Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative with and without metabolic activation (BASF SE, 1997)

HPRT test: negative with and without metabolic activation (BASF SE, 2002)

Chromosomal aberration test: positive with and without metabolic activation (BASF SE, 2002)

Link to relevant study records

Referenceopen allclose all

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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1157
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Metabolic activation was achieved by addition of S-9 mix, which consisted of the S-9 fraction from the liver of male Wistar Hanlbm rats treated with Phenobarbital i.p. and ß-Naphthoflavone p.o., mixed with a series of cofactors.
Test concentrations with justification for top dose:
2.5, 5.0, 10.0, 20.0, 30.0, and 40.0 µg/mL (with and without S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water, nutrient medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethane sulfonate and Cyclophosphamide
Details on test system and experimental conditions:
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 18 hours
- Selection time (if incubation with a selection agent): 14 hours

SELECTION AGENT (mutation assays): Chromosomes

NUMBER OF CELLS EVALUATED: 1E+06

DETERMINATION OF CYTOTOXICITY:
Toxicity of the test substance is indicated by a reduction of mitotic indeces.

S9 mix Preparation:
Metabolic activation was achieved by addition of S-9 mix, which consisted of the S-9 fraction from the liver of male Wistar Hanlbm rats treated with Phenobarbital i.p. and ß-Naphthoflavone p.o., mixed with a series of cofactors (MgCl2, KCl, Glucose-6-phosphate, NADP in sodium-ortho-phosphate buffer).

Dose Selection:
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. 3000 µg/mL of the aqueous test substance solution (corresponds to approx . 10 mM of the active ingredient) were applied as top concentration for treatment of the cultures in the pre-test. Test substance concentrations between 23.4 and 3000 µg/mL (with and without S9-mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, in the absence of S9 mix only precipitation of the test substance after 4 hrs treatment was observed at 1500 µg/mL and above.
Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 hrs treatment with 23.4 µg/mL and above in the absence and the presence of S9 mix. In addition, 24 hrs continuous treatment with 23.4 µg/ml and above in the absence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-test, 40 µg/mL (with and without S9 mix) were chosen as top concentrations in the main experiment. Since the test substance was considered to be clastogenic after 4 hrs treatment a second experiment was not performed.

Negative Controls:
Concurrent negative (culture medium) and solvent controls (deionised water) were performed.

Positive Controls:
- Without metabolic activation: Ethylmethane sulfonate (EMS), purity: >98%, dissolved in: nutrient medium, final conc.: 400 µg/mL = 3.2 mM
- With metabolic activation: Cyclophosphamide (CPA), purity: approx. 98%, dissolved in: nutrient medium, final conc.: 0.7 µg/mL = 2.5 µM
Evaluation criteria:
A test substance is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of the historical control data of the performing laboratory (0.0 - 4.0 % aberrant cells exclusive gaps).

A test substance is classified as clastogenic if :
- the number of induced structural chromosome aberrations are not in the range of the historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

However, both biological and statistical significance should be considered together . If the criteria mentioned above for the test substance are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test substance can be classified as aneugenic if:
- the number of induced numerical aberrations are not in the range of the historical control data (0.0 % - 8.5 % polyploid cells).
Statistics:
Statistical significance was confirmed by means of the Fisher"s exact test (Ricardson C. et al., 1989): p < 0.05.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: 30 µg/mL without S9-mix; 40 µg/mL with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Analysis of Aberrations (200 cells analyzed); preparation interval 18 hrs without S9 mix: exposure period 4 hrs.

Test Substance

Dose

(µg/mL)

% Aberrant cells

(excl. gaps)

Chromatid type

Chromosome type

Break

Exchange

Break

Exchange

Negative Control

-

2.0

2

0

1

1

Solvent Control

Deion. water 10 % (v/v)

0.5

0

1

0

0

Positive Control

EMS 400

47.5

52

47

9

0

Protectol HT

10

7.5*

6

8

2

0

20

25.5*

19

46

5

0

30

43.0*

23

83

15

0

Analysis of Aberrations (200 cells analyzed); preparation interval 18 hrs with S9 mix: exposure period 4 hrs.

Test Substance

Dose

(µg/mL)

% Aberrant cells

(excl. gaps)

Chromatid type

Chromosome type

Break

Exchange

Break

Exchange

Negative Control

-

2.5

3

0

0

0

Solvent Control

Deion. water 10 % (v/v)

2.0

3

0

1

1

Positive Control

CPA 0.7

16.5

15

18

2

0

Protectol HT

5

2.0

2

0

0

0

10

3.0

2

2

2

0

20

15.0*

13

18

4

0

* Aberration frequency statiscally significant higher than corresponding control values.

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:
other: OECD Guideline 471 (Bacterial Reverse Mutation Assay) / OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: PK 95/222

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated
Target gene:
his- or trp-
Species / strain / cell type:
other: Salmonella typhimurium TA1535, TA1537, TA98, TA100, TA102, E. coli WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor 1254 induced rat liver)
Test concentrations with justification for top dose:
25 µg - 7500 µg/plate
Vehicle / solvent:
water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2- Aminoanthracene, N-Methyl-N'-nitro-N-nitrosoguanidine, Mitomycin C, 4-Nitro-o-phenylendiamine, 9-Aminoacridine chloride monohydrate and N-Ethyl-N'-nitro-N-nitrosoguanidine.
Details on test system and experimental conditions:
The test was conducted according to the Ames procedure (standard plate test and preincubation test , with and without metabolic activation).

Number of plates: 3 test plates per dose or per control.

Vehicle: water

The study comprised 6 independent experiments:

1st Experiment:
- Strains: TA 1535, TA 100, TA 1537, TA 98.
- Doses: 0, 100, 500, 2500, 5000 and 7500 µg/plate.
- Test condition: Standard plate test with and without S-9 mix. 

2nd Experiment:
- Strains: TA 1535, TA 100, TA 1537, TA 98.
- Doses: 0, 25, 50, 100, 200 and 400 µg/plate.
- Test condition: Standard plate test with and without S-9 mix.  
 
3rd Experiment:
- Strains: TA 1535, TA 100, TA 1537, TA 98.
- Doses: 0, 25, 50, 100, 200 and 400 µg/plate.
- Test condition: Preincubation test with and without S-9 mix.  

4th Experiment:
- Strains: TA 1535, TA 100, TA 1537, TA 98.
- Doses: 0, 25, 50, 100, 200 and 400 µg/plate.
- Test condition: Standard plate test with and without S-9 mix.  

5th Experiment:
- Strains: TA 102, E. coli WP2 uvrA.
- Doses: 0, 25, 50, 100, 200 and 400 µg/plate.
- Test condition: Preincubation test with and without S-9 mix.  

6th Experiment:
- Strains: TA 102.
- Doses: 0, 25, 50, 100, 200 and 400 µg/plate.
- Test condition: Standard plate test with and without S-9 mix.  

Positive control substance:
- With S-9 mix: 2- Aminoanthracene for the strains TA 100, TA 98, TA 1537, TA 1535, TA 102 and E. coli WP2 uvrA.
- Without S-9 mix: N-Methyl-N' -nitro-N-nitrosoguanidine (strains TA 100 and TA 1535); Mytomycin C (strain TA 102); 4-Nitro-o-phenylendiamine (strain TA 98); 9-Aminoacridine chloride monohydrate (strain 1537) and N-Ethyl-N' -nitro-N-nitrosoguanidine (strain E. coli WP2 uvrA).
Evaluation criteria:
The assessment for the test was based on the number of histidine or tryptophan revertant colonies per plate.
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.
Key result
Species / strain:
other: Salmonella typhimurium TA1535, TA1537, TA98, TA100, TA102, E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: from about 400 µg - 500 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Standard Plate Test:

Mean number of revertants with S-9 mix

Dose/Plate

(µg)

TA 1535

TA 100

TA 1537

TA 98

TA 102

E.coli WP2 uvrA

Test substance

25

18

117

13

35

194

27

50

18

111

5

32

130

34

100

17/17

129/110

8/12

31/23

112

35

200

16

143

11

28

110

30

400

19

169

11

33

112

37

500

14B

79

4

14

-

-

2500

0B

-

-

-

-

-

Positive control

2.5

142/162

1200/1613

167/200

1182/1216

-

-

3.0

-

-

-

-

94

-

60

-

-

-

-

-

70

Vehicle control

 

16/20

145/125

10/12

34/34

229

27

Positive Control: 2 -Aminoanthracene

17/17 = Mean number of revertants from two experiments (1st and 2nd experiment).

B = Reduced his- and trp- background growth. A decrease in the number of his+ or trp+ revertants was observed from about 400 - 500 µg/plate onward.

Mean number of revertants without S-9 mix

Dose/Plate

(µg)

TA 1535

TA 100

TA 1537

TA 98

TA 102

E.coli WP2 uvrA

 

Test substance

25

21

129

10

23

150

31

50

18

140

8

27

154

26

100

19/17

132/98

6/12

28/25

126

23

200

17

119

9

27

102

27

400

11

117

9

23

104

30

500

14B

75

4

10

-

-

2500

0B

-

-

-

-

-

Positive control 1

5

1183/1620

1719/1387

-

-

-

-

Positive control 2

100

-

-

525/538

-

-

-

Positive control 3

10

-

-

-

765/577

-

-

Positive control 4

0.25

 

 

 

 

545

-

Positive control 5

10

 

 

 

 

-

119

Vehicle control

-

23/19

137/117

7/11

31/30

133

21

Positive control 1: N-Methyl-N'-nitro-N-nitrosoguanidine

Positive control 2: 9-aminoacridine chloride monohydrate

Positive control 3: 4 -Nitro-o-phenylendiamine

Positive control 4: Mitomycin C

Positive control 5: N-Ethyl-N'-nitro-N-nitrosoguanidine

Preincubation Test:

Mean number of revertants with S-9 mix

Dose/Plate

(µg)

TA 1535

TA 100

TA 1537

TA 98

TA 102

E.coli WP2 uvrA

 

Test substance

25

20

117

12

36

275

32

50

15

119

10

42

205

35

100

11

116

11

38

233

34

200

9

116

6

33

234

25

400

10

132

4

38

189

23

Positive control 

2.5

80

742

83

389

-

-

3.0

-

-

-

-

728

-

60

-

-

-

-

-

222

Vehicle control

-

19

130

14

42

291

38

Positive Control: 2 -Aminoanthracene

Mean number of revertants without S-9 mix

Dose/Plate

(µg)

TA 1535

TA 100

TA 1537

TA 98

TA 102

E.coli WP2 uvrA

 

Test substance

25

18

114

9

20

186

33

50

21

116

10

16

213

34

100

16

107

9

19

267

30

200

20

107

9

25

248

27

400

17

106

6

37

102

13

Positive control 1

5

705

625

-

-

-

-

Positive control 2

100

-

-

623

-

-

-

Positive control 3

10

-

-

-

445

-

-

Positive control 4

0.25

-

-

-

-

736

-

Positive control 5

10

-

-

-

-

-

264

Vehicle control

-

23

111

12

27

214

30

Positive control 1: N-Methyl-N'-nitro-N-nitrosoguanidine

Positive control 2: 9-aminoacridine chloride monohydrate

Positive control 3: 4 -Nitro-o-phenylendiamine

Positive control 4: Mitomycin C

Positive control 5: N-Ethyl-N'-nitro-N-nitrosoguanidine

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1157
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Metabolic activation was achieved by addition of S-9 mix, which consisted of the S-9 fraction from the liver of male Wistar rats treated with Phenobarbital i.p. and ß-Naphthoflavone p.o., mixed with a series of cofactors.
Test concentrations with justification for top dose:
Experiment I:
1.6, 3.1, 6.3, 12.5, and 25 µg/mL (with and without S9-mix)
Experiment II:
3.1, 6.3, 12.5, 25 and 30 µg/mL (without S9-mix)
3.1, 6.3, 12.5, 20 and 25 µg/mL (with S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, water
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethane sulfonate (without metabolic activation) and 7,12-Dimethylbenz(a)anthracene (with metabolic activation).
Details on test system and experimental conditions:
The essay was performed in 2 independent experiments.

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): about 8 days

SELECTION AGENT (mutation assays): 6TG (6-Thioguanine)

NUMBER OF REPLICATIONS: 5

NUMBER OF CELLS EVALUATED: 1E+06

DETERMINATION OF CYTOTOXICITY:
Toxicity of the test substance is indicated by a reduction of the cloning efficiency.

TEST SYSTEM:
- S9 mix preparation:
Metabolic activation was achieved by addition of S-9 mix, which consisted of the S-9 fraction from the liver of male Wistar Hanlbm rats treated with
Phenobarbital i.p. and ß-Naphthoflavone p.o., mixed with a series of cofactors (MgCl2, KCl, Glucose-6-phosphate, NADP in sodium-ortho-phosphate buffer).

- Dose selection:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. In this pre-test, the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test substance was observed and compared to
the controls. Eight concentrations were chosen ranging from 23.4 to 3000 µg/mL with and without metabolic activation and a treatment duration of 4 hours. Toxic effects occurred down to the lowest concentration with and without metabolic activation. Already at 93.8 µg/mL without and at 46.9 µg/mL with metabolic activation, the cell growth was completely inhibited. Therefore, the maximal concentration of the test substance (25.0 µg/mL) in the first main experiment was based upon the toxicity of the test substance observed in the pre-experiment. The dose range of the second experiment was adjusted according to the results of the first experiment to cover the toxic range more closely.

CONTROLS:
Negative Controls:
Concurrent negative and solvent controls were performed. As negative controls: untreated cells were used (cells were cultivated without interruption) and cells, used throughout the assay without addition of test substance.

Positive Controls:
- Without metabolic activation: Ethylmethane sulfonate (EMS), purity: >98%, dissolved in: nutrient medium, final conc.: 0.15 mg/mL = 1.2 mM
- With metabolic activation: 7,12-Dimethylbenz(a)anthracene (DMBA), purity: approx. 95%, dissolved in: DMSO, final conc.: 2.7 µg/mL = 10.4 µM

METHOD OF APPLICATION: in suspension




Evaluation criteria:
A test substance is classified as positive if it induces a concentration-related increase of the mutant frequency.
A test substance producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows :

A test substance is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment and if this increase exceeds the range of the historical negative or solvent control data of the testing laboratory.

The test substance is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation
may be considered also in the case that a threefold increase of the mutant frèquency is not observed.

However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 - 33.1 mutants per 1E+06 cells) a concentration-related increase of the mutations within this
range has to be discussed.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 25 µg/mL with S9 mix; 25 and 30 µg/mL without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The assay was performed in 2 independent experiments with and without liver microsomal activation and a treatment period of 4 hours. Since experiment I showed no indication of a possible mutagenic potential of the test substance, a second confirmatory experiment was performed with and without metabolic activation. The ratio of S9/cofactor solution was 1:2.0 in experiment I and 1:9.0 in experiment II with metabolic activation.

In the main experiments relevant toxic effects occurred at 25.0 and 30.0 µg/mL in the absence and at 25.0 µg/mL in the presence of metabolic activation. A rather steep gradient of dose dependent toxicity was observed in the second experiment especially in the presence of metabolic activation.

No relevant and reproducible increase in mutant colony numbers 1E+06 cells was observed in the main experiments up to the maximum concentration. Generally, the mutant frequencies remained well within the historical range of negative and solvent controls. The control range was only slightly exceeded in the second culture of the first experiment at 25.0 µg/mL without metabolic activation and at 1.6 µg/mL with metabolic activation. These increases were not dose related and no comparable effect occurred in the parallel culture under identical conditions or in the second experiment.
The induction factor exceeded the threshold of three times the factor of corresponding solvent control in the first culture of experiment II without metabolic activation at 25.0 µg/mL. This effect however, was judged to be based upon the rather low solvent control of 5.2 mutant colonies 1E+06 cells. No relevant and reproducible dose dependent increase below this threshold was observed.

In both experiments of this study (with and without S9 mix), the range of the negative and solvent controls was from 4.5 up to 19.5 mutants per 1 million cells ; the range of the groups treated with the test substance was from 1.2 up to 34.3 mutant colonies per 1 million cells.

EMS (0.15 mg/mL) and DMBA (2.5 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. The historical range of positive controls was not quite reached (91.8 versus 97 .8 colonies per 1 million cells) in the second culture of the first experiment without metabolic activation. Still, the threshold of three times the mutation frequency of the corresponding solvent control was clearly exceeded and the historical range was reached in the parallel culture under identical conditions.

In conclusion, it can be stated that under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Conc. µg/mL

Mean number of mutant colonies found after plating in TG medium with S9-mix

Experiment I

Experiment II

Culture I

Culture II

Culture I

Culture II

test substance

0.8

-

-

-

-

1.6

5.0

8.0

-

-

3.1

4.8

3.2

2.4

3.6

6.3

5.0

8.0

0.4

4.0

12.5

7.6

4.0

3.6

3.8

25.0

8.2

9.0

5.2

5.6

Negative control

-

2.2

4.6

2.2

3.6

Solvent control with water

-

3.8

6.0

2.8

4.6

Solvent control with DMSO

-

4.4

2.6

4.2

2.2

Positive control with DMBA

2.7

176.6

201.2

150.2

131.2

DMBA: 7,12-Dimethylbenz(a)anthracene

Conc. µg/mL

Mean number of mutant colonies found after plating in TG medium without S9-mix

Experiment I

Experiment II

Culture I

Culture II

Culture I

Culture II

test substance

0.8

-

-

-

-

1.6

6.2

4.4

-

-

3.1

4.2

4.6

2.4

2.6

6.3

5.4

3.8

3.6

3.6

12.5

3.8

6.6

6.0

2.6

25.0

8.0

7.8

6.0

3.2

Negative control

-

5.4

5.0

1.6

3.0

Solvent control with water

-

4.8

3.0

1.4

3.0

Positive control with EMS

150.0

37.0

24.4

41.2

34.6

 EMS: Ethylmethane sulfonate

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus test in mice: negative (BASF SE, 2002)
UDS test in rats: negative (BASF SE, 2002)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1157
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, CH-4414 Fuellinsdorf
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: mean weight 39.6 ± 3.0 g
- Housing: single
- Cage type: Makrolon Type II, with wire mesh top (Ehret GmbH, Emmendingen)
- Diet (ad libitum): pelleted standard diet
- Water (ad libitum): tap water
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
intraperitoneal
Vehicle:
The test material was diluted in deionised water and was administered at a volume of 10 mL/kg b.w. The vehicle control animals were administered deionised water at the same volume.
Duration of treatment / exposure:
24 and 48 hours, respectively.
Frequency of treatment:
once
Dose / conc.:
37.5 mg/kg bw (total dose)
Dose / conc.:
75 mg/kg bw (total dose)
Dose / conc.:
150 mg/kg bw (total dose)
No. of animals per sex per dose:
6
Control animals:
yes
Positive control(s):
Cyclophosphamide (CPA), Dosing: 40 mg/kg bw, Volume administered: 10 mL/kg bw
Tissues and cell types examined:
Polychromatic erythrocytes (PCE) in the bone marrow.
Details of tissue and slide preparation:
DOSE SELECTION:
The active ingredient content of the test substance was approximately 75%. Therefore, the doses used in this study and reported presently are
expressed in terms of this indicated content. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT:
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test substance, the vehicle or the positive control substance once ip. Six males were treated per dose group and sampling time. The animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the test substance. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

PREPARATION OF THE ANIMALS
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded . A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.


ANALYSIS OF CELLS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes
(PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes
was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs . The analysis was performed with coded
slides.
Evaluation criteria:
A test substance is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes, which clearly exceeds the negative control range or a relevant positive response for at least one of the test points.
A test substance producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a positive response at any of the test points is considered non-mutagenic in this system.
Statistics:
Statistical significance was evaluated by means of the non-parametric Mann-Whitney test (Krauth J., Annals of Math. Stat., 42: 1949-1956, 1971).
However, the primary point of consideration is the biological relevance of the results.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
150 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Summary of the Micronucleus Test Results:

Test group

Period

Dose

(mg/kg bw)

PCEs with

micronuclei

range

PCE/ NCE

deionised water

24 h

0

0.60

0 – 4

2000/ 1937

CPA

24 h

40

16.90

23 – 55

2000/ 1760

Protectol HT

 

24 h

37.5

0.20

0 – 1

2000/ 2053

24 h

75.0

0.50

0 – 3

2000/ 1792

  24 h

150.0

1.50

1 – 8

2000/ 2368

deionised water

48 h

0

0.40

0 – 4

2000/ 1880

Protectol HT

48 h

 

150.0

0.80

0 – 3

2000/ 2535

CPA: Cyclophosphamide

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
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:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
unscheduled DNA synthesis
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1157
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, CH-4414 Fuellinsdorf
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation: males: 188.6 g (± 27.7 g); females: 173.4 g (± 18.7 g), mean values
- Fasting period before study: The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment) before receiving the test substance, the positive or the vehicle control substance.
- Housing: single
- Cage type: Makrolon Type II, with wire mesh top (Ehret GmbH, Emmendingen)
- Diet (ad libitum): pelleted standard diet
- Water (ad libitum): tap water



ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: gavage
Vehicle:
The test material was diluted in deionised water and was administered at a volume of 10 mL/kg b.w. The vehicle control animals were administered
deionised water at the same volume.
Duration of treatment / exposure:
2 and 16 hours, respectively
Frequency of treatment:
once
Post exposure period:
none
Dose / conc.:
375 mg/kg bw (total dose)
Remarks:
Male animals
Dose / conc.:
750 mg/kg bw (total dose)
Remarks:
Male animals
Dose / conc.:
250 mg/kg bw (total dose)
Remarks:
Female animals
Dose / conc.:
500 mg/kg bw (total dose)
Remarks:
Female animals
No. of animals per sex per dose:
4
Control animals:
no
Positive control(s):
- 2 hours preparation interval: DMH; N,N'-Dimethylhydrazinedihydrochloride (sym.), Purity: >99 %, Dosing: 40 mg/kg bw, Vol. admin.: 10 mL/kg bw
- 16 hours preparation interval: 2-AAF; 2-Acetylaminofluorene, Purity: >99%, Dosing: 100 mg/kg bw, Vol. admin.: 10 mL/kg bw
Details of tissue and slide preparation:
DOSE SELECTION:
The active ingredient content of the test substance was approximately 75%. Therefore, the doses used in this study and reported presently are
expressed in terms of this indicated content. The highest doses used in this test system (750 and 500 mg/kg bw for males and females, respectively) were estimated by the pre-experiments to be suitable. The low doses were half of the high doses.

TREATMENT:
4 male and 4 female rats were assigned to each test group. The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment) before receiving the test substance, the positive or the vehicle control substance. Before the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the body weight of the animals. The animals received the test substance once.

ISOLATION OF THE PRIMARY HEPATOCYTES:
After anaesthetizing the rats with Na-thiopental, the liver was perfused through the vena portae with Hanks' balanced salt solution supplemented with collagenase (0.05 %(w/v), adjusted to pH 7.4 and maintained at 37 °C. The isolated hepatocytes were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh (94 µm) to yield a single cell suspension. The quality of the performed perfusion was determined by the trypan blue dye exclusion method for cell viability. In addition, the number of the cells was determined.

CULTURE CONDITIONS:
The washed hepatocytes were centrifuged and transferred into Williams medium E supplemented with:
Hepes 2.38 mg/mL, L-Glutamine 0.29 mg/mL, Penicillin 100 units/mL, Insulin 0.50 µg/mL, Streptomycin 0.10 mg/mL and Fetal calf serum 100 µL/mL. This complete medium was adjusted to pH 7.6.
At least three cultures were established from each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (2.0xE+05 viable cells/mL) were added to 35 mm six-well dishes containing one 25 mm round plastic coverslip per well coated with gelatine.
After an attachment period of approximately 1.5 h in a 95 % air/ 5% CO2 humidified incubator at 37 °C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently, 3HTdR in 2.0 mL culture medium (WME, 1%(v/v) FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1%(v/v) FCS and 0.25 mM unlabelled thymidine.
Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of
1%(w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 %(v/v) ethanol, and air-dried.

AUTORADIOGRAPHY:
The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 photographic emulsion in the dark.
The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 4 °C. The photographic emulsion was then
developed with KODAK Dektol Developer at room temperature, fixed in TETENAL and stained with hematoxylin/eosin.

EVALUATION OF UDS:
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were
randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the ARTEK 880 or 982 counter. In addition, the number of grains of the most heavily lableled nuclear-sized cytoplasm area adjacent to the nucleus was counted.
At least two slides per animal and 50 cells per slide were evaluated. Heavily radiolabeled cells undergoing replicative DNA synthesis were excluded from counting.
Evaluation criteria:
Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.
A test substance is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.
A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains.
A test substance producing net grains not greater than 0 at anyone of the test points is considered non-effective in this system.
Statistics:
Statistical significance was evaluated by means of the non-parametric Mann-Whitney test (Krauth J., Annals of Math. Stat., 42: 1949-1956, 1971).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Mean Nucleus, Cytoplasmic Area and Net Grains (mean values of 3 animals).

Treatment

Period

Dose

(mg/kg bw)

Grains per nucleus

Grains per cytoplasmatic area

Net grains per nucleus

 

male

female

male

female

male

female

male

female

deionised water

2 h

-

-

13.74

6.24

21.14

11.57

-7.40

-5.33

DMH

2 h

40

40

30.27

28.29

17.50

8.39

12.77

19.90

Protectol HT

2 h

375

250

13.00

6.21

19.60

11.19

-6.60

-4.98

2 h

750

500

13.91

7.18

19.76

11.65

-5.85

-4.47

deionised water

16 h

-

-

9.75

7.83

15.96

14.05

-6.22

-6.21

2-AAF

16 h

100

100

45.28

23.53

19.98

15.12

25.30

8.41

Protectol HT

16 h

375

250

12.38

8.61

22.00

15.23

-9.62

-6.62

16 h

750

500

12.97

7.50

18.77

18.19

-5.80

-10.69

DMH: N,N'-Dimethylhydrazinedihydrochloride

2-AAF: 2-Acetylaminofluorene

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

Additional information

In vitro:

The mutagenic potential of the test substance was investigated in several in vitro studies in compliance with GLP: A Salmonella/microsome assay (Ames test; BASF SE, 1997), a Salmonella assay (Ames test; BASF SE, 1981), a cytogenetic assay for chromosome aberrations in Chinese hamster V79 cells (BASF SE, 2002), and a gene mutation at the HPRT locus in Chinese hamster V79 cells (HPRT test; BASF SE, 2002). All studies were performed both with and without a metabolic activation system to investigate the potential effect of metabolites of the test substance on the test systems. The Ames test and HPRT test gave no indication for a mutagenic potential of the test substance with and without metabolic activation. In contrast, the test substance showed a positive response in the chromosomal aberration test with and without metabolic activation.

These results are supported by further GLP guideline studies: Ames test negative (L.A.C.S.A., 2015), HPRT test negative (L.A.C.S.A., 2015) and in vitro micronucleus test in human peripheral blood positive (L.A.C.S.A., 2015).

In addition, the substance was tested to be not mutagenic in another Ames test (BASF, 1981), whereas ambiguous results were reported in an Ames test performed in course of the US National Toxicology Program (Mortelsmann, 1986). The substance was not genotoxic in an in vitro UDS assay (Troy, 1993).

 

In vivo:

The evidence for a clastogenic potential of the test substance from an in vitro chromosomal aberration test could not be confirmed in an in vivo micronucleus test with intraperitoneal application in mice according to OECD TG 474 in compliance with GLP (BASF SE, 2002). There was not any substantial enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance and with any dose level used (37.5, 75.0, 150.0 mg/kg bw).

The test substance was negative in an UDS test with liver cells in vivo in rats according to OECD TG 486 in compliance with GLP (BASF SE, 2002). In this study with oral administration by gavage, the substance was tested at doses of 375 and 750 mg/kg bw in males, and 250 and 500 mg/kg bw in females. The viability of the hepatocytes was not substantially affected by the in-vivo substance treatment. The test substance did not reveal at any dose level UDS induction in the hepatocytes of the treated animals as compared to the concurrent vehicle (deionised water) controls.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The positive result in the in vitro chromosome aberration assay was not confirmed by two in vivo tests. Thus, the test substance does not need to be classified and labelled according to Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179.

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