tris (4-nonylphenol, branch) phosphorous acid ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

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 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4-(2-methyloctyl)phenyl bis(4-nonylphenyl) phosphite

EC Number:

701-028-2

Cas Number:

26523-78-4

Molecular formula:

C45H69O3P

IUPAC Name:

4-(2-methyloctyl)phenyl bis(4-nonylphenyl) phosphite

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: Chinese hamster ovary (CHO) cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice.

Test concentrations with justification for top dose:

0, 18.75, 37.5, 75, 150, 200 µg/mL (4-hr treatment w/o S9 mix) 0, 6.25, 12.5, 25, 50, 150 (20-hr treatment w/o S9 mix) 0, 18.75, 37.5, 75, 150, 200 (4-hr treatment with S9 mix)

Vehicle / solvent:

The solvent vehicle for the test article (Acetone) was used as the solvent control at the same concentration as that found in the test article-treated groups.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

other: Mitomycin C (MMC) was used as the positive control in the non-activated study at final concentrations of 0.1 and 0.2 µg/mL. Cyclophosphamide (CP) was used as the positive control in the S9 activated study at a final concentration of 10 µg/mL.

Details on test system and experimental conditions:

The chromosome aberration assay was performed by exposing duplicate cultures of CHO cells to the test article as well as positive and solvent controls. For the chromosome aberration assay, CHO cells were seeded at approximately 5E05 cells/25 cm2 flask and were incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 16-24 hours. Treatment was carried out by refeeding duplicate flasks with 5 mL complete medium (McCoy's 5A medium supplemented with 10% FBS, 100 units penicillin and 100 µg streptomycin/mL, and 2 mM L-glutamine) for the non-activated study or 5 mL S9 reaction mixture for the S9 activated study, to which was added 50 µL of dosing solution of test or control article in solvent or solvent alone. The osmolality of the highest concentration of dosing solution in the treatment medium was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape. In the non-activated study, the cells were exposed to the test article for 4 hours or continuously for 20 hours up to the cell harvest at 37±1°C in a humidified atmosphere of 5±1% CO2 in air. In the 4 hour exposure group, after the exposure period, the treatment medium was removed, the cells washed with CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid® was added to duplicate flasks for each treatment condition at a final concentration of 0.1 µg/mL and the flasks returned to the incubator until cell collection. In the S9 activated study, the cells were exposed for 4 hours at 37±1°C in a humidified atmosphere of 5±1% CO2 in air. After the exposure period, the treatment medium was removed, the cells washed with CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid® was added to duplicate flasks for each treatment condition at a final concentration of 0.1 µg/mL and the flasks were returned to the incubator until cell collection. A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems. After cell harvest an aliquot of the cell suspension was removed from each culture and counted using a Coulter counter. The presence of test article precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control. Two hours after the addition of Colcemid®, metaphase cells were harvested for both the non-activated and S9 activated studies by trypsinization. Cells were collected approximately 20 hours after initiation of treatment. The cells were collected by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspended in 2-4 mL 0.075 M potassium chloride (KCl) and allowed to stand at room temperature for 4-8 minutes. The cells were collected by centrifugation, the supernatant aspirated and the cells fixed with two washes of approximately 2 mL Carnoy's fixative (methanol:glacial acetic acid, 3:1, v/v). The cells were stored overnight or longer in fixative at approximately 2-8°C. To prepare slides, the fixed cells were centrifuged at approximately 800 rpm for 5 minutes, the supernatant was aspirated, and 1 mL fresh fixative was added. After additional centrifugation (at approximately 800 rpm for 5 minutes) the supernatant fluid was decanted and the cells resuspended to opalescence in fresh fixative.A sufficient amount of cell suspension was dropped onto the center of a glass slide and allowed to air dry. Slides were identified by the BioReliance study number, date prepared and the treatment condition. The dried slides were stained with 5% Giemsa, air dried and permanently mounted. In the absence of at least 50% toxicity at any dose level, the selection of dose levels for analysis of chromosome aberrations in CHO cells was based upon precipitation of the test article in treatment medium. The highest dose level evaluated was the lowest precipitating dose level, with a sufficient number of scorable metaphase cells. At least two additional lower dose levels were included in the evaluation.

Evaluation criteria:

The frequency of cells with structural chromosome aberrations in the solvent control must be within the range of the historical solvent control. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p<=0.05, Fisher's exact test) relative to the solvent control. Plates/test: Samples were run in duplicate, with and without metabolic activation.

Statistics:

Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell. Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness. All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p <= 0.05). Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative.

Results and discussion

Test results

Metabolic activation:

with and without

Remarks on result:

other: other: Chinese hamster ovary (CHO) cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The osmolalities in (I) the treatment medium of the solvent (acetone), (II) the highest concentration tested (200 µg/mL), (III) the lowest precipitating dose in the absence of S9 (150 µg/mL), and (IV) the highest soluble dose in the non-activated 20 hour continuous exposure group (50 µg/mL) were 292, 290 294, and 302 mmol/kg, respectively. The pH of the highest concentration of test article in treatment medium was approximately 7.

4-hr harvest without metabolic activation: No toxicity (cell growth inhibition relative to the solvent control) was observed with TNPP in CHO cells when treated for 4 hours in the absence of S9. The mitotic index at the highest dose level evaluated for chromosome aberrations, 150 µg/mL, was 24% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 37.5, 75, and 150 µg/mL. The percentage of cells with structural aberrations in the test article-treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of cells with numerical aberrations in the test article-treated groups was significantly increased above that of the solvent control at dose level 150 µg/mL (p=0.01, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p=0.05). However, the percentage of cells with numerical aberrations (5.5%) was within the historical solvent control range of 0% to 8.0%. Therefore, it is not considered biologically significant. The percentage of structurally damaged cells in the MMC group was found to be statistically significant (27.0%).

4-hr harvest with metabolic activation: No toxicity (cell growth inhibition relative to the solvent control) was observed with TNPP in CHO cells when treated for 4 hours in the presence of S9. The mitotic index at the highest dose level evaluated for chromosome aberrations, 75 µg/mL, was 12% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 18.75, 37.5, and 75 µg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated groups was not statistically increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of structurally damaged cells in the CP group was found to be statistically significant (20.0%).

20-hr harvest without metabolic activation: In the absence of a positive response in the non-activated 4 hour exposure group, slides from the non-activated 20 hour exposure group were evaluated for chromosome aberrations. No toxicity (cell growth inhibition relative to the solvent control) was observed with TNPP. The mitotic index at the highest dose level evaluated for chromosome aberrations, 150 µg/mL, was 10% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 25, 50, and 150 µg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of structurally damaged cells in the MMC group was found to be statistically significant (25.0%).

See Table 5.5.1 (Summary of Test Results) in document attached to Chapter 1.11 Additional Remarks.

	S9	Treat-	Mean		Aberrations		Cells With Aberrations
Treatment	Activa-	ment	Mitotic	Cells	Per Cell		Numerical	Structural
(μg/mL)	tion	Time	Index	Scored	(Mean +/- SD)		(%)	(%)
Vehicle (Acetone)	-	4	11.5	200	0.035	±0.184	1.5	3.5
Phenol, nonyl-, phosphite (3:1)
37.5	-	4	9.9	200	0.025	±0.186	4.0	2.0
75	-	4	10.9	200	0.060	±0.342	4.0	4.0
150	-	4	8.7	200	0.015	±0.122	5.5**	1.5
Positive control (MMC)	-	4	11.5	100	0.370	±0.691	3.0	27.0**
0.2
Acetone	+	4	12.6	200	0.030	±0.171	2.5	3.0
Phenol, nonyl-, phosphite (3:1)
18.75	+	4	13.1	200	0.035	±0.184	3.0	3.5
37.5	+	4	10.2	200	0.045	±0.231	3.0	4.0
75	+	4	11.1	200	0.055	±0.250	1.5	5.0
Positive control (CP)	+	4	7.8	200	0.230	±0.498	0.5	20.0**
10
Vehicle (Acetone)	-	20	10.7	200	0.030	±0.171	2.5	3.0
Phenol, nonyl-, phosphite (3:1)
25	-	20	10.8	200	0.065	±0.302	4.5	5.5
50	-	20	8.9	200	0.040	±0.221	6.0	3.5
150	-	20	9.6	200	0.065	±0.267	5.5	6.0
Positive control (MMC)	-	20	9.2	100	0.320	±0.649	5.0	25.0**
0.1

Treatment: Cells from all treatment conditions were harvested at 20 hours after the initiation of the treatments. Aberrations per Cell: Severely damaged cells were counted as 10 aberrations.

Percent Aberrant Cells: *, p</=0.05; **, p</=0.01; using the Fisher's exact test.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):other: Used for data submissionThe positive and solvent controls fulfilled the requirements for a valid test. Under the conditions of the assay described in this report, TNPP was concluded to be negative for the induction of structural and numerical chromosome aberrations in Chinese hamster ovary cells.

Executive summary:

Used for data submission