(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

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:

From July 01, 2004 to Nov. 17, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Remarks:

(according to US FDA and OECD principles of GLP)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

EC Number:

259-134-5

EC Name:

(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

Cas Number:

54381-16-7

Molecular formula:

C10H16N2O2.H2O4S

IUPAC Name:

(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

Constituent 2

Reference substance name:

N,N-Bis(2-hydroxyethyl)-PPD SULF

IUPAC Name:

N,N-Bis(2-hydroxyethyl)-PPD SULF

Details on test material:

- Name of test material N,N-Bis(2-hydroxyethyl)-PPD SULF; (p-ammoniophenyl)bis(2-hydroxyethyl) ammonium sulphate
- TSIN: GTS03849
- Substance type: Pure active substance
- Physical state: Light grey/white powder
- Stability: Results of stability analyses indicated that the test substance formulations (in water) were stable for 30 d at -10 to -30°C. All values were within 7.3% of initial concentration.
- Storage: At room temperature
- Solubility: Soluble in water

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5a culture medium supplemented with approx. 10% heat-inactivated fetal bovine serum, L-glutamine (2 mM), penicillin G (100 units/mL) and streptomycin (100 μg/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes, stock cultures were maintained for up to 8 wks after thawing a sample from a frozen stock.Mycoplasma testing was performed on stock cultures twice during this period to verify there was no mycoplasma contamination.
- Periodically checked for karyotype stability: Yes, to ensure karyotype stability, cell stocks were thawed periodically, recultured twice a week, and then eliminated for use in assays after about 8 weeks of continuous culture. Chromosome modal numbers were continuously checked during the period cells were being used.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9 fraction

Test concentrations with justification for top dose:

Initial toxicity test: 0.167, 0.5, 1.67, 5, 16.7, 50, 167, 500, 1670, 5000 μg/mL
Confirmatory assay without metabolic activation: 1.88, 3.75, 7.5, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 μg/mL
Confirmatory assay with metabolic activation (final trial B3): 50, 100, 200, 250, 300, 350, 375, 400, 425, 450, 475, 500, 550, 600, 700 μg/mL. The confirmatory assay with metabolic activation was conducted three times due to a lack of a high dose with relevant toxicity (Trial B1 and B2), and the final trial (Trial B3) was conducted with above concentrations.

Vehicle / solvent:

- Vehicle used: Cell culture grade water (CCGW)
- Justification for choice of solvent/vehicle: The test substance formed a transparent, light-orange solution in 5% Fischer's Media at 5 mg/mL. The test substance in DMSO formed a heterogeneous, translucent, light-purple suspension at approx. 471 mg/mL; and a transparent, light-purple solution at 256 mg/mL. The formulation in DMSO at 256 mg/mL was dosed into culture medium without cells using a dosing volume of 1% v/v (10 μL/mL). At a dosed concentration of 2560 μg/mL, the culture medium changed to a slightly reddish-purple color with no precipitation occurring. The test substance in water formed a transparent, colorless solution that turned a light-purple color after sitting for approx. 5 min. at 104 mg/mL. The formulations in water at 104 mg/mL were dosed into culture medium without cells using a dosing volume of 10% v/v (100 μL/mL). At a dosed concentration of 10400 μg/mL, the culture medium changed to a reddish-purple color with no precipitation occurring. Based on these results, cell culture grade water was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Cells and culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Cell culture grade water

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

mitomycin C

Remarks:

Stored in freezer set to maintain -10 to -30˚C. Dissolved in sterile deionized water and used at 0.75 μg/ mL for the 4 h treatment and 0.2 μg/ mL for the 20 h treatment

Controls 2

Untreated negative controls:

yes

Remarks:

Cells and culture medium with S9 mixture

Negative solvent / vehicle controls:

yes

Remarks:

Cell culture grade water

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

Stored in freezer set to maintain -10 to -30˚C. Dissolved in sterile deionized water and used at 7.5 μg/mL

Details on test system and experimental conditions:

TEST SYSTEM: Cells were incubated for 24 h at 37°C prior to treatment initiation. Cultures were initiated by seeding approx. 0.3 x 10(6) cells per 25 cm2 flask into a sufficient volume of culture medium so that the final volume was 5 mL (with or without metabolic activation).

METHOD OF APPLICATION: Single monolayer culture of CHO cells (in medium).

DURATION
- Exposure duration: The duration was as follows:
Without metabolic activation: 4 or 20 h
With metabolic activation: 4 h
- Exposure procedure: The cultures were incubated at 37 ± 1˚C for 4 or 20 h (as appropriate) in the presence of the test substance at predetermined concentrations/vehicle/positive controls with or without the S9 reaction mixture. If there was no visible increase in floating cells at the end of the 4 h treatment period, the cultures were washed with phosphate buffered saline, refed with 5 mL of complete McCoy's 5a medium and incubated for the rest of the culture period up to the time of harvest (20 h after initiation of treatment). If there was a visible increase in floating cells at the end of the 4 h treatment period, the medium was collected and centrifuged, the supernatant removed, and the pelleted cells were resuspended in 5 mL of complete McCoy's 5a medium and added back to the flask, after rinsing, the 5 mL of complete McCoy's 5a medium was added and the cultures incubated for the rest of the culture period up to the time of harvest (20 h after initiation of treatment).
- Expression time: Approx. 20 h after initiation of treatment - Fixation time (start of exposure up to fixation or harvest of cells): Harvest of cells 20 h after treatment initiation.

SPINDLE INHIBITOR: Colcemid (0.1 μg/mL) was added approx. 2 h prior to harvest time

STAIN: If there was no visible increase in floating cells, the media from each flask was discarded and the cell monolayer was rinsed and trypsinized. If there was a visible increase in floating cells, the medium from the flask was poured into a centrifuge tube, centrifuged, and added back to the trypsinized cells from the flask (unless there was a precipitate, in which case these were discarded). Slides were stained with 5% Giemsa solution for the analysis of mitotic index and chromosomal aberrations. All slides were then air-dried and mounted permanently.

NUMBER OF REPLICATIONS: At least 2 slides/ flask

NUMBER OF CELLS EVALUATED: 100 metaphase cells (if possible), from each replicate culture from at least three concentrations of the test substance, negative, vehicle and one dose of the positive control cultures were analyzed for the different types of chromosomal aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index, evaluated from the positive and vehicle controls and treatment groups by analyzing the number of mitotic cells in at least 500 cells and the ratio expressed as a percentage of mitotic cells.

OTHER EXAMINATIONS:
- Determination of polyploidy and endoreduplication: Percent polyploidy and endoreduplication were also analyzed by evaluating at least 100 metaphases, if possible, and tabulated.

Evaluation criteria:

- Any significantly increased response was indicated with the corresponding p value, where a p value ≤ 0.05 was considered positive.
- The average number of aberrations per cell was reported but no statistical analysis was applied.
- A notation was made by the aberrations per cell number for treatment groups in which there were unanalyzable cells to indicate that this number is a minimum.
- All conclusions were based on a sound scientific basis taking into account biological relevance; however as a guide to interpretation of the data, the test article was considered to induce a positive response if there was a statistically significant, dose-related increase in the number of aberrations compared to control and one or more concentrations were significantly increased.

Statistics:

- One-tailed Fischer's Exact test was used to analyze percent aberrant cells per treatment group compared to the control (vehicle). The p values were adjusted to take into account multiple dose comparisons using a Bonferroni adjustment.
- If there was a significant increase in the percentage of aberrant cells at one or more doses, then a trend test for the percent aberrant cells was performed to test for evidence of a dose response, using the Cochran-Armitage test.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: The pH of the McCoy’s 5a culture medium was 7.5, and the pH of the preparation containing 2560 μg/mL of test substance in McCoy’s (10% serum) was 6 and pH of the preparation containing 10400 μg/mL of test substance in McCoy’s (10% serum) was 6. The top doses tested in the chromosomal aberration assay were within the specified pH target measurements; the pH of the dosing formulations was adjusted as needed to achieve a pH of 7.0. The details on result of pH determinations are provided in the study report.
- Effects of osmolality: Osmolality of reference solution (290), 5% Fischer Media, Vehicle control (10% water in 5% Fischer Media) and test substance in 5% Fischer’s Media was 291, 310, 279 and 355 mOsm/kg water, respectively. The details on result of osmolality are provided in the study report.
- Precipitation: No precipitation was observed in the Confirmatory Assay.

RANGE-FINDING/SCREENING STUDIES:
- In the assay without metabolic activation with a 4 h treatment, precipitate was visible prior to wash at 1670 and 500 μg/mL. In the assay without metabolic activation with a 20 h treatment, precipitate was visible prior to harvest at 5000 μg/mL.
- Due to a cell count indicating no growth, the cell growth was not calculated for the culture treated at 167 and 500 μg/mL in the assay without metabolic activation (4 h treatment).
- In the assay without metabolic activation (4 h treatment), reductions of 47%, 52%, 39%, 1%, 11% and 93% were observed in the cell growth of the cultures treated with 0.167, 0.5, 1.67, 5, 16.7 and 50 μg/mL, respectively, as compared with the vehicle control cultures.
- In the assay without metabolic activation (20 h treatment), due to a cell count indicating no growth, the cell growth was not calculated for the cultures treated at 50, 167 and 500 μg/mL. Reductions of 22%, 0%, 0%, 3%, and 27% were observed in the cell growth of the cultures treated with 0.167, 0.5, 1.67, 5 and 16.7 μg/mL, respectively, as compared with the vehicle control cultures.
- The assay with metabolic activation was repeated twice in order to get a high dose with relevant toxicity. In the second trial with metabolic activation, precipitate was visible prior to wash at ≥1670 μg/mL. Due to a cell count indicating no growth, the cell growth was not calculated for the culture treated at 500 μg/mL. Reductions of 9%, 2%, 6%, 14%, 27%, 3% and 29% were observed in the cell growth of the cultures treated with 0.167, 0.5, 1.67, 5, 16.7, 50 and 167 μg/mL, respectively, as compared with the vehicle control cultures.

COMPARISON WITH HISTORICAL CONTROL DATA: Vehicle and positive control data in this study were comparable with the historical control data (From July 2003 until Dec. 2003).

Further details on results are provided in ‘Table 1’ and ‘Table 2’ in ‘Any other information on results incl. tables’ section.

Remarks on result:

other: at 200 and 250 μg/mL test concentrations

Any other information on results incl. tables

Table 1: Results of chromosomal aberration of CHO cells after treatment with N,N-Bis(2-hydroxyethyl)-PPD SULF (4 h treatment, approx. 20 h harvest) (Study # 53726)

Treatment	Concentration	Number of cells scored for aberrations	% Cell growth inhibition	Aberrations per cells	Totals*			Statistical significance (+/-)**
					g^-		g^+
	Without metabolic activation
Cell culture grade water (Vehicle)	100 µL/mL	200	0	0.01	1	2.5
MitomycinC (Positive control)	0.750 μg/mL	100	-	1.29	45	49		+
N,N-Bis(2-hydroxyethyl)-PPD SULF	7.50 μg/mL	200	26	0.005	0.5	1		-
	15.0 μg/mL	200	47	0.065	5.5	7.5		-
	35.0 μg/mL	200	57	0.075	5.5	13		-
	With metabolic activation
Cell culture grade water (Vehicle)	100 µL/mL	200	0	0	0	1
Cyclophosphamide (Positive control)	7.50 μg/mL	100	-	2.48	76	76		+
N,N-Bis(2-hydroxyethyl)-PPD SULF	50.0 μg/mL	200	11	0.015	1.5	2.5		-
	100 μg/mL	200	7	0.01	1	2		-
	200 μg/mL	200	3	0.11	11	13.5		+
	250 μg/mL	100	35	2.53	79	79		+

 

Table 2: Results of chromosomal aberration of CHO cells after treatment with N,N-Bis(2-hydroxyethyl)-PPD SULF (20 h treatment, approx. 20 h harvest) (Study # 53726)

Treatment	Concentration	Number of cells scored for aberrations	% Cell growth inhibition	Aberrations per cells	Totals*		Statistical significance (+/-)**
					g^-	g^+
	Without metabolic activation
Cell culture grade water (Vehicle)	100 µL/mL	200	0	0.015	1.5	2.5
MitomycinC (Positive control)	0.200 μg/mL	100	-	1.34	53	55	+
N,N-Bis(2-hydroxyethyl)-PPD SULF	3.75 μg/mL	200	3	0.01	1	4	-
	7.50 μg/mL	200	49	0.05	4	7	-
	20.0 μg/mL	200	59	0.04	3.5	6	-

* = g- = # or % of cells with chromosome aberrations; g+ = # or % of cells with chromosome aberrations + # or % of cells with gaps

** = Significantly greater in -g than the vehicle control, p ≤ 0.05. The p values were adjusted to take into account multiple dose comparisons using a Bonferroni adjustment.

RESULTS OF CONFIRMATORY ASSAY

A) Without metabolic activation

- In the assay without metabolic activation with a 4 h and 20 h treatment, no precipitate was observed.

- In the assay without metabolic activation with a 4 treatment, reductions of 14%, 26%, 47%, 42%, 59%, 34%, 57%, 58%, 90%, 65%, 96%, and 77% were observed in the cell growth of the cultures treated with 3.75, 7.5, 15, 20, 25, 30, 35, 40, 45., 50, 55 and 60 μg/mL, respectively, as compared with the vehicle control cultures.

- Chromosomal aberrations were analyzed from the cultures treated with 7.5, 15 and 35 μg/mL. No statistically significant increase in cells with structural chromosomal aberrations, polyploidy or endoreduplication was observed in the cultures analyzed.

- In the assay without metabolic activation (approx.20 h treatment),  the cell growth was not calculated for the cultures treated at ≥ 45 μg/mL due to cell counts indicating no growth. Reductions of 3%, 49%, 43%, 59%, 61%, 37%, 65%, and 81% were observed in the cell growth of the cultures treated with 3.75, 7.5, 15, 20, 25, 30, 35 and 40 μg/mL, respectively, as compared with the vehicle control cultures. Chromosomal aberrations were analyzed from the cultures treated with 3.75, 7.5, and 20 μg/mL. No statistically significant increase in cells with structural chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.

B) With metabolic activation

- In the assay with metabolic activation, no precipitate was observed.

- Reductions of 11%, 7%, 3%, 35%, 38%, 56%, 60%, 52%, 59%, 71%, 73%, 76%, 73%, 73%, and 77% were observed in the cell growth of the cultures treated with 50, 100, 200, 250, 300, 350, 375, 400, 425, 450, 475, 500, 550, 600, and 700 μg/mL, respectively, as compared with the vehicle control cultures. Chromosomal aberrations were analyzed from the cultures treated with 50, 100, 200, and 250 μg/mL. Statistically significant increases in cells with structural chromosomal aberrations were observed in the cultures analyzed at 200 and 250 μg/mL with metabolic activation; statistically significant increases in cells with endoreduplication were observed in all the cultures analyzed with metabolic activation. The increase in total cells with numerical aberrations was within historical control values so this is not considered biologically relevant.

RESULT OF POSITIVE CONTOL

- The sensitivity of the cell cultures for induction of chromosomal aberrations was shown by the increased frequency of aberrations in the cells exposed to mitomycin C.

- The successful activation by the metabolic system was illustrated by the increased incidence of cells with chromosomal aberrations in the cultures induced with cyclophosphamide.

Applicant's summary and conclusion

Conclusions:

N,N-Bis(2-hydroxyethyl)-PPD SULF was considered negative for inducing structural chromosomal aberrations in Chinese hamster ovary (CHO) cells without S9 metabolic activation for the 4 h and approx. 20 h treatment periods, but positive with S9 metabolic activation for the 4 h treatment period at 200 and 250 μg/mL test concentrations.

Executive summary:

The in vitro Mammalian Chromosome aberration test of N,N-Bis(2-hydroxyethyl)-PPD SULF was determined by following the OECD guideline 473 (In vitro Mammalian Chromosome Aberration Test).

The objective of this in vitro assay was to evaluate the potential of the test substance and its metabolites to induce structural and numerical chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells with and without an exogenous metabolic activation system. Aroclor-induced rat liver S9 fraction was used as the metabolic activation system.

An initial toxicity assay was performed to select the dose levels for the confirmatory assay.Assay was performed with and without metabolic activation at test concentrations of 0.167, 0.500, 1.67, 5.00, 16.7, 50.0, 167, 500, 1670, and 5000 µg/mL. Based upon this initial assay, the test concentrations selected for confirmatory assay were as follows:

With metabolic activation:50.0, 100, 200, 250, 300, 350, 375, 400, 425, 450, 475, 500, 550, 600, and 700 µg/mL for 4 h treatment period

Without metabolic activation: 1.88, 3.75, 7.50, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, and 60.0 µg/mL for both 4 h and approx. 20 h treatment

Cells were incubated for 24 h prior to treatment. Pre-incubated cells were exposed to test /positive/vehicle control treatment for 4 or 20 h, as required. After 18 h of treatment initiation, cells were treated with Colcemid (0.1 μg/mL). Cells were harvested at 20 h after treatment initiation. Cells were analyzed for mitotic index, cell count, cell growth and cell growth inhibition parameters. Slides were prepared and evaluated for chromosomal aberrations.

No test treatment related chromosomal aberrations were observed, in the absence of metabolic activation.

In the presence of metabolic activation, statistically significant increases in cells with structural chromosomal aberrations were observed in the cultures analyzed at 200 and 250 μg/mL. Statistically significant increases in cells with endoreduplication were observed in all the cultures with metabolic activation. The increase in total cells with numerical aberrations and endoreduplication or polyploidy was within historical control values so this is not considered biologically relevant.

Mitomycin C (without metabolic activation) and cyclophosphamide (with metabolic activation) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Based on above, N,N-Bis(2-hydroxyethyl)-PPD SULF was considered negative for inducing structural chromosomal aberrations in Chinese hamster ovary (CHO) cells without S9 metabolic activation for the 4 h and approx. 20 h treatment periods, but positive with S9 metabolic activation for the 4 h treatment period at 200 and 250 µg/mL test concentrations.

This in vitro mammalian chromosome aberration test is classified as acceptable and satisfies the guideline requirements of the OECD 473 method.