Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD471): negative with and without metabolic activation

Chromosome aberration (in-vitro mammalian chromosome aberration test, OECD 473): negative with with and without metabolic activation (result based on read-across to source substance AEPD, CAS 115-70-8, EC 204-101-2)

Gene mutation (mammalian cell gene mutation test, OECD 476): negative with and without metabolic activation (result based on read-across to source substance AEPD, CAS 115-70-8, EC 204-101-2)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar 12 - May 28, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)
TRY operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
his G 428, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
E. coli WP2
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
Test concentrations with justification for top dose:
The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1. Series: 5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate
2. Series: 50, 158, 500, 1580 and 5000 µg/plate
Vehicle / solvent:
Aqua bidest
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
cumene hydroperoxide
other: Daunomycin, 2-Aminoanthracene
Details on test system and experimental conditions:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies.
The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

-----------------------------------------------------------------------------------------
Mean Number of Colonies Maximal Mean Number of Colonies over the Actual
(Solvent Control) Solvent Control
(Test Material)
-----------------------------------------------------------------------------------------

<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment No increase Clear increase

-----------------------------------------------------------------------------------------

All further results, ranging between "no" and "clear", are assessed as "weak increases".
Interpretations:
A test material is defined as non-mutagenic in this assay if "no" or "weak increases" occur in the first and second series of the main experiment.
("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed.
If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Evaluation criteria:
cf. details in results
Statistics:
n.a.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation on agar plates: no
Cytotoxicity: no
Conclusions:
Interpretation of results: negative with and without metabolic activation
With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
Executive summary:

Purpose

The purpose of this assay was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates was not observed. no evidence for toxicity to the bacteria has been obtained.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9 -aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results predetermined in the Study Protocol (cf also "Criteria for negative and positive results"), the test material was not mutagenic under the described experimental conditions.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

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
Study period:
14 Feb - 06 June 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In accordance to the ECHA guidance document "Practical guide: How to use alternatives to animal testing" (Version 2.0, July 2016), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
a detailled justification for the analogue approach can be found in Iuclid section 13 of the dossier.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
CLP Certificate Germany; CLP Certificate The Netherlands; CLP Certifcate India
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Type and identity of media:
- basic medium: Ham´s F-12 medium supplemented with sodium bicarbonate, antibiotics and L-glutamine
- basic medium supplemented with 10% fetal bovine serum was the complete medium and was used for the growth and multiplication of cells as well as in detaching and diluting the cells
- basic medium without serum was the treatment medium used for target cell exposure to the test article and controls
- cloning medium was basic medium supplemented with 20% fetal bovine serum and was used for determination of cell viability or plating/cloning efficiency
- selective medium was basic medium supplemented with 20% fetal bovine serum and the selective agent 6-Thioguanine (6-TG) at 35 µM and was used for the selection of mutants.

Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
initial gene mutation test: 12, 38, 119, 337, 1192 µg/mL with and without metabolic activation
confirmatory gene mutation test: 15, 44, 132, 397, 1192 µg/mL with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water (SDW)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9: 3-Methylcholanthrene at 8 µg/mL; without S9: ethyl methanesulphonate at 600 µg/mL
Details on test system and experimental conditions:
DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: duplicates; two independent experiments (initial gene mutation test and confirmatory gene mutation test: each with and without metabolic activation)

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency (RCE: Effect of the test item on cell multiplication was estimated by expressing cytotoxicity relative to the solvent control cultures.)
Evaluation criteria:
Criteria for determining a positive result: concentration related or reproducible increases in mutant frequencies.
Criteria for data acceptability:
- The cloning efficiency of the solvent controls should not be less than 60%.
- The mean mutant frequency of the solvent controls in each experiment should fall within a range of 0 to 20 mutants per 1E+06 clonable cells.
- The positive controls must induce a statistically significant response.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1192 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test item altered the pH of the test solutions at and above 298 µg/mL with metabolic activation and at and above 149 µg/mL without metabolic activation, therefore, the pH of the test solutions at these concentrations was adjusted to neutrality before exposure to the cells.
- Effects of osmolality: The test substance did not cause any appreciable changes in the osmolarity of the test solutions at the end of 4-h exposure with or without metabolic activation.
- Water solubility: 859-879 g/L
- Precipitation: The test substance did not precipitate in the medium up to the hightest tested concentration with or without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed.

COMPARISON WITH HISTORICAL CONTROL DATA: The frequencies of mutants of the positive controls and of the solvent control were within laboratory historical controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Initial gene mutation assay: There was no evidence of excessive cytotoxicity (i.e. <10% RCE) at any of the tested concentrations either with or without metabolic activation. The RCE values in the presence of metabolic activation, ranged from 38.4 to 81.3% while in the absence of metabolic activation, ranged from 44.6 to 82.3% compared to the solvent control.
Confirmatory gene mutation assay: There was no evidence of excessive cytotoxicity (i.e. <10% RCE) at any of the tested concentrations either with or without metabolic activation. The RCE values in the presence of metabolic activation, ranged from 43.3 to 87.0% while in the absence of metabolic activation, ranged from 58.7 to 77.9% compared to the solvent control.

Table 1: Initial gene mutation assay with metabolic activation

Concentration
[µg/mL]

Toxicity assay:

Relative Cloning Efficiency [%]

Mutation assay: total TGrcolonies per 2E+06 cells

Mutation assay: ACE at selection [%]

TGrmutants per 1E+06 clonable cells

Mean TGrmutants per 1E+06 clonable cells

Mutation factor

0 (SDW)

98.8

25

94.5

13.2

12.1

1.0

0 (SDW)

101.2

20

91.0

11.0

12

81.3

15

89.3

8.4

9.1

0.8

12

79.0

17

86.5

9.8

38

69.1

16

82.8

9.7

8.3

0.7

38

70.2

12

87.8

6.8

119

61.5

18

69.5

12.9

14.9

1.2

119

57.7

22

65.2

16.9

377

45.1

21

61.5

17.1

17.7

1.5

377

43.2

23

63.0

18.3

1192

38.4

24

66.5

18.0

19.8

1.6

1192

41.1

29

67.2

21.6

3-MCA

39.6

284

82.2

172.8

169.2

14.0

3-MCA

41.5

294

88.8

165.5

3-MCA 3-Methylcholanthrene

TGr: 6-Thioguanine resistant

SDW: sterile distilled water

 

Table 2: Initial gene mutation assay without metabolic activation

Concentration
[µg/mL]

Toxicity assay:

Relative Cloning Efficiency [%]

Mutation assay: total TGrcolonies per 2E+06 cells

Mutation assay: ACE at selection [%]

TGrmutants per 1E+06 clonable cells

Mean TGrmutants per 1E+06 clonable cells

Mutation factor

0 (SDW)

100.4

19

93.8

10.1

9.9

1.0

0 (SDW)

99.6

18

93.8

9.6

12

82.3

23

93.0

12.4

11.5

1.2

12

79.5

19

89.7

10.6

38

75.2

28

87.5

16.0

14.8

1.5

38

78.6

24

88.7

13.5

119

67.1

30

82.5

18.2

16.1

1.6

119

67.6

24

86.3

13.9

377

66.1

25

86.8

14.4

13.8

1.4

377

64.9

23

87.5

13.1

1192

51.3

23

81.3

14.1

14.4

1.5

1192

44.6

23

78.8

14.6

EMS

43.5

25

86.7

144.2

159.2

16.1

EMS

48.1

281

80.7

174.2

EMS: Ethyl methane sulphonate

TGr: 6-Thioguanine resistant

SDW: sterile distilled water

Table 3: Confirmatory gene mutation assay with metabolic activation

Concentration
[µg/mL]

Toxicity assay:

Relative Cloning Efficiency [%]

Mutation assay: total TGrcolonies per 2E+06 cells

Mutation assay: ACE at selection [%]

TGrmutants per 1E+06 clonable cells

Mean TGrmutants per 1E+06 clonable cells

Mutation factor

0 (SDW)

102.6

29

93.7

15.5

11.9

1.0

0 (SDW)

97.4

15

91.3

8.2

15

87.0

17

88.5

9.6

9.6

0.8

15

83.9

16

84.3

9.5

44

68.0

26

80.8

16.1

14.3

1.2

44

67.3

20

80.0

12.5

132

71.1

12

81.7

7.3

10.0

0.8

132

64.0

19

75.3

12.6

397

52.7

24

78.8

15.2

12.0

1.0

397

54.5

15

85.2

8.8

1192

43.3

20

74.2

13.5

12.9

1.0

1192

43.7

21

85.2

12.3

3-MCA

45.3

285

85.8

166.0

175.0

14.7

3-MCA

46.1

304

82.7

183.9

3-MCA 3-Methylcholanthrene

TGr: 6-Thioguanine resistant

SDW: sterile distilled water

 

Table 4: Confirmatory gene mutation assay without metabolic activation

Concentration
[µg/mL]

Toxicity assay:

Relative Cloning Efficiency [%]

Mutation assay: total TGrcolonies per 2E+06 cells

Mutation assay: ACE at selection [%]

TGrmutants per 1E+06 clonable cells

Mean TGrmutants per 1E+06 clonable cells

Mutation factor

0 (SDW)

99.4

12

88.3

6.8

8.4

1.0

0 (SDW)

100.6

18

89.8

10.0

15

77.9

20

91.3

10.9

10.9

1.3

15

73.8

18

83.7

10.8

44

65.6

21

80.2

13.1

11.7

1.4

44

65.0

17

83.2

10.2

132

62.4

13

79.8

8.1

6.8

0.8

132

60.9

9

82.2

5.5

397

68.6

16

81.7

9.8

8.1

1.0

397

68.8

10

77.8

6.4

1192

66.9

13

72.0

9.0

11.4

1.4

1192

58.7

21

76.2

13.8

EMS

39.4

299

76.2

196.3

198.1

23.6

EMS

40.9

283

70.8

199.8

EMS   Ethyl methanesulphonate

TGr: 6-Thioguanine resistant

SDW: sterile distilled water

 

Conclusions:
Interpretation of results: negative
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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In accordance to the ECHA guidance document "Practical guide: How to use alternatives to animal testing" (Version 2.0, July 2016), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
a detailled justification for the analogue approach can be found in Iuclid section 13 of the dossier.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung (CHL/IU) cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle´s MEM with 10 vol.% calf serum (deactivated)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
without S9 mix(short-term treatment, continuous treatment 24 h and 48 h): 75, 150, 300, 600 and 1200 μg/mL
with S9 mix(short-term treatment): 75, 150, 300, 600 and 1200 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiol. saline
Untreated negative controls:
yes
Remarks:
medium
Negative solvent / vehicle controls:
yes
Remarks:
physiol. saline
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9 mix: Mitomycin C (0.05 µg/mL), with S9 mix: Cyclophosphamide (15 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 h (short time treatment), 24 and 48 h (continuous treatment)
- Expression time (cells in growth medium): 18 h (for short time treatment)

SPINDLE INHIBITOR (cytogenetic assays): colcemide (final concentration: 0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa stain (2 vol.%)

NUMBER OF REPLICATIONS: 2 plates per test, 1 experiment

NUMBER OF CELLS EVALUATED: 200 metaphase images per dose (100 per plate)

DETERMINATION OF CYTOTOXICITY
- Method: The cell multiplication inhibiting action of the test substance was determined by measuring the cell density using a single-layer cultured cell densitometer and using the ratio of the cell density of the test group and of the negative group.
Evaluation criteria:
The judgment was made according to the criteria of Ishidate et al. (1987). When the frequencies of appearance of the cells with chromosomal aberrations (CA) were < 5%, the results were considered negative; when the frequencies of CA were 5% or > 5%, the results were considered false positive; when the frequencies of CA were 10% or greater, the results were considered positive. Finally, in the cases in which dose dependence or reproducibility was observed the appearance of aberrant cells, the results were considered positive.
Key result
Species / strain:
mammalian cell line, other: Chinese hamster lung (CHL/IU) cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Based on the results of performing cell multiplication inhibition tests, it was decided to use a test concentration range of 75-1200 µg/mL.

No increase in chromosomal aberrations was observed in the test with either the short- term treatment (without S9 mix and with S9 mix) or continuous treatment.

Table 1: Results of experiments

Test item

Concentration

Cell growth ratio [mean in %]*

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

Exposure period 6-18 h, fixation time 24h, without S9 mix

saline

0.9%

100

2.5

1.0

MMC

0.05

79

25

24

Test substance

75

93

2.5

1.0

150

93

3.0

1.5

300

86

2.5

0.5

600

86

2.0

1.0

1200

79

2.0

1.5

Exposure period 6-18 h, fixation time 24h, with S9 mix

saline

0.9%

100

1.0

0

CP

15

93

79.0

79.0

Test substance

75

107

1.5

1.5

150

93

1.0

0.5

300

100

2.0

1.0

600

93

2.0

1.0

1200

85

2.5

1.5

Exposure period 24h, fixation time 24h, without S9 mix

saline

0.9%

100

4.0

2.0

MMC

0.05

89

43.5

42.0

Test substance

75

89

3.0

0.5

150

89

3.0

1.0

300

111

2.5

1.0

600

89

2.5

0.5

1200

67

4.9

3.7

Exposure period 48h, fixation time 48h, without S9 mix

saline

0.9%

100

3.0

0.5

MMC

0.05

94

68.0

66.5

Test substance

75

100

1.5

0

150

106

1.5

0.5

300

106

4.0

1.5

600

100

1.5

0.5

1200

50

4.8

3.6

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

* The mean value showed as a growth ratio against the negative control value.

 

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an Ames test which was conducted according to OECD 471 test guideline with the test material TRIS HCl, information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man is provided (reference 7.6.1 -1). The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates was not observed. No evidence for toxicity to the bacteria has been obtained. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9 -aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results predetermined in the Study Protocol (cf also "Criteria for negative and positive results"), the test material was not mutagenic under the described experimental conditions. With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

There are no data available on cytogenicity in-vitro and mutagenicity in-vitro in mammalian cells of the target substance 2-amino-2-(hydroxymethyl)propane-1,3-diol hydrochloride (TRIS HCl). However, there are reliable data available from the source substance 2-Amino-2-ethyl-1,3-propanediol, (AEPD), considered suitable for read-across using the analogue approach. A detailled justification for the applied analogue approach can be found in Iuclid section 13.

In a study conducted according to OECD 473, the potential of AEPD to induce chromosomal aberrations was tested in cultured Chinese hamster lung (CHL) cells (reference 7.6.1 -3). CHL cells were exposed to AEPD at concentrations up to 1200 µg/mL. No increase in chromosomal aberrations was observed in the experiments with short-term treatment (6 h) in the presence or absence of metabolic activation. No cytotoxic effects were observed and the positive controls were valid. Because of the negative results of the short-term treatment, an additional testing without metabolic activation was performed with continuous treatment (24 and 48 h). After continuous treatment, AEPD did not induce chromosomal aberrations in CHL cells.

AEPD was also tested for its potential to cause gene mutations in an in-vitro mammalian cell mutation assay according to OECD 476 (reference 7.6.1 -2). Chinese hamster ovary (CHO) cells were treated with AEPD at concentrations of up to 1192 µg/mL for 4 h both with and without metabolic activation. After an expression time of 9 days in growth medium, cells were incubated for 10 days with 6-thioguanine as selection agent for forward mutation at the HPRT locus. Both with and without metabolic activation, no increases in mutant frequency were observed in the initial and in the confirmatory gene mutation assay. At the highest tested concentration, AEPD caused cell growth inhibition, evaluated by relative cloning efficiency.

Taking into account all the available data, AEPD showed no evidence of a clastogenic and mutagenic potential with and without metabolic activation in in-vitro test systems.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.