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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1989 to April 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Similar to guideline study, with no major deficiencies. No compounds other than 2-aminoanthracene were used as positive control compounds in the presence of S9. No tests were carried out with E.coli WP2 uvrA or S. typhimurium TA 102. The test substance stability was not tested but this is considered not to have adversely affected the study results since dosing solutions were daily prepared freshly before testing.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: 2-aminoanthracene was the only positive control compound tested with metabolic activation. No test carried out with E.coli WP2 uvrA or S. typhimurium TA 102. Test substance stability was not tested.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
EC Number:
259-709-0
EC Name:
Tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
Cas Number:
55566-30-8
Molecular formula:
C4H12O4P.1/2O4S
IUPAC Name:
tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
Test material form:
other: liquid stored in the dark at ambient temperature
Details on test material:
- Name of test material (as cited in study report): THPS-75
- Physical state: colourless liquid
- Stability under test conditions: not tested
- Storage condition of test material: stored in the dark at ambient temperature

Method

Target gene:
Three mutations in the histidine operon are involved:
- his G 46 in TA1535 and TA100
- his C 3076 in TA1537
- his D 3052 in TA1538 and TA98
All 5 strains contained the deep rough; rfa and uvrB mutations. These 2 deletions included the nitrate reductase (chl) and biotin (bio) genes also.
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium strains TA1535, TA 1537, TA 1538, TA98, TA100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from liver of male Fischer 344 rats induced with Arochlor 1254.
Test concentrations with justification for top dose:
For both experiments, both with and without S9 mix:
Expressed as active substance: 0, 1, 3, 10, 33, 100, 333 µg/plate,
Expressed as main ingredient: 0, 0.75, 2.3, 7.5, 24.9, 75.5, 251.4 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile ultra pure water
- Justification for choice of solvent/vehicle: no data
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile ultra pure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AAN)
Remarks:
with S9 for all bacteria strains
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 for TA 1535 and TA 100
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 for TA 1537
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9 for TA 1538 and TA 98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Incubation period: at 37° for 48 hours.

NUMBER OF REPLICATIONS: 3 plates/dose/strain

DETERMINATION OF CYTOTOXICITY
- Method: background lawn of microcolonies observation

Evaluation criteria:
Acceptance criteria:
A test was considered acceptable if for each strain:
- the bacteria demonstrated their typical responses to crystal violet, ampicillin and u.v. light
- at least 2 of the vehicle control plates were within the following colony ranges number:
TA1535: 4 -30
TA1537: 1 -20
TA98: 10 -60
TA100: 60 -200 and
TA1538: 5 -35
- on a least 2 of the positive control plates, there were x 2 the mean of vehicle control mutant numbers per plate, or in case of TA100; x 1.5
- no toxicity or contamination was observed in at least 4 dose levels
- in case where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number.

A mutagenic response was recorded if:
- the test substance induced a doubling in the number of revertants when compared to that in the vehicle controls, at some concentrations. (for TA100, a 1.5 fold increase over the control value is required)
- a significant dose relationship is shown, although at high dose levels, this relationship could be inverted because of, for example toxicity to bacteria generally, specific toxicity to the mutants or inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
- a reproducible effect in independent tests is demonstrated.
Statistics:
no data

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA1535, 1537, 1538, 98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 251.4 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: There was no precipitation of the test compound


RANGE-FINDING/SCREENING STUDIES: a preliminary toxicity study was carried out in TA100 only at  concentrations of 24.9 to 7550 µg main ingredient/plate (33 to 10000 µg  active substance/plate) with and without S9. Complete killing of the bacteria (ie no background lawn of micro-colonies) occurred at 1000 µg, 3333 µg per plate. Toxicity to the bacteria resulting in a thinning of the background lawn of microcolonies was observed at 333 µg per plate in both the presence and the absence of S9 mix.


COMPARISON WITH HISTORICAL CONTROL DATA: Yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 8.5.1. Gene Mutation Assay in the absence of S9 mix

 

Concentration [µg/plate as main ingredient]

Number of mutant cells

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Test I

Test II

Test I

Test II

Test I

Test II

Test I

Test II

Test I

Test II

0

5

6

9

3

7

12

14

15

112

78

0.75

4

5

10

4

7

13

14

15

94

86

2.3

5

4

7

5

6

8

15

17

114

75

7.5

5

5

11

4

7

8

13

13

95

67

24.9

5

1

9

3

9

9

14

10

99

80

75.5

4

6

7

9

6

11

12

6

118

93

251.4

3*

1*

2*

6*

2*

0*

0*

0*

49*

26*

+ve control

54

40

111

135

177

167

161

100

233

185

* Observation of lawn microcolonies due to compound cytotoxicity

 

Table 8.5.2. Gene Mutation Assay in the presence of S9 mix

 

Concentration [µg/plate as main ingredient]

Number of mutant cells

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Test I

Test II

Test I

Test II

Test I

Test II

Test I

Test II

Test I

Test II

0

9

5

8

5

8

18

14

13

101

106

0.75

12

4

11

3

14

19

16

9

110

108

2.3

8

3

8

1

18

17

11

7

113

85

7.5

10

4

6

2

10

20

16

7

121

89

24.9

8

3

7

6

16

9

13

7

98

77

75.5

13

7

9

4

15

33

14

10

102

87

251.4

5*

1*

3*

1*

7*

8*

9*

3*

46*

9*

+ve control

67

34

79

44

355

209

444

146

802

293

* Observation of lawn microcolonies due to compound cytotoxicity

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, THPS had no mutagenic activity on S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, with and without metabolic activation.
Executive summary:

Tolcide PS75 (75.5% THPS main ingredient) was tested for mutagenic activity in S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 according to the method of Ames et al. (1975), similar to the OECD Guidelines 471.

A preliminary toxicity study was carried out in TA100 only at concentrations of 33 to 10000µg active substance/plate with and without S9, corresponding to 24.9 to 7550 µg main ingredient/plate.

The mutagenicity tests were conducted on agar plates at concentrations ranging from 0.75 to 251.4 µg main ingredient/plate with and without S9 mix from Arochlor 1254 treated rat liver.

Toxicity was seen as thinning of the bacterial lawn at 251.4 µg/plate with and without S9 and total lack of lawn above this concentration. There was no precipitation of the test substance.

The test substance did not induce mutagenic activity in either the presence or absence of S9 mix in any of the 5 bacterial strains used. The positive controls induced the appropriate responses in the corresponding strains.

In conclusion, THPS had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

This study is considered as acceptable as it satisfied the criteria of the OECD guideline N°471.