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Genetic toxicity: in vitro

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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:
from 2014-10-24 to 2015-03-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No control of concentration in treatment solution. and problem of contamination in 2 plates at the intermediary dose in the third assay.
GLP compliance:
yes (incl. certificate)
Remarks:
2013-03-08
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name: THPC urea amine copolymer (or Perform Sti)

Method

Target gene:
The two strains TA1535 and TA100 show a base pair substitution in the his G gene (G-C instead of A-T). These two strains can be used to detect test items which cause reverse base pair substitution. In return, the strain TA102 contains an ochre (T-A-A) mutation in the his G gene permitting to detect several types of mutation not only reverse base pair substitution but also small deletions.

The strain TA1537 has, in the his C gene, a loop of several cytosines which gives rise to a frameshift mutation. The strain TA98 contains, in the his D gene, a sequence of four base pairs of cytosine-guanine (CG) which also give rise to a frameshift mutation. These two strains can be used to detect products causing reverse frameshift mutation.

The five strains also carry two other types of mutation: they are deficient in DNA repair mechanisms (uvrB-) - apart from the strain TA102 which conserves an intact excision repair system - and show a change in the structure of cell-wall lipopolysaccharides (LPS) (rfa-). These two mutations make the bacteria more sensitive to genotoxic agents and more permeable to cell penetration by large molecules.

The R factor (present in strains TA98, TA100 and TA102), which is a pKM 101 ampicillin resistance plasmid, increases sensitivity to mutagenic agents via error-prone repair mechanisms. In addition, the strain TA102 contains the multicopy plasmid, pAQ1, which carries the his G428 mutation and a tetracycline resistance gene (Maron and Ames, 1983).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The bacterial strains obtained from Professor B.N. Ames (University of California, Berkeley - U.S.A.), are kept in the laboratory and preserved in liquid nitrogen.
The strains are regularly checked for their various characteristics:
- Histidine requirements
- Sensitivity to the reference mutagens
- Loss of cell-wall lipopolysaccharides: rfa mutations (sensitivity to crystal violet) for strains Salmonella typhimurium
- uvr B- mutation (sensitivity to uvB), apart from strain TA102
- Presence of pKM 101 plasmid (ampicillin resistance)
- Presence of pAQ1 plasmid (tetracyclin resistance)
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
The bacterial strains obtained from Professor B.N. Ames (University of California, Berkeley - U.S.A.), are kept in the laboratory and preserved in liquid nitrogen.
The strains are regularly checked for their various characteristics:
- Histidine requirements
- Sensitivity to the reference mutagens
- Loss of cell-wall lipopolysaccharides: rfa mutations (sensitivity to crystal violet) for strains Salmonella typhimurium
- Presence of pKM 101 plasmid (ampicillin resistance)
- Presence of pAQ1 plasmid (tetracyclin resistance)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Toxicity assay: 0, 50, 150, 500, 1500 and 5000 µg/plate
Assay 1 and assay 2: 0, 1.5, 5, 15, 50, 150 and 300 µg/plate
assay 3: 0, 15, 50, 100, 150, 200, 250 and 300 µg/plate
Vehicle / solvent:
The substance was diluted n 0.003% (w/v) citric acid (Merck, batch K44221444315), in sterile water for irrigation (Fresenius, batch 13HOP211)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
20.003% (w/v) citric acid, in sterile water for irrigation
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-anthramine
Details on test system and experimental conditions:
Verification of the absence of microorganisms in the test item in the conditions of the assay:
0.1 mL of the three highest concentrations of the test item was added to 2 mL of top agar in a state of superfusion. The mixture was spread out in 3 Petri plates containing 20 mL of minimal agar. The plates were incubated for approximatively 48 hours at ca. 37°C. The plates were examined for any bacterial growth.


Toxicity assay
In order to choose the range of doses for the test, the toxic activity of the test item is determined. The toxicity assay was carried out in all the strains to be tested under the same conditions as the mutagenicity test with and without metabolic activation but using only one plate per dose instead of 3. The plates were incubated for approximatively 48 hours at ca. 37°C, and the revertants were counted.
Toxicity was checked by microscopic examination of the background growth and was noted as follows:
- : non-toxic
+ : slightly toxic
++ : moderately toxic
+++ : strongly toxic
N : no bacterial growth
The limiting factor for maximum dose was thus maximum dose according to OECD Guideline.

MUTAGENICITY TEST
Without metabolic activation
The following technique was performed for each one of the strains used in the test: 0.1 mL of a bacterial suspension from a culture agitated overnight at ca. 37°C and 0.1 mL of the test item at the relevant initial concentrations were successively added to 2 mL of top agar to which 10 % of 0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C, has been added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar. Three plates were used per dose. The plates were then incubated at ca. 37°C for approximately 48 h. At the end of the expression time, colonies of revertants were counted for each plate.

CONTROLS
Solvent controls, Positive controls
At the same time, solvent controls (0.1 mL solvent/plate) were performed under the same conditions but using 6 plates per solvent (Gatehouse et al., 1994). Appropriate positive reference controls were also performed

Sterility of the media
At each assay, 3 Petri plates containing 20 mL of minimal agar receive 2 mL of top agar only and were incubated under the same conditions of assay for the control of media sterility.

With metabolic activation
The method was the same as without metabolic activation except that immediately before spreading in the plates, 0.5 mL of the S9 mix metabolic activation system was added in soft agar.

Repeat test
Without metabolic activation
The test was subsequently repeated in an independent assay. The same method was used.
In strain TA98, a 3rd assay was also performed.
With metabolic activation
The method used in a second assay was the pre-incubation test: The following solutions were added in this order: the bacterial strain to be tested (100 μL), S9-mix (500 μL) and at the end the test item solution (100 μL as an aqueous solvent was used).
The mixture was preincubated with stirring at ca. 37°C for 60 minutes prior to adding soft agar and spreading out in a Petri plate.
In strains TA98 and TA102, a 3rd assay was also performed.

Preparation of the S9 fraction
The S9 fraction was prepared at Institut Pasteur de Lille (IPL).
This preparation is carried out using the method described by Ames et al. (1975) in male rat Sprague Dawley OFA induced by Aroclor 1254 (origin - Monsanto, Saint Louis, U.S.A) according to the standard operating procedures of the Institut Pasteur de Lille.
The S9 batch number used in this study was IPL 13-I
The S9 mix contained per mL:
- S9 0.1 mL
- MgCl2, 0.4M 0.02 mL
-KCl, 1.65M 0.02 mL
- Phosphate buffer, 0.2M pH 7.4 0.5 mL
- NADP, 0.1M 0.04 mL
- Glucose-6-phosphate, 1M 0.005 mL
- H2O 0.315 mL
All the solutions prepared immediately before use were filtered (except for S9) through a sterilizing membrane. The S9 mix was prepared extemporaneously for each assay and preserved in a refrigerated place up to use.

Sterility control of the S9 mix
For each test, the S9 mix alone was added to 3 plates in order to check the sterility.
Evaluation criteria:
After about 48 hours of incubation at ca. 37°C, the prototrophic mutant colonies that have developed in the plates were counted, eventually using a colony counter. The results are expressed as the mean number of revertants per plate and, for each concentration of the test product, the following ratio was established:

Mean number of revertants per plate in presence of the test product / Mean number of revertants per plate without the test product (solvent control)

For the Strains TA1535, TA1537:
A test item causing a positive response proportional to the dose for at least 3 doses with, for the highest increase, a value greater than or equal to 3 times the value for the solvent control, is considered positive in the assay.

For the Strains TA98, TA100, TA102
A test item causing a positive response proportional to the dose for at least 3 doses with, for the highest increase, a value greater than or equal to 2 times the value for the solvent control, is considered positive in the assay.

Reproducibility
If a test item causes a positive response during a single assay and that result cannot be reproduced in at least 1 independent assay, the initial positive result may be considered as not significant.

Comparison to historical control data
In some borderline cases, an additional criterion to be considered is the comparison between the number of revertants induced by the test item and the laboratory historical control data. Indeed, an increase in each individual value that is above the highest value of corresponding historical control data can help supporting a conclusion such as “equivocal” or “weak” mutagen.
All these criteria are not absolute, but they however, help in coming to a decision, which can be conclusive in the majority of the cases (Brusick, 1980).
Statistics:
Data were analysed by means of Dunnett's method (Mahon et al., 1989) allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See results tables
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See results tables
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Verification of the absence of microorganisms in the test item in the conditions of the assay:
No colony was visible. The results were satisfactory

Preliminary toxicity assay
The substance induced an important toxicity at the 2 highest doses of 1500 and 5000 μg/plate with no bacterial growth, and a strong toxicity at 500 μg/plate with the appearance of microcolonies of toxicity both with and without S9-mix. Otherwise, the dose of 150 μg/plate induced a slight toxicity in presence of metabolic activation.
Therefore, the maximum doses retained for the first mutagenicity assay was of 300 μg/plate in all strains both with and without metabolic activation.

Toxicity in mutagenicity assays
In the 1st assay, a slight or a moderate toxicity was noted at the highest dose of 300 μg/plate, depending on the strain and the metabolic activation condition, with sometimes subsequent decreases in the number of revertants.
Under these conditions, the highest dose tested was still 300 μg/plate in all strains both in absence and in presence of metabolic activation in the 2nd assay.
In absence of metabolic activation, slight or moderate toxicity was noted in all strains, except in strain TA102, at the highest dose of 300 μg/plate, with, in strain TA100, no bacterial growth and the appearance of microcolonies of toxicity.
With metabolic activation, the substance induced a slight toxicity at the dose tested of 300 μg/plate in strains TA1537 and TA98, and a moderate toxicity in the 3 other strains with no bacterial growth and the appearance of microcolonies of toxicity.
Under these conditions, the highest dose tested was kept at 300 μg/plate in strain TA98 both in absence and in presence of metabolic activation, and was lowered at 250 μg/plate in strain TA102 in the 3rd assay.
In this assay, a slight and a moderate toxicity was observed in strain TA98 in presence of metabolic activation at the 2 highest tested doses of 250 and 300 μg/plate, respectively. Moreover, a slight toxicity was noted at the 2 highest tested doses of 250 and 300 μg/plate in the same strain without metabolic activation and at the highest tested dose of 250 μg/plate in strain TA102.

Sterility of the media
No colony was observed after around 48 hours at ca. 37°C. The results were satisfactory.

Repeat test
+ Without metabolic activation: a statistically and biologically significant increase in the number of revertants was noted at the intermediary dose of 150 μg/plate.
+ With metabolic activation: negative responses were observed in the first assay. Statistically and/or biologically significant increases in the number of revertants were noted in strains TA98 and TA102 in the second assay. This results were not confirmed with the third assay

Sterility control of the S9 mix
No colony was observed after about 48 hours at ca. 37°C. The results were satisfactory.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results tables

ASSAY 1

Dose in µg / plate

TA 1535

TA 1537

TA 98

TA 100

TA 102

Revertants per plate

Induction ratio (a)

Revertants per plate

Induction ratio (a)

Revertants per plate

Induction ratio (a)

Revertants per plate

Induction ratio (a)

Revertants per plate

Induction ratio (a)

Positive control

(b)

757.3

108.2

1038.7

182.2

461.3

32.2

1032.0

11.1

1274.7

8.1

TEST ITEM

Without S9-mix

0

7.0

-

5.7

-

14.3

-

93.3

-

157.7

 

1.5

7.7

1.1

6.0

1.1

12.3

0.9

98.3

1.1

171.3

1.1

5

12.3

1.8

7.0

1.2

14.3

1.0

91.0

1.0

191.3

1.2

15

6.3

0.9

6.3

1.1

13.3

0.9

94.3

1.0

220.0

1.4

50

9.7

1.4

5.7

1.0

17.7

1.2

89.7

1.0

223.3

1.4

150

5.7

0.8

8.7

1.5

19.0

1.3

109.3

1.2

238.0

1.5

300

3.0

0.4

3.7

0.6

12.7

0.9

38.3

0.4

188.0

1.2

 

 

 

 

 

 

 

 

 

 

 

 

Positive control

(c)

970.7

107.9

976.0

125.1

3152.0

1433

3162.7

30.3

1210.7

5.1

TEST ITEM

With S9-mix

0

9.0

-

7.8

-

22.0

-

104.3

-

239.3

-

1.5

9.3

1.0

6.0

0.8

20.0

0.9

94.7

0.9

265.3

1.1

5

10.7

1.2

6.0

0.8

24.7

1.1

107.0

1.0

286.7

1.2

15

8.7

1.0

7.3

0.9

29.7

1.4

100.0

1.0

289.3

1.2

50

12.3

1.4

6.7

0.9

29.7

1.4

105.0

1.0

324.7

1.4

150

6.7

0.7

8.7

1.1

27.0

1.2

100.0

1.0

319.3

1.3

300

1.7

0.2

3.7

0.5

13.3

0.6

59.7

0.6

315.3

1.3

Solvent: 0.003% (w/v) citric acid, in sterile water for irrigation

a) Induction Ratio = number of revertants in the treated / number of revertants in the control

Reference positive compounds (μg/plate):

·(b) TA1535 and TA 100: Sodium azide w 1 μg/plate; TA1537: 9 -amino-acridined 50 μg/plate; TA98: 2 -nitrofluorened 2 μg/plate; TA102: Mitomycin Cw 0.125 μg/plate

·        (c) TA1535, TA1537, TA98, TA100: 2 -anthramined 2μg/plate; TA102: benzo(a)pyrened 2μg/plate

Solvents used for positive controls:d DMSO;w distilled water

 

ASSAY 2

Dose in µg / plate

TA 1535

TA 1537

TA 98

TA 100

TA 102

Revertants per plate

Induction ratio (a)

Revertants per plate

Induction ratio (a)

Revertants per plate

Induction ratio (a)

Revertants per plate

Induction ratio (a)

Revertants per plate

Induction ratio (a)

Positive control

(b)

642.2

69.9

560.0

98.2

527.3

51.7

597.3

5.9

973.3

6.1

TEST ITEM

Without S9-mix

0

9.2

-

5.7

-

10.2

-

100.5

-

159.0

-

1.5

10.0

1.1

7.7

1.4

15.7

1.5

90.0

0.9

165.7

1.0

5

9.7

1.1

6.7

1.2

13.0

1.3

87.7

0.9

171.0

1.1

15

5.3

0.6

6.3

1.1

15.0

1.5

94.7

0.9

191.3

1.2

50

5.7

0.6

4.3

0.8

12.7

1.2

91.7

0.9

216.7

1.4

150

7.3

0.8

3.3

0.6

24.7

2.4

97.0

1.0

251.3

1.6

300

5.0

0.5

4.0

0.7

12.3

1.2

-*

*

275.3

1.7

 

 

 

 

 

 

 

 

 

 

 

 

Positive control

(c)

152.0

19.7

130.3

17.4

1560.0

61.2

1392.0

16.0

976.0

4.5

TEST ITEM

With S9-mix

0

7.7

-

7.5

-

25.5

-

86.8

-

217.2

-

1.5

4.7

0.6

8.3

1.1

26.3

1.0

98.3

1.1

235.3

1.1

5

9.0

1.2

8.7

1.2

29.3

1.1

91.0

1.0

252.0

1.2

15

5.0

0.6

9.0

1.2

31.0

1.2

104.7

1.2

291.3

1.3

50

8.3

1.1

8.0

1.1

52.3

2.1

121.3

1.4

351.3

1.6

150

3.0

0.4

4.7

0.6

32.3

1.3

75.0

0.9

434.7

2.0

300

*

*

6.3

0.8

22.3

0.9

*

*

*

*

Solvent: 0.003% (w/v) citric acid, in sterile water for irrigation

*: No bacterial growth - No calculations could thus be performed

(a) Induction Ratio = number of revertants in the treated / number of revertants in the control

Reference positive compounds:

·TA 1535 and TA 100 Sodium azidew 1 μg/plate; TA1537: 9 -amino-acridined 50 μg/plate; TA98: 2 -nitrofluorened 2 μg/plate; TA102: Mitomycin Cw 0.125 μg/plate

·TA 1535, TA 1537, TA 98, TA 100: 2 -anthramine d 1 μg/plate; TA102: benzo(a)pyrened 2 μg/plate

Solvents used for positive controls:d DMSO;w distilled water

 

ASSAY 3

Dose in µg/plate

TA 98

TA 102

Revertants per plate

Induction ratio (a)

 

 

Positive control

(b)

220.0

11.4

 

 

TEST ITEM

Without S9-mix

0

19.3

-

 

 

15

20.7

1.1

 

 

50

20.3

1.1

 

 

100

20.7

1.1

 

 

150

21.0

1.1

 

 

200

20.7

1.1

 

 

250

29.3

1.5

 

 

300

18.3

0.9

 

 

 

 

 

 

 

 

Positive control

(c)

3232.0

110.3

1050.7

3.4

TEST ITEM

With S9-mix

0

29.3

-

306.3

-

15

33.3

1.1

292.7

1.0

50

42.7

1.5

362.0

1.2

100

24.0

0.8

426.7

1.4

150

33.3

1.1

436.7

1.4

200

42.7

1.5

463.3

1.5

250

26.3

0.9

443.3

1.4

300

27.7

0.9

462.7

1.5

(a) Induction Ratio = nimber of revertants in the treated / numbers of revertants in the control

Reference positive compounds:

· TA 98: 2 -nitrofluorene d 2 µg/plate

·TA 98: 2 -anthramine d 1 µg/plate; TA102: benzo(a)pyrened 2 µg/plate

Solvents used for positive controls:d DMSOTEST

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The mutagenic activity of the test item Tetrakis[hydroxymethyl]phosphonium chloride oligomeric reaction products with urea and tetradecylamine (CAS No. 359406-89-6) was assessed by means of the Ames’ test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested either in presence or in absence of metabolic activation, in two or three independent assays

The validity criteria for the assay were fulfilled. The study was thus considered as valid.

Under these experimental conditions, no mutagenic activity was revealed.
Executive summary:

The search for any mutagenic activity of Tetrakis[hydroxymethyl]phosphonium chloride oligomeric reaction products with urea and tetradecylamine (CAS No. 359406-89-6) was studied by means of the Ames’ test (Salmonella his-/microsome system) in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 471 using the maximum dose compatible with the toxic activity. Five to six lower dilutions were also tested.

In the second assay, in strain TA98 without S9-mix, a statistically and biologically significant increase in the number of revertants was noted at the intermediary dose of 150 μg/plate with an induction ratio of 2.4 and a mean number of revertants of 24.7, i.e. inside the limits of historical data for negative control (i.e. 2-30). However, this not dose-related increase was not reproducible as the 1st assay was clearly negative.

Moreover, in strain TA98 in presence of metabolic activation, a non-statistically and non dose-related increase in the number of revertants was noted at the intermediary dose of 50 μg/plate with an induction ratio of 2.1 and a mean number of revertants of 52.3, i.e. over the limits of historical data for negative control (i.e. 10-46). However, this increase at this only dose was rather due to an outlier value for the number of revertants in one plate out of 3 with 98 vs. 28 and 31.

A 3rd assay aiming at confirming the negativity of the test item in both these specific conditions was performed, i.e. in strain TA98 with and without metabolic activation with a narrowed range of doses.

In this complementary assay, no statistically or biologically significant increase in the mean number of revertants was noted either with or without metabolic activation. In absence of reproducibility, the effects observed in the second assay were thus considered as not biologically relevant, and the test item was considered as not mutagenic in these conditions.

In both assays in strain TA102 in absence of metabolic activation, statistically but not biologically significant increases in the number of revertants were noted at the doses ranging from 15 to 150 μg/plate or at 150 and 300 μg/plate in the 1st and the 2nd assay, respectively. The induction ratios from 1.4 to 1.5 in the 1st assay or from 1.6 to 1.7 in the 2nd assay, i.e. clearly below the threshold for a positive response set at 2 in this strain.

These effects were thus considered as not biologically relevant, and the test item was considered as not mutagenic in this condition.

In the second assay strain TA102 in presence of metabolic activation following the pre-incubation method, statistically and/or biologically significant increases in the number of revertants were noted at the 2 highest analysable doses of 50 and 150 μg/plate with induction ratios of 1.6 and 2, i.e. equal to the threshold for a positive response. Moreover, this increase was dose-related.

Therefore, a third assay in this experimental condition was performed but with a narrowed range of doses in order to confirm or not this effect.

In this complementary assay, statistically significant increases in the number of revertants were noted at the 5 highest analysable doses ranging from 50 to 250 μg/plate. However, the threshold for a positive response was not reached, with values of 1.4 and 1.5 vs. 2. In absence of biologically significance and reproducibility, the test item was thus considered as not mutagenic in this condition.Otherwise, neither statistically nor biologically significant increase in the mean number of revertants was noted in all the other conditions tested, in the presence of Tetrakis[hydroxymethyl]phosphonium chloride oligomeric reaction products with urea and tetradecylamine (CAS No. 359406-89-6).