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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate has been evaluated in an extensive battery of in vitro genotoxicity tests.

In Salmonella typhimurium TA100, an initial positive finding was not reproduced in another trial. N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate did not either increase mutations at the tk locus in L5178Y mouse lymphoma cells. It induced chromosome aberrations and endoreduplications in Chinese hamster ovary (CHO) cells in the presence of metabolic activation.

In vivo, N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate did not induce micronuclei in polychromatic erythrocytes of mice or DNA damage (measured by unscheduled DNA synthesis) in hepatocytes of rats. Thus, although in vitro assays in E. coli and CHO cells suggest that N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate has genotoxic potential, two in vivo assays indicated no genotoxic activity.


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro transformation study in mammalian cells
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From Aug. 05, 1996 to April 28, 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Qualifier:
equivalent or similar to
Guideline:
EU Method B.21 (In Vitro Mammalian Cell Transformation Test)
Deviations:
no
GLP compliance:
yes
Remarks:
according to US FDA and OECD principles of GLP
Type of assay:
in vitro mammalian cell transformation assay
Species / strain / cell type:
mammalian cell line, other: Syrian hamster embryo cells
Details on mammalian cell type (if applicable):
Target and irradiated (approx. 5000 rads) feeder cryopreserved cell pools were used in this study.
- Source: Genetic and Cellular Toxicology. Both the target and feeder cell pools from embryo cells were derived from timed-pregnant Syrian Golden hamsters at 13 to 13.5 d gestation.
- Type and identity of media: Leboeuf's modification (0.75 g/L NaHCO3, pH 6.7) of Dulbecco's Modified Eagle's Media (DMEM) supplemented with 20% fetal bovine serum and 4 mM L-glutamine
- Properly maintained: Stocks of cells were maintained in liquid nitrogen storage and were used up to 1 year post-isolation.
- Periodically checked for sterility and transformation ability: Yes, each isolate of freshly prepared SHE cells were checked for sterility and evaluated for their ability to undergo morphological changes when treated with known pro-carcinogen, Benzo(a)pyrene (B[a]P). Only those isolates that are free of contamination and respond satisfactorily to Benzo(a)pyrene were used in the assay.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not applicable
Metabolic activation system:
SHE target cells are capable of maintaining a whole range of metabolic activities
Test concentrations with justification for top dose:
3 definitive transformation assays (2 independent experiments in initial transformation assay and 1 experiment in repeat transformation assay) were performed with following test concentrations:
2.0, 4.0, 5.0, 5.313, 5.625 and 6.0 µg/mL for Trial 1 and 2 (Initial assay) of transformation assay.
Three additional dose groups (6.0, 5.625 and 5.313 µg/mL) with target cell seeding density adjusted to control levels based on an estimation of their expected cytotoxicity were run along with the above 6 concentrations.
6.625, 7.0, 7.313, 7.625 and 8.0 µg/mL for Trial 3 (Repeat assay) of transformation assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO) (0.2%)
- Justification for choice of solvent/vehicle: The test material was tested for solubility in complete medium, DMSO and ethanol. Based on solubility test, DMSO was selected as the most appropriate solvent for the test material.
Untreated negative controls:
yes
Remarks:
LeBoeuf's modification of Dulbecco's Modified Eagle's Medium containing 20% fetal bovine serum and 4 mM L-glutamine
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO) (0.2%)
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
at 1.25 and 2.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: In complete medium

EXPERIMENTAL PROCEDURE AND DURATION: The initial transformation assay consisted of 2 independent experiments. A repeat transformation assay was performed with new test material sample (RE0982.03) where only a single trial was set up using approx. twice the number of dishes. This change was made to reduce potential variability in dose response between trials.

- Seeding: Approx. 60-100 target SHE cells (in 2 mL complete medium) were added to each 60 mm petri dish each containing about 4 x 10(4) X-irradiated (approx. 5,000 rads) feeder SHE cells in 2 mL complete medium seeded 24 h earlier. The cultures were then incubated at 37±1°C in 10±0.5% CO2 in humidified air for approx. 24 h.

- Exposure duration: 4 mL of test solution was delivered to each of the dishes containing 4 mL of complete medium, feeder cells and target cells. The cultures were incubated in the presence of test material for approx. 24 h after which the dishes were refed with 8 mL of complete medium and then incubated for a period of 7-8 days.

- Fixation: After the incubation period, the dishes (20 in the initial transformation assay and 40 in the repeat study) were washed once with HBSS, fixed with methanol, stained with about 10% buffered aqueous Giemsa, rinsed with tap water and allowed to air dry. The remaining 5 dishes in each treatment group were washed with calcium and magnesium free HBSS (HBSS-CMF), treated with 0.05% trypsin-0.02% EDTA to detach the cells and then pooled in complete medium.

STAIN USED: 10% buffered aqueous Giemsa

NUMBER OF REPLICATIONS: 25 dishes/treatment group/experiment in the initial transformation assay and 45 dishes/treatment group in the repeat study.

DETERMINATION OF pH AND OSMOLALITY
Prior to performing the definitive transformation assay, an aliquot of the test material was weighed, dissolved in the appropriate solvent and diluted in complete medium to approximate the highest concentration. A portion of this was transferred to a vial and placed in the CO2 incubator for approx. 24 h. The remaining portion of the solution was measured for pH using a portable pH meter and the osmolality was determined using a freezing-point osmometer.
The pH of the incubated sample was determined the following day.

DETERMINATION OF CYTOTOXICITY
- Method: i) Reduction in relative plating efficiency of the treated SHE cells compared with the control ii) Reduction in relative colony density
The average cell density per dish was determined by counting the cells with a hemacytometer or by measuring the absorbance of the resultant cell suspension at A800 with a spectrophotometer and calculating the concentration based on a standard curve. Colony density was calculated by dividing the number of cells/dish by the average number of colonies/dish.

DETERMINATION OF TRANSFORMED CELLS
The stained dishes were screened and individual colonies were evaluated for transformed morphology using a stereomicroscope. The total colony number and the number of colonies with transformed morphology for each test group were recorded and statistically analyzed for treatment related effects using pooled data from the combined experiments.
Criteria for evaluating morphological transformation were:
i) Piled up cells (both at the center and perimeter of the colony).
ii) Extensive random-oriented three dimensional growth.
iii) Crisscrossing cells with increased cytoplasmic basophilia at the perimeter of the colony.
iv) Cells with decreased rates of cytoplasm/nucleus ratios relative to normal cells.
Evaluation criteria:
- A test material was considered to be positive if it causes a statistically significant increase in morphological transformation frequency in at least two doses compared to concurrent controls.
- A test material was considered positive if one dose shows a statistically significant treatment-related increase and there is an indication of a statistically significant (p≤0.05) positive dose trend.
- A test material was considered negative if there is no dose with a statistically significant treatment-related increase and the uppermost dose of test material demonstrates a sufficient level of toxicity (as measured by either a 50% reduction in plating efficiency or the colony density precludes obtaining a higher dosage of test material) unless the maximum soluble dose is run and no toxicity occurs.
- In addition feeder SHE cells must be evident on the stained dishes in order to call a test material negative.
Statistics:
- A one-sided Fisher's Exact Test was employed for judging whether a test material causes a significant treatment related effect.
- In addition an unstratified binomial exact permutation trend test for significant positive dose response trend was conducted if necessary. The trend test was considered positive if the resultant p value is < 0.05.
Species / strain:
mammalian cell line, other: Syrian hamster embryo cells
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
For details refer to 'Additional information on results' section
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 after 24 h incubation was determined to be 6.70 (for a slightly higher concentration than reported for initial experiments i.e. at 7.5 µg/mL).
- Effects of osmolality: The test material solution at 7.5 µg/mL had a measured osmolality of 318 mOSM/kg versus a solvent reference of 316 mOSM/kg.
Considering the fact that the actual top test material concentration was lower than that measured, both pH and osmolality values were considered acceptable.
- Other confounding effects: When the 2X dosing stock test solution was being prepared for use in the initial range finding assay it was noted that the medium turned a ‘pinkish-purple’ color (indicating a slight rise in the pH of the solution). This color gradually shifted toward the color seen in the control dose level such that at the lowest dose there was no difference in color between that and the color of the control. Subsequent studies at lower test concentrations showed no such color change indicating that there was no pH change at these levels.

RANGE-FINDING/SCREENING STUDIES (PRELIMINARY CYTOTOXICITY STUDY):
- Four preliminary range finding assays were conducted to establish the dose range for the original definitive transformation assay series and a fifth range finding assay was performed to verify the expected toxicity of a new test material sample.
- The first preliminary range finding assay doses ranged from 5.0 to 450 µg/mL. These doses resulted in 0 to 100% cytoxicity but produced no dose level clearly identifying 50% toxicity. A second preliminary toxicity assay using doses ranging from 10 to 100 µg /mL was set up but resulted in 100% toxicity throughout the range of doses tested.
- A third range finding assay was then set up using doses ranging from 5.0 to 14 µg/mL. Results from this assay resulted in toxicities which ranged from 11 to 100%, but again produced no dose level clearly identifying 50% toxicity. Therefore a fourth assay was set up using doses ranging from 3.75 to 7.5 µg/mL. The results from this last range finding assay produced sufficient data to identify a dose expected to yield an approximate 50% reduction in relative plating efficiency.
- Based on the fourth range finding assay 6 doses were selected for the initial definitive transformation assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- 3 additional dose groups (6.0, 5.625 and 5.313 µg/mL) were selected to be run in the definitive assay with their target cell seeding densities adjusted to control levels. This was based on an estimation of their expected cytotoxicity as calculated through interpolation of the cytotoxicity assay response of the 6.25 and 5.0 µg/mL dose groups. As a result of a shift in toxicity between that measured in the preliminary and the definitive assays a 50% reduction in plating efficiency was not obtained.
- Because of the steepness of the cytotoxicity curve, it was decided to modify the usual testing approach for the repeat study and to utilize 45 dishes per dose in a single experiment rather than using 25 dishes per dose in 2 experiments.

RESULTS OF TRANSFORMATION ASSAY:
- None of the dose levels tested (both in the original and repeat assays) produced a statistically significant increase in morphological transformation frequency (MTF) relative to concurrent controls when analyzed using the Fisher's Exact test.
- In the second trial of the initial study, treatment with a test concentration of 5.0 µg/mL produced fewer than 500 colonies in that treatment group. This resulted in a total of fewer than 1000 colonies per treatment group (977 colonies). This response did not significantly impact on the transformation frequency obtained for that dose level when the data was pooled and thus had no significant overall impact on the study.
The details on results and individual result tables are provided in the study report.

RESULTS OF CONTROL GROUPS:
All of the controls demonstrated an appropriate response and as a result the assay was considered valid.
Conclusions:
N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate was considered to be negative for its potential to induce morphological transformation following a 24 h exposure in the Syrian hamster embryo cell transformation assay.
Executive summary:

The in-vitro Syrian hamster embryo cell transformation assay of N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate was determined following methods comparable to EU Method B.21 (In Vitro Mammalian Cell Transformation Test). The present study was performed to determine the potential of the test material to induce a statistically significant increase in the frequency of morphologically transformed colonies as compared to controls following a 24 h exposure.

The assay was performed in two independent trials in the initial transformation assay with test sample RE0982.02. A repeat transformation assay with single trial (using twice the number of dishes as in initial assay) was run with the new sample of test material RE0982.03.

Syrian hamster embryo cells were obtained from Genetic and Cellular Toxicology. Approx. 60-100 target SHE cells were seeded in LeBoeuf's modification of Dulbecco's Modified Eagle's Medium containing 20% fetal bovine serum and 4 mM L-glutamine and further treated with following concentrations of test material for an exposure period of 24 h:

Initial transformation assay (Trial 1 and 2): 2.0, 4.0, 5.0, 5.313, 5.625 and 6.0 µg/mL

Three additional dose groups (6.0, 5.625 and 5.313 µg/mL) with target cell seeding density adjusted to control levels based on an estimation of their expected cytotoxicity were run along with the above 6 concentrations.

Repeat transformation assay (Trial 3): 6.625, 7.0, 7.313, 7.625 and 8.0 µg/mL

25 dishes/treatment group/trial were used in the initial transformation assay and 45 dishes/treatment group were used in the repeat study. Untreated cells in complete medium and cells treated with DMSO (vehicle) were employed as negative and solvent controls respectively. Benzo(a)pyrene (at 1.25 and 2.5 µg/mL) served as a positive control.

The total colony number and the number of colonies with transformed morphology for each test group from the successful trials (pooled data from initial and repeat experiments) were recorded and plating efficiencies and transformation frequencies were calculated. The combined data was analyzed for statistically significant treatment-related effects. Reduction in relative plating efficiency of the treated SHE cells compared with the control and reduction in relative colony density were used as indicators to determine cytotoxicity.

None of the dose levels tested (both in the original and repeat assays) produced a statistically significant increase in morphological transformation frequency (MTF) relative to concurrent controls when analyzed using the Fisher's Exact test. Toxicity ranged from 0 to 55%.

All of the controls demonstrated an appropriate response and as a result the assay was considered valid.

Based on above, N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate was considered to be negative for its potential to induce morphological transformation following a 24 h exposure in the Syrian hamster embryo cell transformation assay.

This in-vitro Syrian hamster embryo cell transformation assay is classified as acceptable, and satisfies the guideline requirements of the EU Method B.21.

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
Qualifier:
according to
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
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.
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
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.
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
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

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.

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Remarks:
(according to US FDA and OECD principles of GLP)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase +/- gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The culture media used was RPMI 1640 supplemented with horse serum (10% by volume), Pluronic F68, L-glutamine, sodium pyruvate, penicillin and streptomycin. The treatment media used was Fischer’s medium with same components for culture media except horse serum was reduced to 3%. Cloning medium consisted of the RPMI 1640 culture medium with 10% horse serum, without Pluronic® F68 and with the addition of 0.23% Noble agar to achieve a semisolid state. Selection medium was cloning medium that contained 3 µg/mL of TFT.
- Periodically checked for Mycoplasma contamination: yes, both direct culturing methods and the indirect Hoechst staining method were used. No mycoplasma was observed during the life of the culture.
- Periodically checked for karyotype stability: yes, measured by mean chromosomal number was routinely performed on stock cultures used for assays. Karyotype analysis, including banding, was performed on cells after preparation of a fresh frozen stock culture.
- Periodically "cleansed" against high spontaneous background: Yes, to reduce the frequency of spontaneous TK +/- mutants prior to use in the mutation assay, cell cultures were exposed to conditions that selected against the TK +/- phenotype and then returned to normal culture medium for 3 to 8 d.
- Storage: Stock cultures were obtained from Dr. Donald Clive, Glaxo-Wellcome Inc., Research triangle Park, N.C., (1977) and were stored in liquid nitrogen.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver microsomal enzymes (S9) with Nicotinamide Adenine Dinucleotide Phosphate (NADP, sodium salt) and Isocitrate
Test concentrations with justification for top dose:
INITIAL (PRELIMINARY) TOXICITY ASSAY (TESTS):
- Trial A1: With and without metabolic activation at 10 different concentrations (9.85, 19.7, 39.3, 78.5, 157, 313, 625, 1250, 2500 and 5000 µg/mL) for 4 h duration
- Trial A2: Without metabolic activation at 10 different concentrations (0.197, 0.393, 0.785, 1.57, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL) for 24 h duration

INITIAL AND CONFIRMATROY MUTAGENICITY ASSAYS:
Based upon the initial toxicity tests, initial and confirmatory mutagenicity tests were performed as follows:

a) Without metabolic activation: 4 independent mutagenicity experiments, without metabolic activation, were performed as follows:
- Trial B1 (initial mutation assay): 5 test concentrations were evaluated, at concentrations of 1, 2.5, 3.75, 5 and 7.5 µg/mL, for 4 h treatment duration
- Trial C3 (initial mutation assay): 8 test concentrations were evaluated, at concentrations of 3, 4, 7, 8, 8.5, 9, 9.5 and 10 µg/mL, for 4 h treatment duration.
- Trial C4 (initial mutation assay): 8 test concentrations were evaluated, at concentrations of 4, 6, 6.5, 7, 7.5, 8, 8.5 and 9 µg/mL, for 4 h treatment duration.
- Trial C2 (confirmatory mutation assay): 8 test concentrations were evaluated, at concentrations of 2.25, 2.5, 2.75, 3, 3.25, 3.5, 4 and 4.5 µg/mL, for 24 h treatment duration.
b) With metabolic activation system: Mutation assay was performed with metabolic activation system and no confirmatory assay was performed further.
- Trial B1: 8 test concentrations were evaluated, at concentrations of 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL, for 4 h treatment duration.
Vehicle / solvent:
- Vehicle used: Cell culture grade water (CCGW)
- Justification for choice of vehicle: In solubility testing, the test substance was observed to form a transparent, colorless solution in cell culture grade water at 104 mg/mL. In dimethylsulfoxide (DMSO), the test substance was observed to form a heterogeneous, translucent, light-purple suspension at 471 mg/mL and a transparent, light-purple solution at 256 mg/mL. Based on these results, cell culture grade water was selected as the vehicle.
Positive controls:
yes
Remarks:
(Without metabolic activation)
Positive control substance:
methylmethanesulfonate
Remarks:
13 μg/mL for 4 h duration and 6.5 μg/mL for 24 h duration
Positive controls:
yes
Remarks:
(with metabolic activation)
Positive control substance:
3-methylcholanthrene
Remarks:
2 and 4 µg/mL for 4 h duration
Negative solvent / vehicle controls:
yes
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

EXPERIMENTAL PROCEDURE AND DURATION:
- Cell preparation and exposure: Mouse lymphoma L5178Y cell line (TK+/-) designated as clone 3.7.2C, was used for this assay. Logarithmically growing laboratory stock cultures were seeded into a series of tubes at 6 x 10(6) cells/tube. Cells were pelleted by centrifugation, the culture medium removed, and the cells resuspended to a final volume of 10 mL (4h treatment duration) or 20 mL (24h treatment duration) of treatment medium with a final serum concentration of approx. 3%. Tubes were placed in an orbital shaker incubator at 35-38°C and rotated at 70 ± 10 orbits/min. After an exposure period of approx. 4 or 24 h, cells were centrifuged (approx. 138 g) and the treatment medium removed. Cells were then washed with serum-free culture medium, followed by a wash in medium containing 10% serum, then resuspended in 20 mL of culture medium at a concentration of approx. 3 x 10(5) cells/mL, and returned to the orbital shaker incubator. The assay procedure for metabolic activation assay was identical to the non metabolic activation assay except for the addition of the S9 fraction of rat liver homogenate and necessary cofactors (CORE) during the treatment period of approx 4 h.
- Expression period: A standard expression period of 2 d was used to allow for mutant recovery, growth and expression of the TK +/- phenotype. Cell densities were determined on Day 1 and each culture was adjusted to 3 x 10(5) cells/mL in 20 mL of culture medium. If the cells in a culture failed to multiply to a density of 4 x 10(5) cells/mL on the first day after treatment, the culture was not subcultured. On Day 2, cell counts were again determined, and appropriate cultures were selected for cloning and mutant selection. Cultures with cell densities less than approx. 3 x 10(5) cells/mL on Day 2 were not considered for selection.
- Selection: A total of 3 x 10(6) cells from each selected tube were suspended in selection medium in soft agar to recover TFT-resistant mutants. This sample was distributed into three 100 mm dishes. The absolute selection cloning efficiency was determined by seeding 3 dishes with a total of approx. 600 cells in agar cloning medium without selective agent. All dishes were placed in an incubator at 35-38°C with 4-6% CO2:95% humidified air. After 13-14 d in the incubator for each trial, the colonies were counted with the Loats Associates, Inc. (LAI) High Resolution Colony Counter (HRCC) System for the Mouse Lymphoma Assay, version 2.3.1 build 1.

SELECTION AGENT: Trifluorothymidine (TFT) at a concentration of 3 μg/mL

NUMBER OF REPLICATIONS: Single

NUMBER OF CELLS EVALUATED: 3 x 10(6) cells analyzed

DETERMINATION OF CYTOTOXICITY
- Method: The measurement of the cytotoxicity of each concentration of test substance and control was the relative suspension growth of the cells over the 2 d expression period (for the 4 h treatment) or the relative suspension growth over the 3 d treatment and expression period (for the 24 h treatment) multiplied by the relative cloning efficiency at the time of selection.

DETERMINATION OF MUTATION FREQUENCY
- The mutant frequency was calculated as the ratio of the total number of mutant colonies found in each set of three mutant selection dishes to the total number of cells seeded, adjusted by the absolute selection cloning efficiency.
Evaluation criteria:
- A positive evaluation indicated that the test substance was a mutagen (induces gene/chromosomal mutations) and a negative evaluation indicated the test substance was nonmutagenic in this test system and causes no responses that can be interpreted as positive.

Evaluation of a Positive Response
- The test substance was evaluated as positive if there was a positive dose response and one or more of the doses exhibit a mutant frequency which was greater than or equal to 90 mutants per 10(6) clonable cells over the concurrent background mutant frequency. The background mutant frequency was defined as the average mutant frequency of the vehicle control cultures.

Evaluation of a Negative Response
- Test substances producing fewer than 90 induced mutants per 10(6) clonable cells at all dose levels were be concluded as negative.
- This presentation of the evaluation process may not encompass all test situations, and the Study Director may have used other criteria to arrive at an evaluation. The report provides the reasoning involved when departures from the above description occur.
Statistics:
Not reported (A statistical evaluation of the results is not regarded as necessary according to the OECD 476 guidelines).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the test substance in solution and in culture medium was evaluated using a Beckman pH Meter with a Futura refillable combination electrode. The pH of test substance at 49.9 mg/mL in CCGW was 1.63. The pH measures for the preparations containing approx. 4990 μg/mL in Fischer’s (5% serum) and Fischer’s (serum free) were 3.68 and 2.80, respectively. Values for media with 10 % CCGW ranged from 7.21 to 7.57. The pH values for the test substance in medium were not within specified pH target measurements. While the pH at 4990 μg/mL was measured as acidic, the highest dose cloned in the mutagenicity assays was 100 μg/mL. At this lower concentration, it was observed that appearance of the medium, which contains a pH indicator, did not indicate that the pH was acidic.
- Effects of osmolality: Osmolality of the test substance in the culture medium was evaluated using a Digimatic Osmometer. Sample and control materials were measured in duplicate, at room temperature, using 250 μL in disposable cuvettes. Osmolality of test substance at approx. 4990 μg/mL was evaluated in Fischer’s medium with 5 % horse serum, and serum-free Fischer’s medium. Osmolality of the reference solution of 290 mOsm/kg water was 293 mOsm/kg water, which was within the normal range. Osmolality of the media treated with a 10 % volume of the 49.9 mg/mL test substance solution in CCGW was 294 mOsm/kg water in Fischer’s (5% serum), and 287 mOsm/kg water in Fischer’s (serum free). These values were not significantly different compared to 10 % CCGW in the respective two media preparations.
- Precipitation: Precipitate was observed in the treatment medium in the toxicity test at 5000 μg/mL.

PRELIMINARY TOXICITY STUDIES
- Trial A1 (without S9, 4 h duration): Moderate toxicity at 9.85 μg/mL and excessive toxicity at 19.7 μg/mL and higher concentrations.
- Trial A1 (with S9, 4 h duration): Weak toxicity up to and including 39.3 μg/mL, moderately high toxicity at 78.5 μg/mL and excessive toxicity at 157 μg/mL and higher concentrations.
- Trial A2 (without S9, 24 h duration): No toxicity at 0.197 μg/mL, weak toxicity from 0.393 to 1.57 μg/mL, moderately high toxicity at 3.13 μg/mL and excessive toxicity at 6.25 μg/mL and higher concentrations.

MUTAGENICITY STUDY RESULTS
-The background mutant frequency for the vehicle control in the initial nonactivation assay was 87.9 x 10E-6, therefore a mutant frequency greater than or equal to 177.9 x 10-6 was required for a treatment to be evaluated as exhibiting a positive response
- Trial B1 (Initial mutation assay without S9, 4 h duration): 13 concentrations at 1, 2.5, 3.75, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30 and 35 μg/mL were initiated. Treatments at 10 μg/mL and greater were terminated due to excessive toxicity. The remaining 5 treatments were analyzed for mutant induction and no toxicity to moderately high toxicity (98.7% to 34.2% relative total growth) was induced. Mutant frequencies for the analyzed treatments ranged from 49.1 to 123.3 x 10(-6). None of the analyzed treatments induced a mutant frequency that met criteria for a positive response. However, since sufficient toxicity was not achieved, the assay was repeated.
- Trial B1 (Initial mutation assay with S9, 4 h duration): 8 concentrations ranging from 20 to 100 μg/mL were analyzed for mutant induction and weak to high toxicity was observed (75.8% to 9.7% relative total growth). None of the analyzed treatments induced an increase in the mutant frequency that met the criteria for a positive response. A confirmatory assay was not required.
- Trial C3 (Initial mutation assay without S9, 4 h duration): Eight treatments ranging from 3.00 to 10.00 µg/mL were analyzed for mutant induction and no toxicity to high toxicity was induced (94.4% to 17.6% relative total growth). None of the analyzed treatments induced a mutant frequency that met criteria for a positive response. However, the assay was repeated because the concentration verification results fell outside the acceptable range.
- Trial C4 (Initial mutation assay without S9, 4 h duration): Eight treatments ranging from 4.00 to 9.00 µg/mL were analyzed for mutant induction and weak to high toxicity was induced (68.6% to 11.5% relative total growth). Mutant frequencies for the analyzed treatments ranged from 42.2 to 72.2 x 10(-6). None of the analyzed treatments induced a mutant frequency that met criteria for a positive response.
- Trial C2 (Confirmatory nonactivation (without S9) mutation assay with 24 h treatment period): 8 treatments ranging from 2.25 to 4.50 μg/mL were analyzed for mutant induction and weak to high toxicity was induced (76.3% to 10.7% relative total growth). Mutant frequencies for the treatments analyzed ranged from 81.8 to 122.6 x 10(-6). None of the analyzed treatments induced a mutant frequency that met criteria for a positive response.

COMPARISON WITH HISTORICAL CONTROL DATA: Vehicle and positive control data in this study were comparable with the historical control data (From April 23, 2002 until May 06, 2003).

Further details on results are provided in the study report.

Table 1: Mutation assay with mouse lymphoma L5178Y TK+/-cells (Trial B1, with metabolic activation, 4 h treatment) (Study # 53728)

Test condition

Concentration

Cum. RSG (%)a

Cloning Efficiencyb

Relative Total Growthc(%)

Mutant Frequency (× 10-6)d

Day 1

Day 2

Absolute%

Relative%

Total

Small

Large

Cell Culture Grade Water

10%

100.6

99.3

125.6

98.6

97.9

53.3

34.7

18.5

10%

98.8

99.4

117.3

92.1

91.5

56.1

32.6

23.6

10%

100.6

101.3

139.3

109.3

110.7

57.2

32.1

25.1

Methylcholanthrene

2

60.6

48.5

86.7

68.1

33

374

187.4

186.6

4

50.3

46.2

73.8

57.9

26.8

478

259.6

218.7

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

20

99.7

88.7

108.9

85.5

75.8

60.1

41.4

18.7

40

83.9

81.1

117.3

92.1

74.7

56.4

31.3

25.1

50

52.2

63.7

114.4

89.8

57.2

69.6

46.7

22.9

60

64.3

64.1

111.5

87.5

56.1

88.1

52.9

35.2

70

57.8

55.3

105.8

83.1

46

67.4

34.7

32.6

80

29.8

33.9

112

87.9

29.8

88.6

58.8

29.9

90

13

19.4

63.5

49.8

9.7

93.4

56.7

36.7

100

12.1

13.9

91.1

71.5

10

90.6

57.5

33.1

 

Table 2: Mutation assay with mouse lymphoma L5178Y TK+/-cells (Trial C2, without metabolic activation, 24 h treatment) (Study # 53728)

Test condition

Concentration

Cum. RSG (%)a

Cloning Efficiencyb

Relative Total Growthc(%)

Mutant Frequency (× 10-6)d

Day 1

Day 2

Day 3

Absolute%

Relative%

Total

Small

Large

Cell Culture Grade Water

10%

98

89.5

89.2

122.2

99

88.3

75

54.8

20.2

10%

98.8

108.3

108.9

116.6

94.4

102.8

86.7

60.5

26.2

10%

103.2

102.2

101.8

131.6

106.6

108.6

92

56.6

35.4

Methyl methanesulfonate

6.5

69.4

36.8

20

79.5

64.4

12.8

478.7

342.3

136.4

6.5

72.8

32

19.3

68

55.1

10.6

497.3

361

136.4

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

2.25

83.2

70.8

74.8

126

102.1

76.3

84.8

60.6

24.2

2.5

78

58.8

55.7

128.9

104.4

58.1

93.1

66.6

26.5

2.75

70.2

49.5

55.7

119.3

96.6

53.8

114.9

70.1

44.8

3

65

47.6

42.3

138

111.8

47.3

94.6

63.5

31.1

3.25

44.2

40.7

49

105.5

85.4

41.9

106.6

75.9

30.7

3.5

47.7

31.9

37.5

124.6

100.9

37.8

81.8

50.8

30.9

4

41.6

20.9

20.6

121.1

98.1

20.2

114.7

77.5

37.2

4.5

24.3

14.2

13

101.5

82.2

10.7

122.6

76.7

45.9

 

Table 3: Mutation assay with mouse lymphoma L5178Y TK+/-cells (Trial C4, without metabolic activation, 4 h treatment) (Study # 53728)

Test condition

Concentration

Cum. RSG (%)a

Cloning Efficiencyb

Relative Total Growthc(%)

Mutant Frequency (× 10-6)d

Day 1

Day 2

Absolute%

Relative%

Total

Small

Large

Cell Culture Grade Water

10%

100

96

94

100.5

96.5

51.1

43.7

7.4

10%

98.5

99.1

96.9

103.6

102.7

28.5

26.3

2.3

10%

101.5

104.9

89.6

95.9

100.6

39.8

33.7

6.1

Methyl methanesulfonate

13

14.5

9.3

32.9

35.2

3.3

213.3

205.5

7.7

13

16.8

13.5

24.9

26.6

3.6

292

271.5

20.4

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

4

94.7

76

84.4

90.2

68.6

42.2

36.6

5.6

6

55.7

46.8

110.7

118.4

55.4

53.2

50.6

2.6

6.5

56.5

43.3

86.9

92.9

40.2

53.6

47.7

5.9

7

55

43.1

63.6

68

29.3

66.3

61.7

4.6

7.5

40.5

32.9

92.9

99.4

32.6

63.4

58.7

4.7

8

42.7

31.6

63.8

68.2

21.5

70.7

68.4

2.3

8.5

46.6

27.9

65.8

70.4

19.7

71.8

71.3

0.6

9

41.2

16.4

65.5

70

11.5

72.2

69.4

2.8

aCum. RSG = Cumulative Suspension Growth Relative to the Average Vehicle Control Suspension Growth

bCloning Efficiency = Total Viable Colony Count/Number of Cells Seeded x 100

cRelative Total Growth = (Relative Suspension Growth x Relative Cloning Efficiency) / 100

dMutant Frequency = (Total Mutant Colonies/Total Viable Colonies) x (2 x 10-4)

Results of positive control: Mutant colonies from methyl methanesulfonate (MMS) and methylcholanthrene (MCA) treated cultures showed both small and large colonies. 

Conclusions:
N,N-Bis(2-Hydroxyethyl)-PPD SULF (BHP) was considered non-mutagenic at the Thymidine Kinase +/- locus in L5178Y mouse lymphoma cells, with or without metabolic activation.
Executive summary:

The in-vitro mouse lymphoma mutation test of N,N-Bis(2-Hydroxyethyl)-PPD SULF (BHP) was determined following the OECD guideline 476 (In vitro Mammalian Cell Gene Mutation Test).

The objective of this in vitro assay was to evaluate the potential of test substance to induce forward mutations at the thymidine kinase (TK) locus of cultured L5178Y TK+/-mouse lymphoma cells. The cells were maintained in RPMI 1640 media supplemented with horse serum (10% by volume), Pluronic F68, L-glutamine, sodium pyruvate, penicillin and streptomycin. The cells were periodically checked for mycoplasma contamination and karyotype stability. The microsomal enzymes obtained from liver of rats treated with Aroclor were used as the metabolic activation system (S9).

A preliminary toxicity test was conducted with and without metabolic activation to determine the dose levels for the confirmatory mutagenicity assay. The first toxicity assay was performed for 4 h duration with 10 test substance concentrations (Range: 9.85 to 5000 μg/mL) in the presence and absence of metabolic activation. A second toxicity test was performed without metabolic activation with an extended treatment period of approx. 24 h with 10 test substance concentrations (Range 0.197 to 100 μg/mL). A vehicle control (cell culture grade water) was included under each condition.

The mutagenicity (initial and confirmatory) assays were performed as follows:

a) Without metabolic activation: 4 independent mutagenicity experiments were performed as follows:

Trial B1 (initial mutation assay): 5 test concentrations were evaluated, at concentrations of 1, 2.5, 3.75, 5 and 7.5 µg/mL, for 4 h treatment duration

Trial C3 (initial mutation assay): 8 test concentrations were evaluated, at concentrations of 3, 4, 7, 8, 8.5, 9, 9.5 and 10 µg/mL, for 4 h treatment duration.

Trial C4 (initial mutation assay): 8 test concentrations were evaluated, at concentrations of 4, 6, 6.5, 7, 7.5, 8, 8.5 and 9 µg/mL, for 4 h treatment duration.

Trial C2 (confirmatory mutation assay): 8 test concentrations were evaluated, at concentrations of 2.25, 2.5, 2.75, 3, 3.25, 3.5, 4 and 4.5 µg/mL, for 24 h treatment duration.

b) With metabolic activation system: Mutation assay was performed with metabolic activation system and no confirmatory assay was performed further.

Trial B1: 8 test concentrations were evaluated, at concentrations of 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL, for 4 h treatment duration. 

Methylmethanesulphonate (13 µg/mL for 4 h duration and 6.5 µg/mL for 24 h duration) and 3-methylcholanthrene (2 and 4 µg/mL for 4 h duration) were used as positive controls in absence and presence of metabolic activation system, respectively.

The cytotoxicity of test substance was determined by measuringrelative suspension growth. The mutagenicity potential test substance was determined by calculating mutant frequency.

In the preliminary toxicity test A1 (without S9, 4 h), moderate toxicity at 9.85 μg/mL and excessive toxicity at 19.7 μg/mL and higher concentrations was observed. In toxicity trial A1 (with S9, 4 h), weak toxicity up to and including 39.3 μg/mL, moderately high toxicity at 78.5 μg/mL and excessive toxicity at 157 μg/mL and higher concentrations were observed. In toxicity trial A2 (without S9, 24 h), no toxicity at 0.197 μg/mL, weak toxicity from 0.393 to 1.57 μg/mL, moderately high toxicity at 3.13 μg/mL and excessive toxicity at 6.25 μg/mL and higher concentrations were observed.

In the initial mutation assay (B1) (with S9 metabolic activation system, 4 h duration), none of the analyzed test substance treatments induced an increase in the mutant frequency that met the criteria for a positive response. A confirmatory assay was not required.

In confirmatory assay (C2; without S9, 24 h duration), none of the analyzed test substance treatments induced a mutant frequency that met criteria for a positive response.

In other mutation assays (B1, C3 and C4) in absence of metabolic activation, no positive response was observed. Mutant colonies from all the cultures showed the expected bimodal distribution and mutant colonies from methyl methanesulfonate (MMS) and methylcholanthrene (MCA) treated cultures showed both small and large colonies. Thus, validating the positive controls used in the study.

Vehicle and positive control data in this study were comparable with the historical control data.

Based upon above, N,N-Bis(2-Hydroxyethyl)-PPD SULF (BHP) was considered non-mutagenic at the Thymidine Kinase +/- locus in L5178Y mouse lymphoma cells, with or without metabolic activation (S9).

This mammalian gene mutation test is classified as acceptable, and satisfies the guideline requirements of the OECD 476 method.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo, N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate did not induce micronuclei in polychromatic erythrocytes of mice or DNA damage (measured by unscheduled DNA synthesis) in hepatocytes of rats.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 01, 2005 to Nov. 09, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Remarks:
(according to OECD and US FDA principles of GLP)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: CD-1 (ICR)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina, USA
- Age at study initiation: Young adult, approx. of 9 weeks
- Weight at study initiation: 31-35.8 g
- Assigned to test groups randomly: Yes, animals were randomized into groups using a computer program. Animals were considered acceptable for study use based upon data collected during acclimation. After assignment to groups, the coefficient of variation of the mean weight for each group did not exceed 20%.
- Housing: The animals were housed in sanitary polycarbonate cages containing Sani-Chips Hardwood Chip Laboratory bedding. The animals were housed, separated by gender, up to five animals per cage during acclimation, and by full dose group/harvest timepoint after randomization.
- Diet: PMI Certified Rodent Diet #5002 was available ad libitum.
- Water: Tap water (by water bottle) was available ad libitum. Water samples are routinely analyzed for specified microorganisms and environmental contaminants.
- Acclimation period: At least 5 d prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 17-27°C
- Humidity: 30-70%
- Air changes: 10 or greater air changes/h
- Photoperiod: 12 h light/12 h dark (Cycle may have been interrupted for study-related activities)
Actual temperature and humidity readings were monitored continuously and averaged twice daily.

IN-LIFE DATES: From: March 23, 2005 To: March 25, 2005
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Reverse osmosis water
- Justification for choice of vehicle: The solvent was chosen based on information provided by the Sponsor.
- Concentration of test material in vehicle: 1.5625, 3.125 and 6.25 mg/mL
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prior to dosing, the top stock of the test substance was prepared by adding the appropriate volume of the vehicle, to a pre-weighed quantity of the test substance and mixed, forming a solution. Lower concentrations were obtained by serial dilution with the vehicle. The details on preparation of dosing solutions are provided in the study report.

STORAGE: The formulations were held at room temperature prior to dosing.

TREATMENT SCHEDULE: The treatment schedule was as follows:
Vehicle Control (10 animals): Reverse osmosis water
Low dose Group (5 animals): 15.625 mg/kg bw
Mid dose Group (5 animals): 31.25 mg/kg bw
High dose Group (10 animals): 62.5 mg/kg bw
Positive control (5 animals): 80 mg/kg bw

SACRIFICE: 5 animals from vehicle control and High dose Group were sacrificed at each 24 h and 48 h after treatment initiation. In other treatment groups, all animals (5) were sacrificed at 24 h after treatment. Animals were euthanized by CO2 inhalation.
Duration of treatment / exposure:
24 and 48 h
Frequency of treatment:
Once
Post exposure period:
All animals were examined immediately after dosing, approx. 1 and 4 h after dosing, and at least daily for the duration of the assay (upto 48h after treatment) for signs of clinical toxicity and/or mortality.
Dose / conc.:
15.625 mg/kg bw/day (actual dose received)
Dose / conc.:
31.25 mg/kg bw/day (actual dose received)
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 mice in low (15.625 mg/kg bw), mid dose (31.25 mg/kg bw) test treatment groups and positive control group
10 mice in vehicle control and high dose test treatment group (62.5 mg/kg bw)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): Cyclophosphamide is one of the recommended positive controls of OECD guideline 474.
- Route of administration: oral gavage
- Doses: 80 mg/kg bw
- Concentration in vehicle: 2 mg/mL
Tissues and cell types examined:
Erythrocytes
Tissue: Bone marrow from femur
Cell types: Polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose levels were selected based upon the preliminary toxicity study. The high dose administered in the study was the maximum tolerated dose determined from a preliminary toxicity study.

TREATMENT AND SAMPLING TIMES: Sampling was done at 24 h and 48 h for vehicle and high dose groups (5 animals at each time point). For other treatment groups, samples were collected at 24 h after treatment initiation, 5 animals/treatment group.

BONE MARROW EXTRACTION: Femurs were removed for marrow extraction from the surviving animals in each treatment and control group. For each animal, bone marrow flushed was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (1 tube/animal).
PREPARATION OF SLIDES: Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. Slides were fixed by dipping in methanol, stained in May-Grunwald solution and Giemsa, and protected by mounting with coverslips. For control of bias, all slides were coded prior to analysis. At least 2 slides were prepared from each animal.

METHOD OF ANALYSIS: Slides prepared from the bone marrow collected from the 5 animals/group at the designated harvest time points were scored for micronuclei and the % PCEs. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs/animal and presented per treatment group. The % PCEs were determined by scoring proportion of PCEs to total erythrocytes observed while scoring at least 1000 erythroctyes per animal.

CRITERIA FOR IDENTIFICATION OF MICRONUCLEI: The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stainedand generally round, although almond and ring shaped micronuclei occasionally occurred. Micronuclei were sharp bordered and generally between one twentieth and one fifth the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-gray and red, respectively).

HISTORICAL CONTROL: The historical background frequency of micronucleated cells was expressed as percentage micronucleated cells based on the number of PCEs analyzed. The historical background frequency of micronuclei in the mouse strain at the test laboratory was about 0.0-0.4 %, which was within the range of published data.
Evaluation criteria:
- The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statisticallysignificant dose related response. If the test substance did not induce both of these responses it was considered negative.
- Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevance of the results in the final evaluation.
Statistics:
The following statistical methods were used to analyze the micronucleus data:
- Assay data analysis was performed using an analysis of variance on untransformed proportions of cells with micronuclei per animal and on untransformed % PCEs when the variances were homogenous. Ranked proportions were used for heterogeneous variances.
- If the analysis of variance was statistically significant (p ≤0.05), Dunnett's t-test was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time.
- The test substance groups as well as the positive control group were compared with the vehicle control group at the 5% one tailed probability level.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
(slight hypoactivity in 6/10 animals of the 62.5 mg/kg dose group at the 4 h observation interval)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 62.5-1000 mg/kg
- Mortality: Mortality was observed in 2/3 males and 3/3 females in the 125 mg/kg dose group and in 3/3 males and 3/3 females in the 250, 500 and 1000 mg/kg dose groups.
- Clinical signs of toxicity in test animals: Clinical signs of toxicity observed in the 125, 250, 500 and 1000 mg/kg dose group animals included slight hypoactivity, ataxia, tremors, irregular respiration, hypoactivity, hunched posture, convulsions, and/or squinted eyes. A clinical sign of slight hypoactivity was observed in all animals in the 62.5 mg/kg dose group at the 3 to 3.5 h and 4 h observation intervals. Based on the results, the maximum tolerated dose was estimated to be 62.5 mg/kg bw. This was considered the highest dose that would not cause mortality.
- Harvest times: 2 d post-exposure
- High dose: 1000 mg/kg bw
- Since no relevant differences in toxicity between the sexes were observed in the dose range-finding study, only males were used in the micronucleus assay. The high dose, unless non-toxic, should produce some indication of toxicity, e.g., toxic signs or depression of the % PCEs and should not produce mortality. The use of a high dose increases the likelihood that a weak clastogen would be detected.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The test substance did not induce statistically significant increases in micronucleated PCEs at any dose examined (15.625, 31.25, and 62.5 mg/kg bw).
- Ratio of PCE/NCE: No cytotoxicity to the bone marrow (i.e., no statistically significant decreases in the % PCEs) was observed at any dose of the test substance

Further details on results of micronucleus assay are provided in ‘Table 1’ in 'Any other information on results incl. tables' section.

RESULTS OF VEHICLE AND POSITIVE CONTROL GROUP:
- The vehicle control group had less than approx. 0.5% micronucleated PCEs and the group mean was within the historical control range.
- The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard error of 4 ± 0.18%.

MICRONUCLEUS ASSAY:

Analysis of doing formulation: Results of the high dose formulation analysis were detected at 99.5 % of the target concentration of 6.25 mg/mL. The concentration verification analyses for the test substance indicated that the low dose formulation (1.5625 mg/mL) had no detectable test substance. Analysis of the backup low dose formulation confirmed the initial results. The reason for the 1.625 mg/mL concentration having no detectable test substance was unknown. However, since the high dose formulation of 6.25 mg/mL was at 99.5% of the target concentration, the low dose concentration analysis result was considered to have no significant impact on the scientific integrity or validity of the study.

- Although there were not indications of bone marrow toxicity in the present study, the oral bioavailability of the test substance was evident based on the clinical signs seen at the 62.5 mg/kg dose in the micronucleus study and by the deaths and clinical signs seen at 125 mg/kg and above in the dose range-finding study. Moreover, the rat 14 d oral gavage range finding toxicity study (Covance study # 6114-469, separate study; no data provided in study report), in which a change in blood parameters was observed at a dose of 100/25 mg/kg/d, builds upon the weight of evidence for systemic exposure of the test substance in this in vivo micronucleus study.

Mortality and clinical signs of definitive study:

- Effect on Body Weight: Individual and mean group body weights were recorded. None of the test dose groups had a mean body weight less than 80% of the corresponding vehicle control group.

- Mortality: No mortality was observed in any of treated animals (low (15.625 mg/kg), mid (31.25 mg/kg) or high dose group (62.5 mg/kg)).

- Slight hypoactivity was observed in 6/10 animals in the 62.5 mg/kg dose group at the 4 h observation interval.

Table 1: Result of Micronucleus Assay after treatment with N,N-Bis (2-Hydoxyethyl)-PPD SULF (Study # 53730)

Treatment

Dose

Harvest Time

% Micronucleated PCEs Mean of 2000 per Animal ± S.E. 

% PCE Mean ± S.E.

Vehicle control (Reverse osmosis water)

Water 10 mL/kg

24 h

0.1 ± 0.03

46.72 ± 1.02

48 h

0.05 ± 0

41.4 ± 3.35

Positive control (Cyclophosphamide)

CP 80 mg/kg

24 h

4 ± 0.18*

33.96 ± 1.03**

Test substance (N,N-bis(2-hydroxyethyl)benzene-1,4-diaminium sulfate)

15.625 mg/kg

24 h

0.08 ± 0.02

45.6 ± 2.32

31.25 mg/kg

24 h

0.06 ± 0.02

42.48 ± 1.63

62.5 mg/kg

24 h

0.08 ± 0.01

42.58 ± 3.05

48 H

0.07 ± 0.01

37.14 ± 1.91

* Significantly greater than the corresponding vehicle control, p ≤ 0.01

** Significantly less than the corresponding vehicle control, p ≤ 0.05

CP = Cyclophosphamide

PCE = Polychromatic erythrocyte

% PCE = number of PCEs/total erythrocytes x 100
Conclusions:
N,N-Bis (2-Hydoxyethyl)-PPD SULF was not genotoxic in the in vivo micronucleus assay to mice after a single oral gavage treatment, conducted up to a maximum tolerated dose (62.5 mg/kg bw).
Executive summary:

The in-vivo micronucleus test of N,N-Bis (2-Hydoxyethyl)-PPD SULF on bone marrow cells was determined following OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test).

To select the dose concentrations for in vivo micronucleus assay, a preliminary range finding study was performed on male and female mice (3/sex/dose). The doses selected were 62.5, 125, 250, 500 and 1000 mg/kg bw. Animals were treated once orally by gavage and observed for mortality and clinical signs. Based upon the results of this range finding study, 62.5 mg/kg bw was used as maximum tolerated dose. 

For the definitive micronucleus assay, male CD-1 (ICR) BR mice weighing 31-35.8 g from Charles River Laboratories were used in the study. Animals were acclimated for at least 5 d prior to treatment. The animals were housed, up to 5 animals/sex/cage during acclimation. The mice were separated by dose group/harvest time after randomization and maintained under standard laboratory conditions (temperature: 17 - 27°C, humidity: 30-70%; artificial light: 12 h cycle). The test formulations were prepared in water (reverse osmosis). Reverse osmosis water was served as vehicle control and cyclophosphamide was used as positive control.

The treatment schedule was as follows:

Vehicle Control (10 animals): Reverse osmosis water 

Low dose Group (5 animals): 15.625 mg/kg bw (10 mL/kg bw)

Mid dose Group (5 animals): 31.25 mg/kg bw (10 mL/kg bw)

High dose Group (10 animals): 62.5 mg/kg bw (10 mL/kg bw)

Positive control (5 animals): 80 mg/kg bw (10 mL/kg bw)

5 animals from vehicle control and high dose group were sacrificed at 24 h and 48 h after initiation of treatment to collect bone marrow samples. In other treatment groups, all animals were sacrificed 24 h after initiation of treatment to collect bone marrow samples. Animals were euthanized by CO2 inhalation. The bone marrow was collected from femurs and was processed. Slides were prepared and analyzed for micronuclei and the % PCEs.

None of the test dose groups had a mean body weight less than 80% of the corresponding vehicle control group.

The test substance did not induce statistically significant increases in micronucleated PCEs at any dose examined (15.625, 31.25, and 62.5 mg/kg bw). No cytotoxicity to the bone marrow (i.e., no statistically significant decreases in the % PCEs) was observed at any dose of the test substance.

The vehicle control group had less than 05% micronucleated PCEs (0.10 and 0.05% micronucleated PCEs at 24 and 48 h respectively) and the group mean was within the historical control range. The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard error of 4 ± 0.18%.

Based on above, N,N-Bis (2-Hydoxyethyl)-PPD SULF was not genotoxic in the in vivo micronucleus assay to mice after a single oral gavage treatment, conducted up to a maximum tolerated dose (62.5 mg/kg bw).

This Mammalian Erythrocyte Micronucleus Test is classified as acceptable, and satisfies the guideline requirements of the OECD 474 method.
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 23, 2005 to June 16, 2005.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
GLP compliance:
yes
Remarks:
(according to US-FDA and OECD principles of GLP)
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Male Sprague-Dawley rats were obtained from Harlan Sprague-Dawley, Inc., Frederick, MD, USA.
- Age at study: 8 wks 3 d at the time of randomization
- Weight at study: 234.7 to 257.3 g at the time of randomization
- Assigned to test groups randomly: Yes; animals were assigned to eight experimental groups (5 males each) according to a computer-generated program which was based on distribution according to body weight.
- Housing: Rats were housed up to five per rodent Micro-Barrier cage. Cages were placed in the racks equipped with Micro-VENT full ventilation, HEPA filtered system. The purpose of this system was to supply uninterrupted positive air to each individual rodent Micro-Barrier cage and to capture the effluent air from each cage and re-filter the air (HEPA) prior to introducing the air back into the room. Heat-treated hardwood chips were used for bedding.
- Diet: Certified laboratory rodent chow, ad libitum
- Water: Tap deionized distilled water, ad libitum
- Acclimation period: At least 5 d
Animals were housed in an AAALAC-accredited facility. Diet and water were analyzed for environmental contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3˚F
- Humidity: 50 ± 20%
- Air changes: At least 10 changes of fresh HEPA-filtered air/h
- Photoperiod: 12 h light/12 h dark

IN-LIFE DATES: From: May 23, 2005 To: May 24, 2005
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Deionized distilled water
- Justification for choice of solvent/vehicle: The solvent was chosen based on information provided by the Sponsor and compatibility with the test system.
- Concentration of test material in vehicle: 10 and 20 mg/mL
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solution was prepared in the distilled water. Each dose preparation was swirled by hand for less than 1 min until it went into solution.
Duration of treatment / exposure:
2-4 h and 12-16 h
Frequency of treatment:
Once
Post exposure period:
The animals were observed for clinical signs at time of treatment and harvest.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Dose volume: 10 mL/kg bw
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Dose volume: 10 mL/kg bw
No. of animals per sex per dose:
5/sacrifice time point/dose (only the first 3 rats with successful perfusion from each group per sacrifice time point were used for analysis of UDS)
Control animals:
yes, concurrent vehicle
Positive control(s):
Dimethylnitrosamine (DMN)
- Justification for choice of positive control(s): Not reported
- Route of administration: Oral gavage
- Doses / concentrations: 35 mg/kg bw
- Dose volume: 10 mL/kg bw
Tissues and cell types examined:
Hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on information supplied by the Sponsor, the high dose level was selected as the maximum tolerated dose (MTD) level producing signs of toxicity such that a higher dose level would be expected to produce lethality. Based on the information supplied by the Sponsor 200 mg/kg bw was estimated to be the MTD. Based on the OECD guideline a second dose that was 50 % of the top dose was selected as the next dose.

TREATMENT AND SAMPLING TIMES: 5 animals were exposed for each 2-4 h and 12-16 h after treatment. Hepatocytes were isolated from 3 animals for both exposure durations, 2-4 h and 12-16 h.

DETAILS OF SLIDE PREPARATION:
- Preparation of Hepatocyte Cultures: Each rat was anesthetized by inhalation of isoflurane and a midventral incision was made to expose the liver. The liver was perfused with 0.5 mM ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) solution followed by collagenase solution (80-100 units Type I collagenase/mL culture medium). The liver was removed, transected, and shaken in a dilute collagenase solution to release the hepatocytes. The cells were pelleted by centrifugation, resuspended in complete Williams' medium E (WME; buffered with 0.01 M HEPES, supplemented with 2mM L-glutamine, 50 µg/mL gentamicin and 10% fetal bovine serum). Approx. 5 × 10(5) viable cells (determined by trypan blue exclusion) were seeded into each of six 35 mm tissue culture dishes containing 25 mm coverslips and preconditioned complete WME (i.e., complete WME medium in 35 mm tissue culture dishes incubated overnight in a humidified atmosphere of 5 ± 1% CO2 and 37 ±1˚C). A minimum of 6 cultures were set up for each rat. The hepatocyte cultures were maintained in a humidified atmosphere of 5 ± 1% CO2 and 37 ± 1˚C. - Treatment with 3H-Thymidine: 90 to 180 min after plating, the cells were washed once with complete WME and refed with serum free WME containing 10 µCi 3H-thymidine/mL. 4 h later, the radioactive medium was removed, the cultures were washed three times in serum free WME containing 0.25 mM thymidine, and then refed with serum free WME containing 0.25 mM non-tritiated thymidine and incubated for 17-20 h.
- Washing: 17 to 20 h after exposure to thymidine, the coverslips bearing cultures were washed once in serum free WME.
- Fixation and Labelling of Slides: The nuclei were swelled in 1% sodium citrate solution and the cultures fixed in three changes of ethanol glacial acetic acid fixative (3:1, v/v). The coverslips were allowed to air dry for at least 1 h mounting cell side up on glass slides. The slides were labeled with the study number and a code to identify the animal number. At least three of the six slides for each rat were dipped in NTB emulsion diluted 1:1 in deionized distilled water, at 43-45˚C, allowed to drain and dry for at least 1.5 h at room temperature and were stored for 3 d (3 d was found to be the optimal time for storing the sides as the NTB emulsion was much more sensitive than NTB-2 emulsion it replaced) at 2-8˚C in light tight boxes with a desiccant.
- Staining: Slides were developed in Kodak D-19 developer (diluted 1:1 in deionized water), fixed in Kodak fixer, and stained with hematoxylin-eosin stain.

METHOD OF ANALYSIS: All coded slides were read without knowledge of treatment group. The slides were viewed microscopically under a 100X oil immersion lens. A computer equipped with image analyzing software was interfaced through a video camera with the microscope so that silver grains within each nuclei and the surrounding cytoplasm could be counted. ProtoCOL system Version 3.07 with accompanying support software was used for grain counts. First the number of grains in a nucleus was counted. The number of grains in three separate nuclear-sized cytoplasmic areas (taken from the area adjacent to the nucleus and which appeared to have the highest grain counts) were counted. The counts were captured directly by the software and stored as raw data in anelectronic data file created by software. 50 nuclei were scored from each of 3 replicate cultures for a total of 150 nuclei from each rat. Replicative DNA synthesis was evidenced by nuclei completely blackened with grains, and such cells were not counted. Cells exhibiting toxic effects of treatments, such as irregularly shaped or very darkly stained nuclei, were not counted. A net nuclear grain count was calculated from each nucleus scored by subtracting the mean cytoplasmic area count from the nuclear area count. The net nuclear counts were averaged and the mean ± standard deviation (SD) calculated for each treatment slide. The average mean and SD for each animal and treatment group as well as the percent of cells in repair (cells with ≥ 5 net nuclear grains) were reported.
Evaluation criteria:
- All conclusions were based on sound scientific judgment; however the following was offered as a guide to interpretation of the data.
- Based on laboratory historical control data, any group average of the mean net nuclear count which was increased by at least five counts over the group average of the vehicle control was considered significant.
- The test substance was judged positive if it induced a dose related increase in the group average mean net nuclear grain count with one dose group significantly elevated above the vehicle control.
- A significant increase in the group average of the mean net nuclear grain count in both doses in the absence of a dose response was considered positive.
- The test substance was considered negative if no significant increase in the group average of the net nuclear grain counts at any treatment was observed.
- The percentage of cells in repair (cells with ≥ 5 net nuclear grains) was also reported. These data may have also been used by the Study Director in making a final evaluation of the activity of the test substance.
Statistics:
Not reported
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF MORTALITY AND CLINICAL SIGNS:
- All animals treated for 2 to 4 h with the positive and vehicle controls and test substance concentrations of 100 and 200 mg/kg bw appeared normal immediately following dosing and prior to harvest.
- All animals treated for 12 to 16 h with vehicle and positive controls were observed to be normal immediately following dosing and prior to harvest.
- The 100 mg/kg bw 12 to 16 h-treated animals was observed to be normal immediately following dosing but one animal appeared to have a crusty nose prior to harvest.
- The 200 mg/kg bw 12 to 16 h-treated animals was observed to be normal immediately following dosing and was observed to have crusty eyes and crusty noses prior to harvest. One 200 mg/kg bw 12 to 16 h-treated animals was found dead at time of harvest. The cause of death of the animal was unknown.

RESULT OF UDS ASSAY:
a) Results of UDS assay for 2-4 h post-exposure:
- Viability exceeded 50% in all hepatocyte cultures isolated from the vehicle control animals. Prior to scoring, slides were examined for cytotoxicity such as irregularly shaped or very darkly stained nuclei of which none was detected.
- The mean net nuclear grain count for the vehicle control group was - 0.1.
- The mean net nuclear grain count for 100 and 200 mg/kg bw treatment groups were -0.2 and 0.2, respectively. None of the test substance doses caused a significant increase in the mean net nuclear counts compared to vehicle control group.
- The positive control compound, DMN, at 35 mg/kg bw, induced an increase in the average mean net nuclear grain counts of +10.6 over that of the vehicle control. According to the criteria set for evaluating the test results, the induced increase was considered to be significant in the DMN-treated animals.

b) Results of UDS assay for 12-16 h post-exposure:
- Viability exceeded 50% in all hepatocyte cultures isolated from the vehicle control group.
- The mean net nuclear grain count for the vehicle control group was -0.3.
- The mean net nuclear grain count for 100 and 200 mg/kg bw treatment groups were -0.1 and 0, respectively. None of the test substance doses caused a significant increase in the mean net nuclear counts.
- The mean net nuclear grain count for the positive control group was 10.4. The positive control compound, DMN, at 35 mg/kg bw, induced an increase in the average mean net nuclear grain counts of +10.7 over that of the vehicle control. According to the criteria set for evaluating the test results, the induced increase was considered to be significant in the DMN treated animals.
- One 200 mg/kg bw animal was unexpectantly found dead at the time of harvest. The cause of death of the animal was unknown.

Detailed results of UDS assay are provided in “Table 1” of “Any other information on results incl. tables” section.

Dosing Solution Analysis: The formulations used in the UDS assay were within the acceptable limits of ± 10% of theoretical concentration. Results from the stability analysis of the test substance in water were within ± 10% of initial concentrations for up to 30 d at concentrations of 0.05, and 100 mg/mL when frozen at -20 ± 10˚C. Stability analysis was performed on solutions prepared for a separate study and not used in the UDS assay.

Table 1: Summary of UDS Assay with N,N-Bis(2-hydroxyethyl)-PPD SULF (Study # 54975)  

Groups

Dose

Mean Net Grain Counts ± SD1/treatment group

Cells in Repair/treatment group

 

2-4 h post exposure

Water (Vehicle)

10 mL/kg bw

-0.1 ± 0.1

2%

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

100 mg/kg bw

-0.2 ± 0.5

1%

200 mg/kg bw

0.2 ± 0.4

3%

Dimethylnitrosamine (positive control)

35 mg/kg bw

10.5 ± 0.8*

99%

 

12-16 h post exposure

Water (Vehicle)

10 mL/kg bw

-0.3 ± 0.3

1%

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

100 mg/kg bw

-0.1 ± 0.1

1%

200 mg/kg bw

0 ± 0.3

1%

Dimethylnitrosamine (positive control)

35 mg/kg bw

10.4 ± 1.1*

96%

1Standard deviation reflecting variation between animals

2S D= Standard deviation reflecting variation between animals

*= Significant

Conclusions:
N,N-Bis(2-hydroxyethyl)-PPD SULF when administered once orally to rats at 100 and 200 mg/kg bw (for 2-4 h and 12-16 h exposure periods) did not induce a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the vehicle control) in hepatocytes isolated from treated animals and was concluded to be negative in vivo unscheduled DNA synthesis assay.
Executive summary:

The in vivo DNA damage and repair assay of N,N-Bis(2-hydroxyethyl)-PPD SULF was determined following the OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo).

Male Sprague-Dawley rats obtained from Harlan Sprague-Dawley, Inc., Frederick, MD, USA were used in the study. The age and body weight of rats at time of randomization was 8 weeks and 234.7 to 257.3 g. Animals were acclimated for minimum of 5 d prior to treatment. Animals were housed up to 5/rodent micro-Barrier cage and placed in the racks equipped with Micro-VENT full ventilation (HEPA filtered system). Animals were maintained under standard laboratory conditions (temperature: 72 ±3°F, humidity: 50 ± 20%; 12 h light/12 h dark cycle, air changes: at least 10 changes of fresh HEPA-filtered air/h). Deionized distilled water was used as the vehicle for preparation of dosing solution of test substance. Deionized distilled water served as vehicle control.

Based upon the toxicity information, two dose levels of test substance were selected for study. Dimethylnitrosamine (35 mg/kg bw; Dose volume: 10 mL/kg bw) served as positive control.

Two independent experiments with two sampling times (2-4 and 12-16 h) were performed in this study. Animals were treated once orally by gavage. The following dose levels with 5 males per dose group were investigated:

 

Experiment 1 (2-4 h sampling time): 100and 200 mg/ kg bw (Dose volume: 10 mL/kg bw)

 

Experiment 2 (12-16 h sampling time): 100and 200 mg/ kg bw (Dose volume: 10 mL/kg bw)

 

After treatment, hepatocytes were isolated using collagenase solution. Hepatocytes from only 3 animals from each treatment group were used for evaluation. Hepatocytes were processed and treated with radiolabelled thymidine (3H-Thymidine). After treatment with thymidine, hepatocytes were washed and slides were prepared. The slides were stained with hematoxylin-eosin stain. The slides were observed microscopically for the alterations in net nuclear count of hepatocytes along with % cells in repair.

Animals were observed for clinical signs and mortality during the study period.

The 200 mg/kg bw (for 12 to 16 h) treated animals were observed to be normal immediately following dosing and were observed to have crusty eyes and crusty noses prior to harvest. One 200 mg/kg bw (for 12 to 16 h) treated animal was found dead at time of harvest. The cause of death of the animal was unknown. The 100 mg/kg bw (for 12 to 16 h) treated animals were observed to be normal immediately following dosing but one animal appeared to have a crusty nose prior to harvest.

No signs of toxicity were observed in any of the other test substance treated animals.

The viability of hepatocytes at both 2-4 and 12-16 h exposure period was more than 50%. None of the test substance treatments caused a significant increase in the mean net nuclear counts compared to vehicle control group at both exposure periods (2-4 and 12-16 h).

According to the criteria set for evaluating the test results, the induced increase was considered to be significant in the DMN treated animals at both exposure periods (2-4 and 12-16 h). The assay met the all validity criteria.

Based on above, N,N-Bis(2-hydroxyethyl)-PPD SULF when administered once orally to rats at 100 and 200 mg/kg bw (for 2-4 h and 12-16 h) did not induce a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the vehicle control) in hepatocytes isolated from treated animals and was concluded to be negative in vivo unscheduled DNA synthesis assay.

This in vivo DNA damage and repair assay is classified as acceptable, and satisfies the guideline requirements of the OECD 486 method.

Additional information

N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate has been evaluated in an extensive battery of state of the art genotoxicity tests that cover the relevant endpoints including a) gene mutations (bacteria and mammalian mouse lymphoma assays), b) clastogenicity (mouse lymphoma assay, Chinese hamster ovary cell cytogenetic assay and in vivo mouse micronucleus assay), c) aneugenicity (in vivo mouse micronucleus assay) and d) DNA damage (in vivo unscheduled DNA synthesis assay). The mutagenicity data for N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate indicates that it is genotoxic in some assays in vitro but is not genotoxic in vivo.

N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate was positive in the Ames assay without S9 activation and in the in vitro chromosome aberration assay in Chinese hamster ovary cells with S9 activation but was negative in the in vitro mouse lymphoma (L5178Y TK+/-) mutation assay in both the presence and absence of metabolic activation, indicating that this material is not a mammalian cell mutagen. N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate was negative in an in vivo micronucleus assay in ICR mice following single oral doses up to a maximum tolerated dose of 62.5 mg/kg body weight in reverse osmosis water. N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate is therefore not considered to be an in vivo clastogen/aneugen. Negative results were also obtained in an in vivo unscheduled DNA synthesis assay in Sprague Dawley rats, up to a maximum tolerated dose of 200 mg/kg body weight in deionised distilled water, indicating N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate does not induce DNA damage repair in vivo. There was evidence for bioavailability of the test article in these in vivo tests and from other related toxicity studies. Overall, N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate is considered not to be genotoxic in vivo in robust tests conducted to maximum tolerated doses.

Justification for classification or non-classification

Thus, although in vitro assays in E. coli and CHO cells suggest that N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate has genotoxic potential, two in vivo assays indicated no genotoxic activity.