Ammonium 6H-dibenzo[c,e][1,2]oxaphosphinin-6-olate 6-oxide

Genetic toxicity: in vitro

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:

26 July 2011 to 3 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

DOPO-OX-Ammonium

IUPAC Name:

DOPO-OX-Ammonium

Details on test material:

Name of test material (as cited in study report): "DOPO-OX-Ammonium"
Chemical name: 9,10-dihydro-10-hydroxy-9-oxa-10-phosphaphenanthren-10-on- resp. 10-oxid-ammonium salt, corrected later to: 9,10-dihydro-10-hydroxy-9-oxa-10-phosphaphenanthren-10-oxid-ammonium salt
Trade name: DOPO-OX-Ammonium
Hill formula: C12H12NO3P
Lot No.: DAS-1101
Appearance: White to slight yellow solid
Conditions of storage: Room temperature
Expiry date: According to the sponsor, the stability of the test item is guaranteed throughout the study period when stored under the conditions stated.

Method

Target gene:

his- (s. typhimurium)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 5

Species / strain / cell type:

other: S. typhimurium TA97a

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from Aroclor 1254 induced microsomes of rat liver

Test concentrations with justification for top dose:

The test substance was suspended in DMSO. The following concentrations were tested:
62, 185, 556, 1667 and 5000 µg per plate without external metabolisation, and
62, 185, 556, 1667 and 5000 µg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene; 7,12-Dimethylbenz[a]anthracene ; 1,8-Dihydroxy-anthraquinone; 2-Nitrofluorene; Sodium azide; 4-Nitro-o-phenylenediamine; t-Butyl-hydroperoxide

Details on test system and experimental conditions:

For the preliminary toxicity test the following solutions/suspensions were combined:
Strain TA100 (overnight culture): 0.1 mL,
S9-mix or phosphate buffered saline: 0.5 mL,
Test substance solution: 0.1 mL,
Top agar: 2.0 mL.
Concentrations of test substance solutions used: 5000, 1667, 556, 185, 62 and 21 µg/plate.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C for 2 days and the growth of the bacterial background and the density of revertant colonies were determined.

The exposure for the first experiment was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state.
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution and
• 2 mL of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).
The exposure for the second experiment was performed according to the 'Preincubation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate with preceding incubation in the liquid state.
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution.
The solutions were preincubated for 20 minutes at 37 °C using a shaker, afterwards combined with 2 mL of top agar and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

Evaluation criteria:

Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 12/3 fold of the amount of the spontaneous revertants.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

other: S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRECIPITATION:
No precipitation of the test substance was seen in any of the concentration groups.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary test and in the main test no toxicity was seen up to 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
The numbers of spontaneous revertants were comparable with the historic control data for the negative controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was seen up to 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

According to the results of this investigation, "DOPO-OX-Ammonium" is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 5000 µg/plate, which is the limit concentration for this kind of test.

Executive summary:

"DOPO-OX-Ammonium" was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and the Council Regulation (EC) No 440/2008, Method B.13/14.

The test substance was suspended in DMSO. The following concentrations were tested:

62, 185, 556, 1667 and 5000µg per plate without external metabolisation, and

62, 185, 556, 1667 and 5000µg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.

In the first experiment the test was performed according to the "direct plate incorporation method", in the second experiment according to the "preincubation method". As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included.

 

Results

Positive controls:

All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

 

Test substance:

Toxicity:

No toxicity of the test substance to the bacteria was observed up to 5000 µg per plate.

 

Solubility:

No precipitation of the test substance was seen in any of the concentration groups.

Mutagenicity:

In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.