, and TA 1535 without metabolic activation and in Salmonella typhimurium TA 100 and TA 1535 with metabolic activation system.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed in accordance with the guidelines for screening mutagenicity testing of chemicals (chenical substances control law of Japan) and OECD TG 471. The study was performed in accordance with GLP standard.
Reason / purpose:
reference to same study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
no data
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell lines (if applicable):
NA
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
male rat, liver, S9 mix induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
TA 100: 4.88, 9.77, 19.5, 39.1, 78.1, and 156 µg /plate (with and without S9 mix)
TA 1535: 0.61, 1.22, 2.44, 4.88, 9.77, and 19.5 µg/plate (with S9 mix)
Ta1535: 0.61, 1.22, 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate (without S9 mix)
TA 98 and TA 1537: 313, 625, 1,250, 2,500, and 5,000 µg/plate (with and without S9 mix)
WP2 uvrA: 39.1, 78.1, 156, 313, 625, and 1,250 µg/plate (with and without S9 mix)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Negative controls:
yes
Remarks:
solvent
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 9-Aminoacridine hydrochloride, and 2-Aminoanthracene
Details on test system and conditions:
Description of follow up repeat study: Concentration range finding experiment was carried out using concentration levels with 3-fold intervals of 2.29, 6.6, 20.6, 61.7, 185, 556, 1,667, and 5,000 µg/plate both in the absence and in the presence of metabolic activation system.
Evaluation criteria:
Criteria for determining a positive result is the number of revertant colonies increased double over in at least one strain at one or more concentrations and the data set showed a reproducible increase or a dose related correlation.
Statistics:
no data
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Additional information on results:
CYTOTOXIC CONCENTRATION:
Without and with metabolic activation: not observed

GENOTOXIC EFFECT:
Without and with metabolic activation: positive in TA 100, TA 1535 and WP2 uvrA

Table1.Results of bacterial reverse assay of 4,4’-oxybis(benzenesulfonylhydrazide) [non-activation method: -S9mix]

Compound

Concentration

(mg/plate)

Revertant coloniesperplate (Mean±S.D.)

TA 100

TA1535

WP2uvrA

TA98

TA1537

DMSOa)

0

115±17

14±0

15±6

27±6

6±1

Testsubstance

2.44

 

21±3

 

 

 

4.88

143±12

35±4

 

 

 

9.77

146±13

53±6

 

 

 

19.5

191±15

89±10

 

 

 

39.1

246±12

139±22

25±1

 

 

78.1

327±18

209±22

21±7

 

 

156

443±25

 

27±4

 

 

313

 

 

62±5

32±2

7±1

625

 

 

96±2

37±8

11±4

1,250

 

 

124±5

36±4

6±3

 2,500+

 

 

 

44±14

11±2

 5,000+

 

 

 

38±3

14±5

Positive control

 

734±21b)

459±3c)

173±26b)

644±8d)

196±19e)

a) Negative control (Dimethyl sulfoxide)   b) AF-2; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 0.01mg/plate c) NaN3;Sodium azide 0.5mg/plate   d)AF-2, 0.1mg/plate   e)9-AA; 9-Aminoacridinehydrochloride,80mg/plate

  +: Visible precipitation was shown at the end of exposure period

Table2.Results of bacterial reverse assay of 4,4’-oxybis(benzenesulfonylhydrazide) [Activation method: +S9mix]

Compound

Concentration

(mg/plate)

Revertant coloniesperplate (Mean±S.D.)

TA 100

TA1535

WP2uvrA

TA98

TA1537

DMSOa)

0

113±5

9±4

20±3

35±6

14±2

Testsubstance

 0.61

 

17±8

 

 

 

1.22

 

21±6

 

 

 

2.44

 

30±3

 

 

 

4.88

140±26

65±3

 

 

 

9.77

173±21

105±1

 

 

 

19.5

220±29

164±11

 

 

 

39.1

283±20

 

22±5

 

 

78.1

337±24

 

27±10

 

 

156

385±25

 

32±4

 

 

313

 

 

37±7

30±9

16±4

625

 

 

63±6

31±6

13±5

1,250

 

 

107±13

40±9

20±2

      2,500+

 

 

 

27±8

17±4

 5,000+

 

 

 

39±7

13±2

Positive control

 

920±13b)

268±17c)

659±61d)

414±22e)

149±19c)

a) Negative control (Dimethyl sulfoxide)                                                                            

b) 2-AA; 2-Aminoanthracene, 1mg/plate   c) 2-AA, 1mg/plate     d) 2-AA, 10mg/plate    e) 2-AA, 0.5mg/plate

+: Visible precipitation was shown at the end of exposure period

Conclusions:
Interpretation of results (migrated information):
positive

OBSH showed mutagenic effects in Salmonella typhimurium TA 100, TA 1535, and WP2 uvrA without and with metabolic activation system.
Executive summary:

The genetic toxicity in vitro of OBSH was evaluation in a bacterial reverse mutation assay.

The study was performed in accordance with the guideline for screening mutagenicity testing of chemicals (chenical substances control law of Japan) and the OECD guideline 471. OBSH showed mutagenic effects in Salmonella typhimurium TA 100, TA 1535, and WP2 uvrA without and with metabolic activation system.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed in accordance with the OECD guideline 473, and in compliance with GLP standard.
Reason / purpose:
reference to same study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Target gene:
NA
Species / strain:
other: Chinese Hamster Lung (CHL/IU) Cells
Details on mammalian cell lines (if applicable):
NA
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat, malem, liver, 30% S9 mix induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Absence metabolic system; 0, 557, 696, 870, 1,088, 1,360, and 1,700 µg/mL
Presence metabolic system; 0, 357, 446, 557, 696, 870, and 1,088 µg/mL
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Negative controls:
yes
Remarks:
solvent
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and conditions:
Culture establishment: CHL cells in logarithmic were to maintain low aberration frequencies. Subcultured at low density, before over growth occurs. The cells were incubated at 37 C in atmosphere of 5 % (v/v) CO2 and 100 % humidity.

Description of follow up repeat study: Series of seven and ten concentration levels with 1.25-fold intervals were tested in the absence and the presence of metabolic activation system.
Evaluation criteria:
The test was considered as positive in this assay if; 1) Statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at one or more concentration levels 2) 1) exceed the normal range, 3) If the result of 1) is reproducible.
Statistics:
Fisher’s exact test and Chochran Armitage’s trend test (p-value: 0.05)
Species / strain:
other: Chinese Hamster Lung (CHL/IU) Cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
-S9: at 1360 µg/mL and above. +S9: at 696µg/mL and above
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
None

Table 1. Chromosome aberration test on CHL cells treated with 4,4’-oxybis(benzenesulfonylhydrazide) [short-term treatment: -S9 mix]

Compound

Concentration (mg/mL)

Time of exposure (hr)

Relative cell growth (%)

No. of cells analyzed

No. of cells with aberrations –gap (%)

No. of cells analyzed for polyploid

No. of polyploid cells (%)

Final Judgment

DMSOa)

0

6

100.0

200

0 (0.0)#

200

1 (0.5)

 

Test substance

557

6

47.7

200

8 (4.0)*

200

1 (0.5)

 

696

6

37.7

200

21 (10.5)*

200

4 (2.0)

 

870

6

33.1

200

27 (13.5)*

200

0 (0.0)

Positive

1088

6

18.0

200

6 (3.0)*

200

2 (1.0)

 

1360c)

6

9.1

Toxic

 

 

 

 

1700c)

6

3.0

Toxic

 

 

 

 

MMCb)

0.1

6

57.2

200

88 (44.0)*

200

0 (0.0)

Positive

-gap: total number of cells with aberrations except gap

# p≤0.05: Significant difference by trend test (Cochran-Armitage trend test)

* p≤0.05: Significant difference from the negative control group (Fisher’s exact test)

a) Negative control (Dimethyl sulfoxide),                  b) Positive control (Mitomycin C)

c) Visible precipitation was shown at the end of exposure period

Table 2. Chromosome aberration test on CHL cells treated with 4,4’-oxybis(benzenesulfonylhydrazide) [short-term treatment: +S9 mix]

Compound

Concentration (mg/mL)

Time of exposure (hr)

Relative cell growth (%)

No. of cells analyzed

No. of cells with aberrations –gap (%)

No. of cells analyzed for polyploid

No. of polyploid cells (%)

Final Judgment

DMSOa)

0

6

100.0

200

0 (0.0)#

200

2(1.0)

 

Test substance

357

6

58.7

200

3 (1.5)*

200

3(1.5)

 

446

6

   46.9

200

6 (3.0)*

200

2(1.0)

 

557

6

18.2

200

29 (14.5)*

200

0(0.0)

Positive

696

6

4.8

Toxic

 

 

 

 

870

6

3.3

Toxic

 

 

 

 

1088

6

2.4

Toxic

 

 

 

 

CPb)

12.5

6

85.4

200

66 (33.0)*

200

1(0.5)

Positive

-gap: total number of cells with aberrations except gap

# p≤0.05: Significant difference by trend test (Cochran-Armitage trend test)

* p≤0.05: Significant difference from the negative control group (Fisher’s exact test)

a) Negative control (Dimethyl sulfoxide),                   b) Positive control (Cyclophosphamide)

Conclusions:
Interpretation of results (migrated information):
positive

The genetic toxicity of OBSH was evaluated in an in vitro Mammalian chromosome aberration test on chinese hamster ling cells. The study was performed in accordance with the OECD guideline 473. OBSH exhibited clastogenic activity on cultured Chinese Hamster Lung cells.
Executive summary:

The genetic toxicity of OBSH was evaluated in an in vitro Mammalian chromosome aberration test on chinese hamster lung cells. The study was performed in accordance with the OECD guideline 473.

Significant dose-dependent increases in the proportion of cells with structural aberrations were observed in cultures both in the absence (557 to 870mg/mL) and presence (446 to 557mg/mL) metabolic activation system. Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all concentration levels.

In conclusion, OBSH exhibited clastogenic activity on cultured Chinese Hamster Lung cells both with and without S9.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Score evaluated in the OECD SIDS and considered to be well documented meeting generally scientific principles and acceptable for assessment
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
not specified
GLP compliance:
not specified
Remarks:
Study by OECD indicated to be well documented meeting generally scientific principles and acceptable for assessment
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Test material information:
Composition 1
Test concentrations with justification for top dose:
0.001; 0.0001; 0.00001 M
positive control: N-2-fluorenylacetamide in rat hepatocyte assay
positive control: 9,10.dimethyl-1,2-benz(a)anthracene in mouse hepatocyte assay
Vehicle:
DMSO
Negative controls:
not specified
Solvent controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
other: N-2-fluorenylacetamide
Details on test system and conditions:
Hepatocytes were isolated from the livers of ACI/N rats weighing 200 ~ 250 g and C3H/HeN mice weighing about 20 g. The isolated hepatocytes were allowed to attach for 2 hr to plastic cover slips in primary culture using William Medium E. The culture were then washed and exposed to the test compounds and [Me-3H] thymidine for 20 hr.

- Solvent: Dimethyl sulfoxide
- Number of replicate: 2
- Positive control: N-2-fluorenylacetamide was used in the assay with rat hepatocytes, and 9,10-dimethyl-1,2-benz[a]anthracene was used in the assay with mouse hepatocytes.
- Description of follow up study: At the end of incubation, the cultures were washed in Williams’ Medium E, and the coverslips were mounted on glass slides. The slides were dipped in Sakura NR-M2 photographic emulsions and exposed for 14 days. Autoradiographic grains were counted on a CRT display with a microscopic attachment.

Evaluation criteria:
Criteria for evaluating results: Each cell nucleus was considered positive when the net nuclear grain count was more than 5 grains above the cytoplasmic background. The test compounds were judged positive when the mean nuclear grain count was more than 5 grains or the positive cells were more than 33 %.
Statistics:
NA
Species / strain:
hepatocytes:
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: heaptocytes
Remarks:
Migrated from field 'Test system'.
Additional information on results:
In the DNA repair test with rat hepatocytes, the test substance elicited positive DNA repair synthesis and the nuclear grain counts were roughly related to the doses. OBSH was positive in the DNA repair test with mouse hepatocytes.
The test substance was positive in the DNA repair test with hepatocytes of rat and mouse.
Conclusions:
In the DNA repair test with rat hepatocytes, the test substance elicited positive DNA repair synthesis and the nuclear grain counts were roughly related to the doses.
OBSH was positive in the DNA repair test with mouse hepatocytes.
The test substance was positive in the DNA repair test with hepatocytes of rat and mouse.
Executive summary:

OBSH was tested in a rat + mouse heaptocyte/ DNA repair test using concentrations of 0.001; 0.0001; 0.00001 M of OBSH in a vehicle of DMSO.

Positive control: N-2-fluorenylacetamide was used in the assay with rat hepatocytes, and 9,10-dimethyl-1,2-benz[a]anthracene was used in the assay with mouse hepatocytes.

In the DNA repair test with rat hepatocytes, the test substance elicited positive DNA repair synthesis and the nuclear grain counts were roughly related to the doses.

OBSH was positive in the DNA repair test with mouse hepatocytes.

The test substance was positive in the DNA repair test with hepatocytes of rat and mouse.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Principles of method if other than guideline:
NA
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
no data
Species / strain:
S. typhimurium TA 100
Details on mammalian cell lines (if applicable):
NA
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 mix, male Sprague Dawley rats, induced with Aroclor 1254
Test concentrations with justification for top dose:
100-10000 µg/plate
Vehicle:
No data
Negative controls:
not specified
Solvent controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and conditions:
N/A
Evaluation criteria:
To be considered positive, it had to induce at least a doubling in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose
response to increasing concentrations of the test chemical.
Statistics:
no data
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
at 100 µg/plate
Cytotoxicity:
not specified
Additional information on results:
none
Conclusions:
p-toluenesulfonic hydrazide showed mutagenic effects in Salmonella typhimurium TA 100 at 100 µg/plate with and without metabolic activation system.
Executive summary:

The genetic toxicity of p-toluenesulfonic hydrazide (p-TSH) was evaluation in a bacterial reverse mutation assay. The study was performed in equivalence to OECD guideline 471.

p-TSH showed mutagenic effects in Salmonella typhimurium TA 100 at 100  µg/plate with and without metabolic activation system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
other: TA 97, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Trial 1: control (DSMO), 5, 10, 50, 100, 500, 100
Trial 2: control (Acetone), 7.5, 25, 75, 250, 750, 2500
Vehicle:
- Vehicle(s)/solvent(s) used:
Trial 1: DMSO
Trial 2: acetone
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Species / strain:
other: TA 97, TA98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Positive controls valid:
yes
Conclusions:
In this study the mutagenic acitivity of TSH was determined in the salmonella/mammalian microsome assay. TSH induced gene mutations both with and without metabolic activation. This study indicates that TSH is mutagenic in vitro.
Executive summary:

In this study the mutagenic acitivity of TSH was determined in the salmonella/mammalian microsome assay. TSH induced gene mutations both with and without metabolic activation. This study indicates that TSH is mutagenic in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254-induced rat liver homogenate (S9)
Test concentrations with justification for top dose:
Trial 1 (10 h): 0, 0.1, 0.3, 0.6, 1.0 mM
Trial 2 (16 h): 0, 0.1, 0.3, 0.6, 1.0 mM

The top dose was the highest dose, at which an adequate number of metaphase cells could be recovered
Vehicle:
serum free complete medium
Negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Negative controls valid:
yes
Positive controls valid:
yes

In two seperate trials, there were no significant increase in the percentage of cells with chromosomal aberrations or the number of chromosomal aberrations per cell measured either 10 or 16 h following treatment. In all experiments, the top dose was the highest dose at which an adequate number of metaphase cells could be recovered

Conclusions:
TSH does not induce chromosomal aberrations in CHO cells.
Executive summary:

In this study the mutagenic acitivity of TSH was determined in the in vitro chromosomal aberration assay using Chinese hamster ovary (CHO) cells. TSH did not induce chromosomal aberrations in the CHO cells. In two seperate trials, there were no significant increase in the percentage of cells with chromosomal aberrations or the number of chromosomal aberrations per cell measured either 10 or 16 h following treatment. In all experiments, the top dose was the highest dose at which an adequate number of metaphase cells could be recovered. The results demonstrate that TSH does not induce chromosomal aberrations in CHO cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Type of assay:
other: Mouse lymphoma mutagenicity assay
Test material information:
Composition 1
Specific details on test material used for the study:
No data
Target gene:
N/A
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 miix, male rat liver, Aroclor 1254 induced
Test concentrations with justification for top dose:
100-2400 µg/mL
Vehicle:
Not specified
Negative controls:
not specified
Solvent controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Evaluation criteria:
Mutant frequencies were expressed as mutants per 106 surviving cells. Although there are several different methods for evaluating mouse lymphoma data, results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 1000 µg/mL
Cytotoxicity:
not specified
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity:
not determined
Conclusions:
p-toluenesulfonic hydrazide showed mutagenic effects in mouse lymphoma L5178Y at 1000 µg/mL without S9 metabolic activation system. The results were equivocal with S9 metabolic activation system
Executive summary:

The genetic toxicity of p-toluenesulfonic hydrazide (p-TSH)was evaluation in a Mouse lymphoma mutagenicity assay. The study was performed in equivalence to OECD guideline 476.

p-TSH showed mutagenic effects in mouse lymphoma L5178Y at 1000 µg/mL without S9 metabolic activation system. The results were equivocal with S9 metabolic activation system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed in accordance with the OECD guideline 474, and in compliace with GLP standard.
Reason / purpose:
reference to same study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Principles of method if other than guideline:
NA
GLP compliance:
yes
Type of assay:
micronucleus assay
Test material information:
Composition 1
Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: 8 weeks
- Weight at study initiation: 28.5 - 36.0 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
no data

IN-LIFE DATES: no data
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: MC (methyl cellulose)
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): 0.5% MC
- Type and concentration of dispersant aid (if powder): no data
- Lot/batch no. (if required): 126H0394
- Purity: no data
Details on exposure:
no data
Duration of treatment / exposure:
two treatments at 24 hour intervals
Frequency of treatment:
duplicate treatments (positive control group was dosed once)
Post exposure period:
24 hours
Remarks:
Doses / Concentrations:
0, 375, 750 and 1500 mg/kg bw/day
Basis:

No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide.H2O
- Justification for choice of positive control(s): no data
- Route of administration: intraperitoneal injection
- Doses / concentrations: 70mg/kg
Tissues and cell types examined:
erythrocytes
Details of tissue and slide preparation:
no data
Evaluation criteria:
At least 2,000 polychromatic erythrocytes per animals were scored for the incidence of micronuclei.
There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time.
Statistics:
To compare the results of the control and treatment groups, Kruskal-Wallis’ H-test was carried out. If this result was significant, Dunnett’s t-test was conducted to find out significance. If there was a significant difference, Cochran-Armitage trend test was conducted to confirm the dose-dependency. Mann-Whitney’s U-test was carried out to compare the results of the control and positive controls.
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
All animals dosed with OBSH exhibited similar PCE/(PCE + NCE) ratios and MNPCE frequencies compared to those of negative control animals (p > 0.01). All frequencies of MNPCE in the negative control groups fell within acceptable ranges, while the positive control groups induced clear increase in the frequencies of MNPCE.

Table. Summary of PCE/(PCE+NCE) ratio and MNPCE frequency

Treatment group

Dose (mg/kg)

No. of Animal

 PCE/(PCE+NCE)

(mean±S.D.)

MNPCE per2,000 PCE

(mean±S.D.)

Vehicle(0.5 % methyl cellulose)

      0

6

0.43 ( 0.11

0.67 ( 0.82

OBSH

  375

6

0.47 ( 0.04

0.67 ( 0.82

 

  750

6

0.47 ( 0.05

1.00 ( 0.89

 

1,500

6

0.45 ( 0.03

1.33 ( 0.82

Cyclophosphamide

    70

6

0.37( 0.04

75.50 ( 22.41*

*: Significant difference from the control at p < 0.01

MNPCE; Micronucleated polychromatic erythrocyte,

PCE; Polychromatic erythrocyte,

NCE; Normochromatic erythrocyte,
Conclusions:
Interpretation of results (migrated information): negative
The genetic toxicity in vivo of OBSH was evaluated in a Mammalian erythrocyte micronucleus test. The test was performed in accordance with the OECD guideline 474. It was concluded that OBSH did not induce micronuclei in the mice bone marrow cells.
Executive summary:

The genetic toxicity in vivo of OBSH was evaluated in a Mammalian erythrocyte micronucleus test. The test was performed in accordance with the OECD guideline 474.

All animals dosed with OBSH exhibited similar PCE/(PCE + NCE) ratios and MNPCE frequencies compared to those of negative control animals (p > 0.01). All frequencies of MNPCE in the negative control groups fell within acceptable ranges, while the positive control groups induced clear increase in the frequencies of MNPCE.

It was concluded that OBSH did not induce micronuclei in the mice bone marrow cells.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read-across from micronucleus study on one closely structural and physicochemical related substance: OBSH (4,4'-Oxybis (benzenesulfonyl hydrazide)).
For further read-across justification se document attached in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Executive summary:

No data is available for Toluene-4-sulphonohydrazide (T).

The genetic toxicity in vivo of OBSH was evaluated in accordance with the OECD guideline 474 in a Mammalian erythrocyte micronucleus test.

All animals dosed with OBSH exhibited similar PCE/(PCE + NCE) ratios and MNPCE frequencies compared to those of negative control animals (p > 0.01). All frequencies of MNPCE in the negative control groups fell within acceptable ranges, while the positive control groups induced clear increase in the frequencies of MNPCE.

It was concluded that OBSH did not induce micronuclei in the mice bone marrow cells.

Due to the close chemical similarity to Toluene-4-sulphonohydrazide this value may be considered relevant for this substances as well.

See justification for read-across attached in section 13.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

OBSH was shown to be mutagenic in two reliable reverse mutation assays (OECD 471) with Salmonella typhimurium and Escherichia coli with and wihtout S9.

Further a reliable a mammalian chromosome aberation test (OECD 473) was positive with and without S9 and further a hepatocyte DNA repair test was positive in rat as well as mouse heaptocytes.

The genetic toxicity of OBSH in vivo was evaluated in a mammalian erythrocyte micronucleus test (OECD 474). It was concluded that OBSH did not induce micronuclei in mouse erythrocytes (negative).

Although no structural anomalies were found in this vivo test OBSH must be suspeced to be a mutagen due to the clear positive in vitro tests showing direct mutagenic response, clastogenic response and response on DNA repair.

Hence


Short description of key information:
In in vitro bacterial reverse mutation tests (OECD TG 471), OBSH showed positive results in Salmonella typhimurium strains TA 98, TA 100, TA 1535 and/or Escherichia coli (WP2 uvrA) with or without S9 mix. In a chromosomal aberration test (OECD TG 473) with CHL cells and in a DNA repair test with rat and mouse hepatocytes, OBSH elicited positive results. However, in an in vivo mammalian erythrocyte micronucleus assay (OECD TG 474), OBSH did not exhibit mutagenic effects in mouse bone marrow cells at doses ranging from 375 to 1,500 mg/kg bw.

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

In summary, OBSH was shown to be mutagenic in two reliable reverse mutation assays (OECD 471) with Salmonella typhimurium and Escherichia coli with and wihtout S9.

Further a reliable a mammalian chromosome aberation test (OECD 473) was positive with and without S9 and also a hepatocyte DNA repair test was positive in rat as well as mouse heaptocytes.

In an in vivo OECD 474 OBSH did not induce micronuclei and chromosome aberations in mouse erythrocytes. Although no structural anomalies were found in this vivo test, OBSH with its reactive hydrazide structure most still be highly suspected to be a mutagen due to the clear positive in vitro tests showing direct mutagenic response, clastogenic repsonse and impact on DNA repair.

Based on this it is considered most appropriate to assign a classification with Muta 2 H341 to OBSH (S1), as further more targeted in vivo testing on the direct mutagenic properties is considered superfluous as positive response is considered to be the most probable outcome.

Based on read-across to these data Toluene-4-sulphonohydrazide (T) should also be classified with Muta 2 H341, as well.