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Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: N,N-Bis (2-Hydoxyethyl)-PPD SULF; (p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate
- TSIN: GTS03849
- Substance type: Pure active substance
- Physical state: Light grey/white powder
- Storage condition of test material: At room temperature
- Stability: The test substance formulations were stable for 30 d at -10 to -30°C and all values were within 7.3% of initial concentration.
- Solubility: Soluble in water

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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%.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.