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EC number: 259-134-5
CAS number: 54381-16-7
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
- 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
- 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)
% Micronucleated PCEs Mean of 2000 per Animal ± S.E.
% PCE Mean ± S.E.
Vehicle control (Reverse osmosis water)
Water 10 mL/kg
0.1 ± 0.03
46.72 ± 1.02
0.05 ± 0
41.4 ± 3.35
Positive control (Cyclophosphamide)
CP 80 mg/kg
4 ± 0.18*
33.96 ± 1.03**
Test substance (N,N-bis(2-hydroxyethyl)benzene-1,4-diaminium sulfate)
0.08 ± 0.02
45.6 ± 2.32
0.06 ± 0.02
42.48 ± 1.63
0.08 ± 0.01
42.58 ± 3.05
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
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
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
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).
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