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Genetic toxicity in vitro

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of test substance (as cited in study report): Bumetrizole
- Analytical purity: 99.9 % w/w
- Lot/batch No.: 01721IW4
- Stability under test conditions: stable
- Storage condition of test material: stored sealed in a cabinet at 20.0 - 25.3 °C
- Other: Supplier: Ciba Specialty Chemicals, Osaka, Japan
Target gene:
not applicable
Species / strain / cell type:
mammalian cell line, other: CHL/IU
Details on mammalian cell type (if applicable):
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver induced with phenobarbital and 5,6-benzoflavone (see table 1)
Test concentrations with justification for top dose:
Cell growth inhibition test:
Short treatment test (6 hr test/18 hr recovery): 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1600 or 3200 µg/mL
Continuous treatment test (24 hour test/0 hour recovery): 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1600 or 3200 µg/mL

Chromosome aberration test:
Short treatment test (6 hr test/18 hr recovery): 150, 300, 600, 1200 or 2400 µg/mL
Continuous treatment test (24 hour test/0 hour recovery): 75, 150, 300, 600 or 1200 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO is commonly used for the reverse mutagenicity test on bacteria and it forms a good suspension.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: see table 2
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Pulse treatment: 6 hours; Continuous treatment: 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 in total per dose

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
Evaluation criteria:
The test result was considered negative when the occurrence rate of cells with numerical or structural aberrations was below 5% and false positive for a rate between 5-10 % and positive for above 10 % with a dose related increase. The occurence rate of cells with structural aberrations was calculated with/without gaps and judged by the occurrence rate without gaps.
Statistics:
none
Species / strain:
mammalian cell line, other: CHL/IU
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 1200 µg/plate (pulse treatment), > 600 µg/plate (continuous treatment)
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

1. Short Treatment Method (results of the tests are shown in Tables 3 and 4.)

The rate of cells with numerical aberrations was below 1.5% for all concentrations with and without S9 mix. The rate of cells with structural aberrations was below 1.0% in all concentrations with and without S9 mix.

The cell survival rate in plates with S9 mix was more than 90% for 150 and 300 µg/mL. At concentrations above 600 µg/mL, survival rate decreased proportionally to concentrations, reaching 34% at the maximum concentration of 2400 µg/mL. The cell survival rate without S9 mix was more than 90% for 150 and 300 µg/mL. At concentrations above 600 µg/mL the value decreased in proportion to higher concentration, attaining 34% at the maximum concentration of 2400 µg/mL.

A white oil filmy precipitate and white impalpable precipitates were observed in all concentrations.

In the negative control, 0% and 0.5% of cells carried numerical aberrations in the absence of S9 mix and presence of S9 mix, respectively. The occurrence rate of cells with structural aberrations was 0% in the presence of S9 mix and 0.5% in the absence of S9 mix. In the positive controls, the occurrence rate of cells with numerical aberrations with (DMN; 500 µg/mL) and without S9 mix (MMC;0.1 µg/mL) was 0%. The occurrence rate of cells with structural aberrations was 72.5% with S9 mix and 52.5% without S9 mix. In conclusion, the induction rate of chromosomal aberrations in the negative and positive control groups was within the range of background thus satisfying the crieria of a valid test.

2. Continuous Treatment Method (test results are shown in Table 5)

The occurrence rate of cells with numerical aberrations in the bumetrizole treatment group was below 1.5% for all concentrations. Likewise, the occurrence rate of cells with structural aberrations was below 1.0% for all concentrations.

The survival rate of cells was above 90% for 75 and 150 µg/mL. However, above 300 µg/mL, survival decreases in proportion to higher concentration, and was 34% for the maximum concentration of 1200 µg/mL.

White filmy oil precipitates and white impalpable precipitates were observed for all concentrations when dosing preparations were added to culture medium in the beginning and also at the end of treatment.

In the negative control, the percentage of cells with numerical aberrations and structural aberrations was 0% and 0.5%, respectively. In the positive controls, the percentage of cells with numerical aberrations and structural aberrations (MMC: 0.05 µg/mL) was 0%, and 43.5%, respectively. Hence, the induction rate of chromosomal aberrations in negative and positive control groups was within the range of background, thus satisfying the criteria of a valid test.

Table 3: Chromosome aberration test results (short term treatment) -S9

Treatment period (h)

S9 mix

Dosage of test substance (mg/mL)

Number of cells showing structural chromosome aberration (incidence, %)

Number of gap appearances

Cell growth index

(%)

Number of cells showing numerical chromosome aberration (incidence, %)

Number of cells observed

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Other

Total number of aberrations (%)

Number of cells observed

Polyploid

Others

Total numberof aberrant cells (%)

6-18

-

Negative control

(DMSO )

200

1

0

0

0

0

1 (0.5)

0

100

200

0

0

0 (0)

6-18

-

0.150 *

200

0

0

0

0

0

0 (0)

0

95

200

1

0

1 (0.5)

6-18

-

0.300 *

200

1

0

0

0

0

1 (0.5)

0

95

200

0

0

0 (0)

6-18

-

0.600 *

200

0

0

0

0

0

0 (0)

0

89

200

0

0

0 (0)

6-18

-

1.200 *

200

1

0

0

0

0

1 (0.5)

0

48

200

3

0

3 (1.5)

6-18

-

2.400 *

200

0

1

0

0

0

1 (0.5)

0

34

200

2

0

2 (1.0)

6-18

-

Positive control (MMC)

0.0001

200

53

75

0

0

0

105 (52.5)

0

88

200

0

0

0 (0)

*: Precipitate observed

Table 4: Chromosome aberration test results (short term treatment) +S9

Treatment period (h)

S9 mix

Dosage of test substance (mg/ml)

Number of cells showing structural chromosome aberration (incidence, %)

Number of gap appearances

Cell growth index

(%)

Number of cells showing numerical chromosome aberration (incidence, %)

Number of cells observed

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Other

Total number of aberrations (%)

Number of cells observed

Polyploid

Others

Total numberof aberrant cells (%)

6-18

+

Negative control

(DMSO )

200

0

0

0

0

0

0 (0)

0

100

200

1

0

1 (0.5)

6-18

+

0.150 *

200

0

0

0

0

0

0 (0)

0

96

200

1

0

1 (0.5)

6-18

+

0.300 *

200

1

1

0

0

0

2 (1.0)

0

91

200

0

0

0 (0)

6-18

+

0.600 *

200

1

0

0

0

0

1 (0.5)

0

85

200

0

0

0 (0)

6-18

+

1.200 *

200

0

0

0

0

0

0 (0)

0

51

200

3

0

3 (1.5)

6-18

+

2.400 *

200

0

0

0

0

0

0 (0)

0

34

200

3

0

1 (1.5)

6-18

+

Positive control (DMN)

0.500

200

66

124

0

2

0

145 (72.5)

0

85

200

0

0

0 (0)

*: Precipitate observed

Table 5: Chromosome aberration test results (continuous treatment)

Treatment period (h)

Dosage of test substance (mg/ml)

Number of cells showing structural chromosome aberration (incidence, %)

Number of gap appearances

Cell growth index

(%)

Number of cells showing numerical chromosome aberration (incidence, %)

Number of cells observed

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Other

Total number of aberrations (%)

Number of cells observed

Polyploid

Others

Total numberof aberrant cells (%)

24-0

Negative control

(DMSO )

200

0

1

0

0

0

1 (0.5)

0

100

200

0

0

0 (0)

24-0

0.075*

200

0

0

0

0

0

0 (0)

0

97

200

0

0

0 (0)

24-0

0.150*

200

1

0

0

1

0

2 (1.0)

0

92

200

2

0

2 (1.0)

24-0

0.300*

200

0

1

0

0

0

1 (0.5)

0

88

200

1

0

1 (0.5)

24-0

0.600*

200

1

0

0

0

0

1 (0.5)

0

62

200

3

0

3(1.5)

24-0

1.200*

200

0

0

0

0

0

0 (0)

0

34

200

2

0

2 (1.0)

24-0

Positive control (MMC)

0.00005

200

40

56

0

0

0

87 (43.5)

0

88

200

0

0

0 (0)

*: Precipitate observed

Conclusions:
In conclusion, the in vitro chromosome aberration assay resulted in the test substance being negative (with and without metabolic activation) for inducing genotoxicity and thus, is considered to be non-clastogenic.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only 2-aminoanthracene as positive control with metabolic activation)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test substance (as cited in study report): Bumtrizole
- Analytical purity: 99.9 % w/w
- Lot/batch No.: 01721IW4
- Stability under test conditions: stable
- Storage condition of test material: stored sealed in a cabinet at 20.0 - 25.3 °C
- Other: Supplier: Ciba Specialty Chemicals, Osaka, Japan
Target gene:
His- and Trp-operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver induced with phenobarbital and 5,6-benzoflavone (see table 1)
Test concentrations with justification for top dose:
Range-finding test: 0.305, 1.22, 4.88, 19.5, 78.1, 312.5, 1250 or 5000 µg/plate for all strains +/- S9 mix
Main test 1:
-S9 mix:
TA 100: 9.77, 19.5, 39.1, 78.1, 156.3 or 312.5 µg/plate
TA 1535 and WP2uvrA: 39.1, 78.1, 156.3, 312.5, 625 or 1250 µg/plate
TA 98 and TA 1537: 0.61, 1.22, 2.44, 4.88, 9.77, 19.5 µg/plate

+S9 mix:
TA 100, TA 1535, TA 98 and TA 1537: 9.77, 19.5, 39.1, 78.1, 156.3 or 312.5 µg/plate
WP2uvrA: 39.1, 78.1, 156.3, 312.5, 625 or 1250 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO is commonly used for the reverse mutagenicity test on bacteria and it forms a good suspension.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see table 2
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37 °C
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: 3 plates per test

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
When the revertant colony number on the plate with test substance was larger than twice that of the negative control substance, the test was judged to be positive.
Statistics:
Revertant colony numbers were calculated as average and standard deviation. No significance test was performed.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(depending on strain from 9.77 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 1250 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Range-finding test:
Precipitation:
-S9 mix: Seen as white oily membrane or fine white particles at concentrations above 78.1 µg/plate at the start and above 19.5 µg/plate at the end of incubation
+S9 mix: White fine precipitation was observed at concentrations above 312.5 µg/plate at the start and above 78.1 µg/plate at the end of incubation.

Main test 1 & 2:
Precipitation:
-S9 mix: Precipitatation was observed at concentrations above 39.1 µg/plate at the start and above 19.5 µg/plate at the end of incubation as a white oily membrane or fine white particles.
+S9 mix: Fine white particles were observed at concentrations above 156.5 µg/plate at the start and above 78.1 µg/plate at the end of the incubation period.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Reverse mutation test I

S9 mix

Concentration (µg/plate)

Number of revertants

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

- S9

Negative control

105 ± 18

13 ± 2

24 ± 6

14 ± 1

11 ± 5

0.61

---

---

---

18 ± 1

6 ± 4

1.22

---

---

---

20 ± 6

10 ± 1

2.44

---

---

---

21 ± 4

10 ± 1

4.88

---

---

---

22 ± 5

11 ± 2

9.77

99 ± 11

---

---

21* ± 2

10* ± 3

19.5

107 ± 14

---

---

21* ± 3

10* ± 5

39.1

95 ± 3

10 ± 2

28 ± 9

---

---

78.1

103 ± 5

13

27 ± 3

---

---

156.3

104* ± 11

16 ± 5

31 ± 5

---

---

312.5

90* ± 9

14 ± 7

30 ± 6

---

---

625

---

15* ± 2

31 ± 6

---

---

1250

---

9* ± 4

27* ± 6

---

---

Positive control

Name

AF-2

NaN3

AF-2

AF-2

9AA

Concentration (µg/plate)

0.01

0.5

0.01

0.1

80

Number of colonies/plate

449 ± 31

618 ± 39

124 ± 18

371 ± 5

410 ± 18

+ S9

Negative control

116 ± 15

13 ± 2

34 ± 1

29 ± 4

18 ± 1

9.77

112 ± 8

16 ± 9

---

28 ± 4

23 ± 1

19.5

118 ± 11

11 ± 6

---

25 ± 6

18 ± 2

39.1

104 ± 16

10 ± 2

33 ± 3

26 ± 4

15 ± 4

78.1

92 ± 14

15 ± 3

26 ± 5

27 ± 3

14 ± 2

156.3

108 ± 15

15 ± 3

29 ± 1

24 ± 5

18 ± 4

312.5

101* ± 17

17* ± 6

26 ± 5

25* ± 2

18* ± 2

625

---

---

25 ± 7

---

---

1250

---

---

27* ± 7

---

---

Positive control

Name

2-AA

Concentration (µg/plate)

1

2

10

0.5

2

Number of colonies/plate

887 ± 30

331 ± 15

760 ± 21

413 ± 23

136 ± 7

AF-2: 2-(2-Furyl)-3-(5-nitro-2furyl)acrylamide; NaN3: sodium azide; 9AA: 9-aminoacridine hydrochloride; 2AA: 2-Aminoanthracene

*) Bacterial growth inhibition was observed.

 

 Table 2: Reverse mutation test II

S9 mix

Concentration (µg/plate)

Number of revertants

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

- S9

Negative control

104 ± 6

6 ± 1

31 ± 3

19 ± 3

7 ± 2

0.61

---

---

---

23 ± 3

9 ± 2

1.22

---

---

---

20 ± 7

11 ± 1

2.44

---

---

---

20 ± 2

9 ± 2

4.88

---

---

---

21 ± 7

8 ± 1

9.77

114 ± 6

---

---

23* ± 5

7* ± 1

19.5

108 ± 10

---

---

20* ± 8

7* ± 3

39.1

108 ± 8

8 ± 4

24 ± 3

---

---

78.1

98 ± 5

10 ± 1

24 ± 5

---

---

156.3

117* ± 8

10 ± 5

27 ± 3

---

---

312.5

100* ± 10

9 ± 3

22 ± 6

---

---

625

---

11* ± 3

24 ± 10

---

---

1250

---

10* ± 1

23* ± 4

---

---

Positive control

Name

AF-2

NaN3

AF-2

AF-2

9AA

Concentration (µg/plate)

0.01

0.5

0.01

0.1

80

Number of colonies/plate

496 ± 73

551 ± 45

127 ± 4

463 ± 9

434 ± 153

+ S9

Negative control

121 ± 6

10 ± 3

27 ± 4

28 ± 7

17 ± 5

9.77

106 ± 2

10 ± 2

---

22 ± 2

13 ± 2

19.5

113 ± 17

8 ± 3

---

22 ± 2

16 ± 4

39.1

112 ± 8

10 ± 1

26 ± 7

27 ± 7

17 ± 5

78.1

112 ± 8

6 ± 2

32 ± 6

28 ± 4

20 ± 5

156.3

116 ± 2

8 ± 1

27 ± 5

25 ± 4

13 ± 8

312.5

107* ± 9

6* ± 1

29 ± 7

26* ± 9

11* ± 3

625

---

---

30 ± 3

---

---

1250

---

---

34* ± 4

---

---

Positive control

Name

2-AA

Concentration (µg/plate)

1

2

10

0.5

2

Number of colonies/plate

933 ± 31

372 ± 41

785 ± 14

452 ± 22

132 ± 15

AF-2: 2-(2-Furyl)-3-(5-nitro-2furyl)acrylamide; NaN3: sodium azide; 9AA: 9-aminoacridine hydrochloride; 2AA: 2-Aminoanthracene

*) Bacterial growth inhibition was observed.

 

Growth inhibition:

-S9 mix: Growth inhibition was observed at concentrations above 156.3 µg/plate for TA 100, above 625 µg/plate for TA 1535, above 1250 µg/plate for WP2uvrA and above 9.77 µg/plate for TA 98 and TA 1537.

+S9 mix: Growth inhibition was observed at concentrations above 312.5 µg/plate for TA 100, TA 1535, TA 98 and TA 1537 and above 1250 µg/plate for WP2uvrA.

Revertant colony number: The number of revertant colonies, both with and without S9 mix, was within 2 times of the solvent control for all strains used.

Range-finding test:

Growth inhibition:

-S9 mix: Growth inhibition was observed at concentrations above 312.5 µg/plate for TA 100, above 1250 µg/plate for TA 1535 and WP2uvrA, above 19.5 µg/plate for TA 98 and TA 1537.

+S9 mix: Growth inhibition was observed at concentrations above 312.5 µg/plate for TA 100, TA1535, TA 98 and TA 1537 and above 1250 µg/plate for WP2uvrA.

Revertant colony number: For all strains, revertant colony numbers, with and without S9 mix, were less than twice the colony number of the solvent controls.

Conclusions:
The Ames test generated negative (with and without metabolic activation) results for the test substance, thus the test substance is considered to be non-mutagenic.
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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(incomplete characterization of the test substance e.g. no data on test material purity. - Strains capable of detecting oxidizing agents/crosslinker are not tested, reliabilty check with S9 activation performed in one strain only)
Principles of method if other than guideline:
Ames Test
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): TK 10048
- Lot/batch No.: EN 26565
Target gene:
His-operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 induced with Aroclor 1254 (cofactors = MgCl, KCl, glucose-6-phosphate, NADP and phosphate buffer, pH 7.4)
Test concentrations with justification for top dose:
2, 6, 18, 54 or 162 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: phosphate buffer
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
phosphate buffer
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37°C

NUMBER OF REPLICATIONS: 3/dose (control and treated groups), 2/dose (positive controls)





Evaluation criteria:
The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration.
Statistics:
When the colonies had been counted, the arithmetic mean was calculated.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Occurred at the highest dose level tested (162 µg/plate) in all strains +/- S9

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Results of the bacterial mutation test

 

Concentration µg/plate

Number of revertant colonies

Strain

TA 98

TA 100

TA 1535

TA 1537

- S9

Neg. control

21

191

17

6

2

26

199

23

9

6

24

228

17

8

18

26

188

20

8

54

12

222

22

4

162

23

241

26

10

Positive control

substance

DB

4-NQO

MNNG

9AAC

Highest dose tested (µg/plate)

10

0.25

5

100

Revertants

494 (16)

1037 (221)

> 1500 (22)

> 1100 (4)

+ S9

Neg. control

27

207

13

7

2

30

220

17

8

6

25

221

14

7

18

28

235

17

8

54

22

214

12

8

162

30

193

16

9

Positive control

substance

---

---

CP

---

Highest dose tested (µg/plate)

---

---

250

---

revertants

---

---

611 (21)

---

DB: daunoblastin; 4-NQO: 4-nitroquinoline-N-oxide; MNNG: N-methyl-N’-nitro-N-nitroso-guanidine; 9AAC: 9-aminoacridinehydrochloride; CP: cyclophosphamide

values in parenthensis = negative control values

Conclusions:
In conclusion, the present Ames test generated negative results (with and without metabolic activation), and thus the test substance is considered to be non-mutagenic.
Endpoint:
in vitro gene mutation 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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH


1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across justification is based on the hypothesis that 2-tert-Butyl-6-(5-chloro-2H-benzotriazol-2-yl)-4-methylphenol (CAS 3896-11-5) and the source chemical 2-(2H-Benzotriazol-2-yl)-p-cresol (CAS 2440-22-4) have similar properties because the chemical structure of these substances is comparable and because the source substance represents a worst-case scenario as it has a higher solubility in water and some systemic toxicity.
The target substance is non hazardous in local and repeated dose oral toxicity studies. Its poor water solubility of 0.004 mg/L indicates a low potential to interact with cells. In contrast, the target substance has a water solubility of 0.17 mg/L and is a skin sensitizer indicating a protein binding potential.

More information is provided in the attached read-across justification document.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Purity of the target substance: 99%
Identity of source substance: CAS 2440 - 22-4: Purity of the source substance: 99.6%


3. ANALOGUE APPROACH JUSTIFICATION
This information is provided in the attached read-across justification document.

4. DATA MATRIX
This information is provided in the attached read-across justification document.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
not applicable
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Deviations:
yes
Remarks:
(only two dose levels were applied)
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): TK10048
- Lot/batch No.: EN 26580(1/75)
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1.0
- Humidity (%): 50 ± 5
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Amount of vehicle: 0.2 mL/10 g bw
Duration of treatment / exposure:
Single dose application
Frequency of treatment:
Single dose
Post exposure period:
Males: 6 weeks
Females (not exposed): day 0 of gestation (vaginal plug); sacrificed on day 14 of gestation
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
3 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 males
40 females
Control animals:
yes, concurrent vehicle
Positive control(s):
No positive controls were used in this particular study. However, the same laboratory that conducted this study also conducted studies on positive control substances at the same facility using the same strain of mice. The results of these tests have been published (see, for example, Fritz H et al (1973) Agents and Actions, 3, 35). This study clearly demonstrates that dominal lethal effects can be chemically induced in NMRI mice.
Tissues and cell types examined:
Number of live embryos and embryonic resorptions/deaths
Statistics:
Total number of implantations - the t-test or Mann-Whitney's U-test
Total numbers of mated and pregnant dams or embryonic deaths - x squared test or Fisher's exact test.
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not specified

No evidence of dominant lethal effect was observed in the progeny of male mice treated with the test substance.

Table 1: Dominant lethal assay results

Dose Group

(mg/kg)

No. females mated*

No. of pregnant females

No. of implantations

Live embryos

Embryonic deaths

Total

%

Total

%

Total

Mean

SD

Total

%

Total

%

Mating Period I

0

38

95.0

36

94.7

408

11.33

3.80

365

89.5

43

10.5

1000

39

97.5

34

87.2

391

11.50

4.32

370

94.6

21

5.4

3000

33

82.5

21

63.6

230

10.95

3.90

213

92.6

17

7.4

Mating Period II

0

37

92.5

34

91.9

399

11.74

4.19

373

93.5

26

6.5

1000

40

100

36

90.0

481

13.36

2.32

455

94.6

26

5.4

3000

38

95.0

30

78.9

383

12.77

3.18

352

91.9

31

8.1

Mating Period III

0

40

100

34

85.0

413

12.15

3.11

388

94.0

25

6.0

1000

39

97.5

33

84.6

434

13.15

1.73

414

95.4

20

4.6

3000

40

100

32

80.0

373

11.66

2.62

358

96.0

15

4.0

Mating Period IV

0

38

95.0

30

78.9

388

12.93

1.53

357

92.0

31

8.0

1000

37

94.9

34

91.9

455

13.38

1.39

433

95.2

22

4.8

3000

35

87.5

30

85.7

384

12.80

1.67

354

92.2

30

7.8

Mating Period V

0

36

90.0

33

91.7

390

11.82

2.56

371

95.1

19

4.9

1000

38

95.0

34

89.5

442

13.00

1.74

415

93.9

27

6.1

3000

34

85.0

31

91.2

388

12.52

1.52

358

92.3

30

7.7

Mating Period VI

0

36

90.0

29

80.6

364

12.55

2.89

344

94.5

20

5.5

1000

36

90.0

32

88.9

400

12.50

1.97

371

92.8

29

7.2

3000

39

97.5

31

79.5

363

11.71

2.75

333

91.7

30

8.3

Cumulative control

328

-

281

85.7

3242

11.54

2.58

2998

92.5

244

7.5

* 40 females/group

Conclusions:
In conclusion, in the present study the test substance did not induce chromosome aberrations and thus, is considered to be non-clastogenic.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
(test substance incompletely characterized, only 1000 cells scored for micronuclei, micronuclei scored in interphase cells instead of metaphase arrested cells, PCE to NCE proportion not determined)
GLP compliance:
no
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): TK 10048
- Lot/batch No.: EN 26580 1/75
Species:
hamster, Chinese
Strain:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Further characterization: Cricetulus griseus
- Source: Fa. THOMAE, D-Biberach a.d. Riss
- Weight at study initiation: 24 - 37 g
- Diet: NAFAG no 196
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1.0
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose); 2% aqueous solution
- Dosing volume: 20 mL/kg bw/d
Duration of treatment / exposure:
48 hours
Frequency of treatment:
once daily for 2 consecutive days
Post exposure period:
24 h
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from both femurs using a pipette filled with 0.5 µL rat serum. In order to receive a homogeneous suspension the bone marrow was aspirated gently three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and air dried before staining with undiluted May-Gruenwald solution for 2 min then in May-Gruenwald solution/water (1:1) for 2 min and then in Giemsa's 40% for 20 min. The slides were rinsed in methanol (55%) and washed twice in water. They were then immersed in water for approximately 2 min. After rinsing with distilled water and air drying the slides were cleared in Xylol and mounted in Eukitt.


Evaluation criteria:
1000 bone marrow cells were scored per animal and the following anomalies were registered:
1) Single Jolly bodies
2) Fragments of nuclei in erythrocytes
3) Micronuclei in erythroblasts
4) Micronuclei in leucopoietic cells
5) Polyploid cells
Statistics:
The significance of difference was assessed by x squared-test
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg) yielded a marked increase of the percentage of cells with anomalies.
Here the mean percentage of anomlies was 6.92, whereas the negative control yielded a percentage of 0.10. The difference is highly significant (p<0.05).

Table 1: The effect of the test substance on bone marrow cells of Chinese hamster sacrificed 24 hours after last application (percent of interphase cells with anomalies of nuclei).

Test substance

Number/sex of animals

Single Jolly bodies

Fragment of nuclei in erythrocytes

Micronuclei in erythroblast

Micronuclei in leucopoietic cells

Polyploidy cells

Total

Control (2% CMC)

1♀

 

 

 

 

 

0.0

2♀

 

 

 

 

0.1

0.1

3♀

 

 

 

 

 

0.0

4♂

0.1

 

 

 

 

0.1

5♂

0.2

 

 

 

0.1

0.3

6♂

0.1

 

 

 

 

0.1

Cyclophophamide (128 mg/kg bw)

1♀

3.1

0.2

0.4

0.1

0.2

4.0

2♀

6.6

1.0

0.4

 

0.9

8.9

3♀

4.3

0.2

0.4

0.4

0.7

6.0

4♂

6.1

1.1

1.6

 

0.9

9.7

5♂

2.9

0.4

1.7

 

0.3

53

6♂

5.1

0.4

1.3

0.2

0.6

7.6

500 mg/kg bw

1♀

0.2

 

 

0.1

 

0.3

2♀

0.1

 

 

 

 

0.1

3♀

0.1

 

 

 

 

0.1

4♂

0.2

 

 

 

 

0.2

5♂

 

 

 

 

 

0.0

6♂

0.1

 

 

 

 

0.1

1000 mg/kg bw

1♀

 

 

 

 

 

0.0

2♀

 

 

 

 

 

0.0

3♀

0.1

 

 

 

 

0.2

4♂

0.1

 

 

 

 

0.1

5♂

 

 

 

 

 

0.0

6♂

0.2

 

 

 

 

0.2

2000 mg/kg bw

1♀

0.1

 

 

 

0.1

0.2

2♀

 

 

 

 

 

0.0

3♀

0.1

 

 

 

 

0.1

4♂

0.1

 

 

 

 

0.1

5♂

 

 

 

 

 

0.0

6♂

0.1

 

 

 

0.1

0.2

 

 

Conclusions:
In conclusion, it can be stated that during the study described, the test substance did not induce micronuclei. Therefore, the test substance is considered to be non-clastogenic and non-aneugenic.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
(only 4 animals/sex/dose were used. Test substance was administered on two consecutive days. Only one sampling time was performed)
Principles of method if other than guideline:
Chinese Hamsters (male/female, 4/sex/dose) were given a single dose of the substance by gavage at 0, 500, 1000 or 2000 mg/kg bw/day on each of two consecutive days. The animals were injected intraperitoneally with colcemide (10 mg/kg bw) two hours after the second dose, and sacrificed four hours later. Chromosome preparations were made from the femoral bone marrow of two males and two females from each group and 100 metaphases from each animal analysed for chromatid-type and chromosome-type aberrations.
GLP compliance:
no
Type of assay:
chromosome aberration assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): TK 10 048
- Lot/batch No.: EN. 26580 1/75
Species:
hamster, Chinese
Strain:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Further characterization: Cricetulus griseus
- Weight at study initiation: 25 - 36 g
- Housing: no data
- Diet: NAFAG no 924; ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1.0
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle used:CMC (carboxymethyl cellulose) (2% aqueous solution)
Details on exposure:
DOSING VOLUME: 20 mL/kg bw
Duration of treatment / exposure:
48 hours
Frequency of treatment:
once daily on two consecutive days.
Post exposure period:
4h after the second exposure
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 64 mg/kg
- dosing volume: 20 mL/kg bw in 2.0% CMC
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the oral LD 50 in Chinese hamster which was found to be > 6000 mg/kg bw in both sexes.

DETAILS OF SLIDE PREPARATION:
Bone marrow harvested from both femurs was suspended in balance salt solution and diluted with distilled water to induce hypotonicity, kept in a water bath at 4 to 6°C for 23 min and centrifuged for 10 min at 200 g. The pellets were fixed in methanol-acetic acid for 30 min. After resuspension and centrifugation, the fixative was changed and the specimen stored overnight at 4°C. The pellet was resuspended, centrifuged and resuspended in 0.5 mL fixative. Specimens were pipetted onto wet slides and stained with acetic-orcein.

METHOD OF ANALYSIS:
The slides of two female and two male animals per group were examined. 100 metaphase plates from each animal were analysed.
Evaluation criteria:
1) chromatid-type aberration (including breaks and exchanges)
2) chromosome type aberrations
3) chromatid gaps
4) chromosome pulverizations
Statistics:
No statistical analysis was necessary.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

In the negative control group as well as in the groups treated with the various doses of the test substance neither chromosome-type nor chromatid-type aberrations were detected.

In contrast the positive control caused a marked increase in all types of aberrations (chromatid type aberrations 10.75%, chromosome aberrations type 0.5%). 5.5% of the cells showed pulverisations.

Conclusions:
In conclusion, in the present study the test substance did not induce chromosome aberrations and thus, is considered to be non-clastogenic.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Gene Mutation

In vitro studies

Studies in bacterial cells:

In a reverse gene mutation assay, bacterial strains TA 1535, TA 1537, TA 98, and TA 100 of S. typhimurium as well as the bacterial strain E. Coli WP2 uvrA were exposed to the test substance (99% pure) in DMSO at concentrations between 0.61 – 1250 µg/plate without metabolic activation and between 9.7 -1250 µg/plate in the presence of metabolic activation. The preincubation method was the method of choice. Each concentration was tested in triplicate cultures per experiment. Two experiments were performed.

Cytotoxicty measured as relative cell growth and precipitation were evident depending on the tested strain and testing conditions. In cells deprived of metabolic activation, growth inhibition was observed at concentrations higher than 156.3 µg/plate for TA 100, above 625 µg/plate for TA 1535, above 1250 µg/plate for WP2uvrA and above 9.77 µg/plate for TA 98 and TA 1537. Precipitation was seen from 19 µg/plate and above. In the presence of metabolic activation, growth inhibition occurred at concentrations above 312.5 µg/plate for TA 100, TA 1535, TA 98 and TA 1537 and above 1250 µg/plate for WP2uvrA. Precipitation in agar was seen from 78.1 µg/plate and above. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background (Nihon Bioresearch Center Inc., 2006).

This is acceptable for assessment as it satisfies the requirements of OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data and was performed under GLP. This study hence rated as key study for the assessment of mutagenic effects in bacteria.

The result of second reverse gene mutation assay in bacteria is in line with findings of the key study. In this study carried out using a protocol equivalent to guideline standards, there was no evidence of induced mutant colonies over background when bacterial strains TA 1535, TA 1537, TA 98, and TA 100 of S. typhimurium were exposed to the test substance (no data on analytical purity) at concentrations of 2, 6, 18, 54 or 162 µg/plate in phosphate buffer. Cytotoxicity was not evident, but tests were performed up to precipitating concentrations (162 µg/plate). Positive controls induced the appropriate responses in the corresponding strains (Ciba Geigy Ltd. 1978).

Studies in mammalian cells:

A valid gene mutation test performed with mammalian cell is not available for the substance itself. Read-across is done to the structural analogue CAS 2440 -22 -4 and the substance is considered to be non genotoxic.

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and ß-naphthoflavone induced rats (exogenous metabolic activation).

According to two initial range-finding cytotoxicity tests for the determination of the experimental doses and based on the solubility properties of the test substance in culture medium the following concentrations were tested. Test groups printed in bold type were evaluated for mutant frequencies:

1st Experiment

without S9 mix

0; 1.56;3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL

with S9 mix

0; 0.78; 1.56;3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL

2nd Experiment

without S9 mix

0; 2.50;5.00; 10.00; 20.00; 25.00; 30.00; 40.00; 80.00 µg/mL

with S9 mix

0; 2.50;5.00; 10.00; 20.00; 25.00; 30.00; 40.00; 80.00 µg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

Based on the solubility properties of the test substance in culture medium 100.0 µg/mL was the highest applied concentration for genotoxicity testing. In both experiments, test substance precipitation occured in the highest applied concentrations (25.00 µg/mL onward) in culture medium 4 hours at the end of exposure period.

In both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.

In the 1st experiment in the absence of a metabolizing system a dose related increase in the mutant frequencies of cultures treated was observed. The highest evaluated value of the test group 25.00 µg/mL (MFcorr.: 6.33 per 10^6 cells) exceeded the upper limit of the 95 % level of the historical control values (MFcorr.: 5.97 per 10^6 cells). However, the value obtained at 25.00 µg/mL was not statistically significant as compared to the corresponding control value and was within the range of our historical negative control data (MFcorr.: 0.00 - 6.48 per 10^6 cells). In the 2nd experiment in the absence of S9 mix, the observed concentration related increase as well as the observed increase in the mutation frequency at 25.00 µg/mL was not observed. Thus, the increase observed in the 1st experiment at 25.00 µg/mL is considered as biologically irrelevant.

In the presence of a metabolizing system the corrected mutation frequencies ranged between 1.14 - 6.73 per 10^6 cells and 0.69 - 4.36 per 10^6 cells in the 1st and 2nd experiment, respectively. The vehicle control values were (corrected mutation frequency: 3.82 and 0.31 per 10^6 cells) in the 1st and 2nd experiment, respectively. In the 2nd experiment, the corrected mutation frequency obtained at 5.00 µg/mL (4.36 mutants per 10^6 cells) was statistically significant. However, this observation is not biologically relevant, since it is neither concentration related nor exceeds the 95 % level of the historical control data.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under the in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

 

Chromosomal Aberration

In vitro studies

In a mammalian cell cytogenetics assay (Chromosome aberration),CHL/IUcell cultures were exposed to the test substance (99.9% pure) in DMSO at concentrations of 150, 300, 600, 1200 or 2400 µg/mL for 6 hours (with and without metabolic activation) and at concentrations of 75, 150, 300, 600 or 1200 µg/mL for 24 hours (without metabolic activation). Rat liver induced with phenobarbital and 5, 6-benzoflavone was employed for metabolic activation.

Precipitation was seen at all tested concentrations with and without metabolic activation. Cytotoxicity was detected in the two highest concentrations (with and without S9) in pulse treatments and from 600 µg/plate and above in continuous treatments. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background; neither of structural nor of numerical nature (Nihon Bioresearch Center Inc., 2006).

This study is classified as acceptable for assessment of chromosomal aberration. It satisfies the requirement for Test Guideline OECD 473 (In vitro Mammalian Chromosome Aberration Test) for in vitro cytogenetic mutagenicity data. In addition it was conducted under GLP standards.

 

In vivo studies

In a dominant lethal assay, the test substance (no data on purity) was administered in a single application by gavage to 20 male NMRI mice/dose at dose levels of 1000 and 3000 mg/kg bw in 2% aqueous carboxymethyl cellulose. Control animals were treated with the vehicle only. Each male animal was placed with two females for 1 week, at the end of the week the females were replaced with a further two animals. The procedure was continued for six consecutive weeks. In total 40 females were used. Females were autopsied at day 14 of pregnancy. The number of live embryos and embryonic deaths were listed. In addition, early embryonic resorptions were evaluated.

There was no evidence of dominant lethal effect was observed in the progeny of male mice treated with the test substance (Ciba Geigy Ltd. 1977).

This study does not fully satisfy the guideline requirement for a dominant lethal test (OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test) as only two doses were tested and the test substance was not completely characterized e.g. no data on test material purity is given, but it is nonetheless sufficiently detailed and is considered acceptable for assessment.

There was also no evidence of structural chromosomal aberrations when in the bone marrow chromosomal aberration test, 4 Chinese Hamster (Cricetulus griseus) per sex per dose were treated once daily for two consecutive days with doses of 500, 1000 and 2000 mg/kg bw in 2% carboxymethyl cellulose aqueous solution. Control animals were treated with the vehicle. Bone marrow was harvested 4 hours after the last treatment, prepared and 100 metaphases were scored. No signs of systemic toxicity were observed in the treated animals. The positive control substance (cyclophosphamide) induced the appropriate response (Ciba Geigy Ltd. 1979).

This study does not fully satisfy the guideline requirement for OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test) as the dosing schedule spanned two days with only one sampling time, only 4 animals were employed per sex per dose and the test substance was not completely characterized e.g. no data on test material purity. The study is nevertheless considered acceptable for assessment as there are no other major deviations from the guideline standard.

In a Cricetulus griseus Chinese Hamster bone marrow nucleus anomaly assay, 6 animals per sex per dose were treated with the test substance (no data on purity) at doses of 500, 1000 and 2000 mg/kg bw in 2% aqueous solution of carboxymethylcellulose. The control animals received the vehicle. Single dose applications were performed once daily for two consecutive days via gavage. Animals were sacrificed 24 hours after the second application and bone marrow cells were harvested. 1000 bone marrow cells were scored per animal and following parameters were evaluated: single jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells and polyploid cells.

There were no signs of overt systemic toxicity up to the highest dose tested. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated bone marrow cells after treatment with the test substance (Ciba Geigy Ltd. 1972).

This study does not fully satisfy the requirements of OECD 474 for in vivo mutagenicity (Mammalian Erythrocyte Micronucleus Test) data since only 1000 PCEs instead of 2000 PCEs were scored per animal per dose. In addition, the proportion of PCE to NCE was not evaluated. In addition micronuclei were scored in interphase cells instead of metaphase arrested cells. The study is nevertheless considered as acceptable for assessment as there are no other major deviations from the guideline standard.

 


Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse findings on genotoxicity were observed in in vitro or in vivo studies. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.