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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:
From 15 February to 19 March 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 471 guideline study in compliance with the GLP. No deviation from the protocol of the study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2011-08-31
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Extremely pale yellow viscous liquid
Details on test material:
- Name of test material (as cited in study report): Isobutyl acid phosphate (IBAP)
- Physical state: pale yellow viscous liquid
- Stability under test conditions: the test item is assumed to be stable by the sponsor.
- Storage condition of test material: room temperature in the dark.

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
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):
Not applicable.
Additional strain / cell type characteristics:
other: see Table 7.6.1/1
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
other: see Table 7.6.1/1
Metabolic activation:
with and without
Metabolic activation system:
S9 from induced phenobarbitone/beta­naphthoflavone rat liver.
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment I: 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment II: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Formulated concentrations were adjusted to allow for the stated water/impurity content (4%) of the test item.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO).
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 25 and 50 mg/ml but was fully miscible in
dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controls
Untreated negative controls:
yes
Remarks:
(untreated)
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
See Table 7.6.1/2
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- In agar (plate incorporation): in the Range Finding Test as well as in the Experiment 1.
- Pre-incubation: in Experiment 2

DURATION
- Incubation period: at 37°C for 48 hours
- Preincubation period: 20 min at 37°C

NUMBER OF REPLICATIONS: Triplicate plating. Two independent experiments were performed.

DETERMINATION OF CYTOTOXICITY
- Method: the cytotoxicity of the test material was determined using TA100 and WP2uvrA in the presence and absence of metabolic activation system with appropriate untreated and a vehicle control (DMSO). The concentrations examined were 5000, 1500, 500, 150, 50, 15, 5, 1.5, 0.5, 0.15 and 0 µg/plate. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures should be in the approximate range of 0.9 to 9 x 1000000000 bacteria per ml.
- Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic
sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There should be a minimum of four non-toxic test material dose levels.
- There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
- Criteria for a Positive Response:
A test item was considered mutagenic if:
- A dose-related increase in revertant frequency over the dose range tested and/or;
- A reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation.
- Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

- Criteria for a Negative Response
A test material will be considered non-mutagenic in the test system if the above criteria are not met.
Statistics:
Standard deviation
Dunnett's Linear Regression Analysis

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
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:
cytotoxicity
Remarks:
at 5000 µg/plate
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:
cytotoxicity
Remarks:
at 5000 µg/plate
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:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was immiscible in sterile distilled water at 25 and 50 mg/ml but was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item exhibited weakened background lawns to both strains (TA100 and WP2uvrA) at 5000 µg/plate in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first experiment (plate incorporation method) the test item caused a visible reduction in the
growth of the bacterial background lawns of all of the tester strains at 5000 µg/plate in both the absence and presence of S9-mix. In the second
experiment (pre-incubation method) the test item induced a weaker toxic response with weakened bacterial background lawns noted at
5000 µg/plate to TA1537 only in both the absence and presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test
item varied between strain type and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate.

Any other information on results incl. tables

Table 7.6.1/3:Summary Table of the Experiment I (with and without metabolic activation)

Concentrations
(µg/plate)

Mean
values of revertants / Standard deviation (SD)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

Mean

18

29

97

109

27

13.0

9

10

31

36

     SD

3.2

6.1

16.5

2.3

2.5

1.2

3.1

3.5

0.6

1.5

15

Mean

20

29

87

111

21

9

8

11

32

38

SD

1.7

5.3

6.0

8.7

5.5

1.2

3.5

4.5

3.5

7.0

50

Mean

19

17

80

100

15

12

8

9

30

32

SD

5.3

4.4

16.8

7.8

3.2

6.2

4.0

4.7

4.0

4.6

150

Mean

25

22

100

105

21

12

7

12

29

37

SD

4.0

2.5

15.9

14.7

4.0

6.2

2.1

5.0

2.1

0.6

500

Mean

14

24

80

84

16

13

8

12

31

41

SD

6.1

7.8

5.1

11.0

2.5

5.9

1.5

3.2

4.9

2.0

1500

Mean

14

18

90

79

21

9

5

9

37

34

SD

1.7

2.5

7.2

5.0

0.6

0.6

2.1

1.0

2.1

4.9

5000

Mean

15

16

117

79

14

13

5

5

30

27

SD

2.3

2.0

9.1

5.0

4.6

2.5

2.3

0.6

1.0

1.2

ENNG

Mean

-

-

407

-

85

-

-

-

518

-

SD

-

-

19.1

-

3.6

-

-

-

45.2

-

4NQO (0.2 µg/plate)

Mean

103

--

--

--

--

--

--

--

--

--

SD

11.1

--

--

--

--

--

--

--

--

--

9AA (80 µg/plate)

Mean

--

-

--

-

--

-

577

-

--

--

SD

--

-

--

-

--

-

111.4

-

--

--

2AA

Mean

--

--

--

1384

--

319

--

234

--

349

MF

--

--

--

66.9

--

18.6

--

37.1

--

23.5

BP (5µg/plate)

Mean

--

225

-

--

-

--

--

--

--

--

MF

--

12.7

-

--

-

--

--

--

--

--

Notes: ENNG; N-ethyl-N’-nitro-N-nitrosoguanidine; 4NQO; 4-Nitroquinoline-1-oxide; 2AA: 2-aminoanthracene; 9AA: 9-aminoacridine; BP; Benzo(a)pyrene;

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative (with and without metabolic activation)

Under the test conditions of this study, the test item Reaction Mass of "Diisobutyl hydrogen phosphate and Isobutyl phosphate" (IBAP) had no mutagenic activity in the applied bacterium tester strains.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 guideline and in compliance with GLP, the Reaction Mass of "Diisobutyl hydrogen phosphate and Isobutyl phosphate" (IBAP) diluted in Dimethyl sulphoxide (DMSO) was tested in S. typhimurium TA1535, TA1537, TA100 and TA98 and in E. coli WP2 uvr A in the presence and the absence of mammalian metabolic activation (S9) using the direct incorporation or the preincubation method. Six known mutagens (4-nitroquinoline-N-oxide; 9-Aminoacridine; N-ethyl-N-nitro-N-nitrosoguanidine; 2-Aminoanthracene and Benzoapyrene), dissolved in dimethylsulfoxide, were used to check the sensitivity of the test system. 

A preliminary study (one plate/concentration) was performed in order to determine the appropriate concentrations for the two independent main studies (3 plates/concentration). In the preliminary study, the bacterial strains TA100 and WP2 uvrA were exposed to the test substance at the following concentrations: 0,0.15,0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test item was freely soluble up to the highest tested concentration. Cytotoxicity, assessed by the decrease in the number of revertants and/or the thinning of the bacterial lawn, was observed in all strains. The test item exhibited weakened background lawns to both strains (TA100 and WP2uvrA) at 5000 µg/plate in the absence and presence of S9-mix. Therefore, 5000 µg/plate was chosen as the highest tested concentration for the main study.

In the first experiment (plate incorporation method; 15, 50, 150, 500, 1500 and 5000 µg/plate) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains at 5000 µg/plate in both the absence and presence of S9-mix. In the second experiment (pre-incubation method with S9 mix, direct plate incorporation without S9 mix; 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) the test item induced a weaker toxic response with weakened bacterial background lawns noted at 5000 µg/plate to TA1537 only in both the absence and presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Under the test conditions, the Reaction Mass of "Diisobutyl hydrogen phosphate and Isobutyl phosphate" (IBAP) did not show any mutagenic activity in the bacterial reverse mutation test using S. typhimurium and E. coli.

This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 471.