tris (4-nonylphenol, branch) phosphorous acid ester

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:

2001

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tris (4-nonylphenol, branch) phosphorous acid ester

EC Number:

701-028-2

Cas Number:

26523-78-4

Molecular formula:

C45H69O3P

IUPAC Name:

tris (4-nonylphenol, branch) phosphorous acid ester

Constituent 2

Reference substance name:

Nonylphenol

EC Number:

246-672-0

EC Name:

Nonylphenol

Cas Number:

25154-52-3

IUPAC Name:

2-nonylphenol

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

0, 75, 200, 600, 1800, 5000 ug/plate

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

yes

Remarks:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

other: 1) all Salmonella strains and WP2 urvA with S9 : 2-aminoanthracene 2) TA98 without S9 : 2-nitrofluorene 3) TA100 and TA1535 without S9 : sodium azide 4) TA1537 without S9 : 9-amino-acridine 5) WP2 urvA without S9 : methyl methanesulfonate.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: Samples were run in triplicate, with and without metabolic activation.METHOD OF APPLICATION- On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 µM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water produced by the Milli-Q Reagent Water System. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5% (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5% (W/V) agar and supplemented with 2.5% (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5% (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain, and activation, as described in detail in BioReliance's Standard Operating.- Procedures. Test article dilutions were prepared immediately before use. One-half (0.5) mL of S9 or Sham mix, 100 µL of tester strain and 50 µL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted. The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

Evaluation criteria:

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.All criteria for a valid test were met. To meet this criteria, all Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10-50; TA100, 80-240; TA1535, 5-45; TA1537, 3-21; WP2uvrA, 10-60. To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3E09 cells/mL. The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels are required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the background lawn.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test results

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: With metabolic activation: > 5000 ug/plate Without metabolic activation: > 5000 ug/plate

Remarks on result:

other: other: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

For a summary of results: See Table 5.5.2 (Summary of Results) in document attached to Chapter 1.11 Additional Remarks.

Dose (mg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Liver Microsomes: None
Vehicle	17±3	96±11	13±2	5±3	17±3
75	16±3	99±10	9±4	5±0	15±3
200	18±2	105±16	9±3	5±3	14±2
600	15±3	108±15	11±1	5±1	15±2
1800	15±3	89±18	14±4	6±1	11±2
5000	11±3	95±13	11±2	6±1	13±2
Positive	91±13	332±10	195±7	448±50	77±10
Dose (mg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Liver Microsomes: Rat Liver S9
Vehicle	18±4	90±4	8±4	7±1	11±2
75	16±6	88±10	10±1	6±3	11±2
200	16±5	97±7	10±3	6±1	14±2
600	20±1	92±11	10±3	5±3	11±1
1800	15±3	85±7	11±2	5±3	12±1
5000	15±3	84±9	10±2	4±2	10±1
Positive	279±134	356±19	50±8	32±11	86±8

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negativeUsed for data submission

Executive summary:

Used for data submission