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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 October 2018 - 31 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
Diammonium dihydroxybis[lactato(2-)-O1,O2]titanate(2-)
EC Number:
265-409-0
EC Name:
Diammonium dihydroxybis[lactato(2-)-O1,O2]titanate(2-)
Cas Number:
65104-06-5
Molecular formula:
C6H18N2O8Ti
IUPAC Name:
Diammonium dihydroxybis[lactato(2-)-O1,O2]titanate(2-)
Test material form:
solid: particulate/powder

Method

Target gene:
Gene of histidine in Salmonella typhimurium and gene of tryptophan in Escherichia coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa w all mutation and delation of uvr B gene: presence of R factor plasmid pKM101 in strains TA98 and TA 100 to further increase the sensitivity of these strains to some mutagens
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat-liver homogenate activation system (S-9)
Test concentrations with justification for top dose:
The test substance was tested in the tester strain TA100 with concentrations of (a) 50, (b) 100, (c) 200, (d) 400, (e) 800, (f) 1600, (g) 3200 and (h) 5000 µg/plate in the absence and presence of S9-mix.
Based on the results of the dose range finding test, the test item was tested with the concentrations of a) 50, (b) 158, (c) 500, (d) 1581, and (e) 5000 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed using the direct plate incorporation method and the second mutation experiment was conducted using the pre-incubation method.
Vehicle / solvent:
Sterile water

- Justification for choice of solvent/vehicle:
Water is one of the vehicles compatible with this test system. Hence, based on the results of the solubility test, SW was selected as the vehicle of choice in the mutation assay.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
4 ug/plate
Positive control substance:
other: 2-Aminoanthracene
Remarks:
used for assay w ith metabolic activation for all tester strains
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate
Positive control substance:
sodium azide
Remarks:
used for assay w ith metabolic activation for strain TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2 µg/plate
Positive control substance:
2-nitrofluorene
Remarks:
used for assay w ith metabolic activation for strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
50 µg/plate
Positive control substance:
9-aminoacridine
Remarks:
used for assay with metabolic activation for strain TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
4 µg/plate
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
used for assay w ith metabolic activation for strain WP2uvrA
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF APPLICATION:
In the initial mutation assay, which was a plate incorporation mode of exposure, the bacterial suspensions were exposed to the test item, vehicle and the positive controls in the presence and absence of an exogenous metabolic activation system. These bacterial suspensions were then mixed with overlay agar and plated immediately onto minimal medium viz., his- for Salmonella typhimurium and trp- for Escherichia coli, respectively.

In the confirmatory assay, which was a pre-incubation mode of exposure, the test constituents were mixed with the bacteria inside a tube, incubated in an incubator shaker, mixed with overlay agar and plated immediately onto minimal medium his- for Salmonella typhimurium and trp- for Escherichia coli, respectively.

DURATION
- Exposure duration: 67 hrs
-Temperature: 37.0 ± 1.0 degrees

NUMBER OF REPLICATIONS: three

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of the test item toxicity using the code system
Code Description Characteristics
0 Absent Distinguished by a complete lack of any micro-colony lawn over > 90% of the plate compared with the vehicle control plates
1+ SeverelyReduced Distinguished by an extreme thinning of the micro-colony lawn resulting in an increase in the size of the micro-colonies compared with the vehicle control plates such that the micro-colony lawn is visible to the unaided eye as isolated colonies
2+ Moderately reduced Distinguished by a marked thinning of the micro-colony lawn resulting in a pronounced increase in the size of the micro-colonies compared with the vehicle control plates
3+ Slightlyreduced Distinguished by a noticeable thinning of the micro-colony lawn and possibly a slight increase in the size of the micro-colonies compared with the vehicle control plates
4+ Normal Distinguished by a healthy background lawn comparable to vehicle control plates
NP Non-interfering precipitate Distinguished by precipitate on the plate that is visible to the naked eye and is not interfering with the evaluation of bacterial lawn
IP Interfering precipitate Distinguished by precipitate on the plate that is visible to the naked eye and is interfering with the evaluation of bacterial lawn
OP Obscured by particulate The background lawn cannot be accurately evaluated due to microscopic test item particulate
NPO No precipitation observed Precipitation not observed


Evaluation criteria:
To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.

The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test resultsopen allclose all
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
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
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
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
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item did not cause precipitation on the basal agar plates at any of the tested doses.

RANGE-FINDING/SCREENING STUDIES: The test item did not show toxicity in any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies was comparable to the vehicle control plates. Based on these observations, the OECD 471-recommended highest test dose of 5000 µg/plate was tested in the mutation assay.

COMPARISON WITH HISTORICAL CONTROL DATA: Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 demonstrated the requirement of histidine amino acid for their growth. Escherichia coli strain WP2uvrA (pKM101) demonstrated the requirement of tryptophan amino acid for its growth. Ampicillin resistance was demonstrated by the strains TA98, TA100 and WP2uvrA (pKM101) which carry R-factor plasmids. The presence of characteristic mutations like the rfa mutation was demonstrated by all the Salmonella typhimurium strains by their sensitivity to crystal violet. The uvrA mutation in the
Escherichia coli strain and the uvrB mutation in the Salmonella typhimurium strains were demonstrated through their sensitivity to ultraviolet light. Finally, all these tester strains produced spontaneous revertant colonies which were within the frequency ranges of the test facility’s historical control data.

Any other information on results incl. tables

TABLE 1.       Summary Results of Initial Mutation Assay in the Presence of Metabolic Activation

Treatment

(µg/plate)

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

SW

26

2

NA

97

6

NA

14

1

NA

12

2

NA

141

6

NA

 

50

24

2

0.91

100

7

1.04

13

1

0.90

10

2

0.83

134

7

0.95

 

158

26

3

0.99

95

7

0.98

13

1

0.93

10

2

0.89

134

8

0.95

 

500

23

1

0.90

96

3

0.99

13

2

0.95

10

1

0.83

142

5

1.01

 

1581

25

2

0.95

90

6

0.93

13

1

0.90

9

1

0.77

136

6

0.96

 

5000

24

2

0.91

94

5

0.98

13

1

0.93

11

1

0.94

142

4

1.01

 

Positive control

571c

19c

21.95c

873c

26c

9.03c

169c

17c

12.10c

175c

26c

15.03c

567d

20d

4.02d

aValues are means of three replicatescalculated from individual values of Appendix 3and are rounded off to the nearest whole number                            

bRatio of treated/Vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table.

cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)

dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate) SD: Standard deviation       NA: Not applicable

 

 

 

 

TABLE 2.       Summary Results of Initial Mutation Assay in the Absence of Metabolic Activation

Treatment

(µg/plate)

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

SW

25

2

NA

98

8

NA

13

1

NA

12

2

NA

134

5

NA

 

50

24

3

0.96

93

5

0.95

13

2

0.98

10

1

0.86

137

3

1.02

 

158

25

2

1.00

95

6

0.97

12

1

0.90

10

1

0.83

132

4

0.98

 

500

24

2

0.96

93

5

0.95

12

2

0.93

9

2

0.80

139

5

1.04

 

1581

23

2

0.92

96

5

0.98

13

1

0.98

10

2

0.89

131

5

0.98

 

5000

24

2

0.95

92

5

0.94

13

1

0.98

10

2

0.89

132

7

0.98

 

Positive control

275c

15c

10.87c

576d

9d

5.87d

171d

21d

12.80d

179e

17e

15.34e

578f

13f

4.31f

aValues are means of three replicatescalculated from individual values of Appendix 4and are rounded off to the nearest whole number                

bRatio of treated/Vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table.

cTA98: 2-Nitrofluorene (2 µg/plate),                                                                                               

dTA100, TA1535: Sodium azide (1 µg/plate)                                 eTA1537: 9-Aminoacridine (50 µg/plate)                                                                                                                                                          

fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)                 SD: Standard deviation        NA: Not applicable

 

 

 

 

 

TABLE 3.       Summary Results of Confirmatory Mutation Assayin the Presence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA (pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

SW

26

3

NA

96

3

NA

14

1

NA

11

2

NA

140

8

NA

 

50

25

2

0.97

96

9

1.00

13

1

0.93

11

2

0.94

144

5

1.03

 

158

25

3

0.95

97

5

1.01

12

1

0.88

10

2

0.91

136

7

0.97

 

500

24

2

0.94

89

4

0.93

14

1

1.00

10

1

0.85

132

8

0.94

 

1581

23

1

0.88

98

3

1.02

13

1

0.93

10

2

0.88

132

5

0.94

 

5000

24

2

0.94

95

3

0.99

13

1

0.93

10

1

0.88

142

9

1.01

 

Positive control

571c

26c

21.95c

880c

12c

9.16c

182c

16c

12.98c

167c

21c

14.76c

582d

19d

4.16d

aValues are means of three replicatescalculated from individual values of Appendix 5 and are rounded off to the nearest whole number                            

bRatio of treated/Vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table.            

cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)

dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate) SD: Standard deviation       NA: Not applicable

 

 

 

 

 

 

 

TABLE 4.       Summary Results of Confirmatory Mutation Assay in the Absence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

SW

26

2

NA

98

7

NA

14

1

NA

11

2

NA

145

5

NA

 

50

25

2

0.95

99

10

1.01

14

2

1.00

10

2

0.91

143

5

0.99

 

158

25

3

0.95

95

6

0.96

13

2

0.98

10

2

0.85

136

6

0.94

 

500

23

2

0.90

91

4

0.93

13

1

0.93

10

1

0.88

145

5

1.00

 

1581

25

3

0.97

91

7

0.93

13

1

0.95

10

1

0.85

136

9

0.94

 

5000

24

2

0.92

96

6

0.97

13

2

0.98

10

2

0.91

140

8

0.97

 

Positive control

272c

25c

10.46c

573d

22d

5.82d

175d

14d

12.78d

184e

12e

16.26e

568f

20f

3.92f

aValues are means of three replicatescalculated from individual values of Appendix 6and are rounded off to the nearest whole number                            

bRatio of treated/Vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table.

cTA98: 2-Nitrofluorene (2 µg/plate),                                                                          

dTA100, TA1535: Sodium azide (1 µg/plate)                                  eTA1537: 9-Aminoacridine (50 µg/plate)                                                                                                                                      

fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)           SD: Standard deviation                                  NA: Not applicable 

 

Applicant's summary and conclusion

Conclusions:
All criteria for a valid study were met as described in the study plan. It is concluded that the test item, Tyzor LA, was not mutagenic in this bacterial reverse mutation test up to the OECD 471-recommended top dose of 5000 µg/plate under the conditions of testing employed.

Executive summary:

Both the Salmonella typhimurium and Escherichia coli tester strains were found to be reliable and responsive to the different genotypic characterization tests like the amino acid requirement, rfa mutation, uvr mutation and the R-factor plasmids. Similarly, the spontaneous revertant counts of the vehicle control groups of these tester strains were in the ranges of the test facility’s historical control data.

The positive controls produced a more than 3-fold increase in the mean numbers of revertant colonies when compared to the respective vehicle controls, demonstrating the sensitivity of the assay procedure.

The test item at doses up to 5000 µg/plate did not cause a two fold increase in the mean numbers of revertant colonies in the strains TA98, TA100 and WP2uvrA (pKM101) or three fold increase in the mean numbers of revertant colonies in the strains TA1535 and TA1537 either in the presence or absence of the metabolic activation system when compared to the respective vehicle control plates.