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EC number: 220-562-2
CAS number: 2814-77-9
This study was performed to investigate the potential of C.I. Pigment
Red 4 to induce gene mutations according to the plate incorporation
assay with rat liver S9 (experiment I), and the pre-incubation test with
hamster liver S9 (experiment II) using the Salmonella typhimurium
strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli
strain WP2 uvrA.
The assay was performed in two complete experiments with and without
liver microsomal activation and one additional experiment solely with
strain TA 98 in the presence of metabolic activation. The results of the
additional experiment are reported as part of experiment I. Each
concentration, including the controls, was tested in triplicate. The
test item was tested at the following concentrations:
Experiment I (including the additional experiment with strain TA 98): 3;
10; 33; 100; 333; 1000; 2500; and 5000mg/plate.
Experiment II: 33; 100; 333; 1000; 2500; and 5000mg/plate
The plates incubated with the test item showed normal background growth
up to 5000mg/plate with and without metabolic activation in
bath independent experiments.
No toxic effects, evident as a reduction in the number of revertants,
occurred in the test groups with and without metabolic activation. No
substantial increase in revertant colony numbers was observed following
treatment with C.I Pigment Red 4 at any dose level, neither in the
presence nor absence of metabolic activation (S9 mix) in strains TA
1535, TA 1537, TA 100, and WP2 uvrA. However, a substantial increase was
observed in strain TA 98 in the presence of metabolic activation in both
independent experiments. The required threshold of two times the
mutation frequency of the corresponding solvent control was well
exceeded at concentrations as low as 3mg/plate in
experiment I and 33mg/plate in experiment II. To verify the
results of experiment I, an additional plate incorporation test was
performed with strain TA 98 in the presence of metabolic activation. In
this additional experiment the result of the first experiment was
Appropriate reference mutagens were used as positive controls and showed
a distinct increase of induced revertant colonies.
In this study the potential of C.I. Pigment Red 4 (Batch No. BRAB
036744) to induce gene mutations at the HPRT locus in V 79 cells of the
Chinese hamster lung in vitro was investigated.
The test item was suspended in cell culture medium and tested at the
First main experiment:
without S9-mix: 6.4, 12.9,25.7, 51.3, 102.5,204.9,409.7, 819.4, 1638.7
and 3277.3* ug/ml
with S9-mix: 6.4, 12.9,25.7, 51.3, 102.5,204.9,409.7, 819.4, 1638.7 and
Second main experiment:
without S9-mix: 78.1, 156.3, 312.5, 625 ,937.5, 1250.0, 1875, 2500 and
with S9-mix: 78.1, 156.3, 3 l2.5, 625, 937.5, 1250.0, 1875, 2500 and
*= 10 mM
at 6.4 = no selection was performed because higher concentrations were
The concentration ranges were based on the results of preliminary
testing for solubility and toxicity. The highest concentration produced
no severe toxic effects with and without metabolic activation in both
Higher concentrations were not applied because of the 10 mM limitation
The test item produced microscopic precipitation down to a concentration
of 25.7 ug/ml after treatment time. Macroscopic precipitation of the
test item was observed at 204.9 ug/ml after treatment time and higher
This GLP study was experimentally initiated on 23-May-2005.
Up to the highest investigated dose the test item induced no relevant of
dose-dependent increase in mutant colony numbers in two independent
experiments. Appropriate reference mutagens used as positive controls
showed a significant increase in mutant colonies, thus indicating the
sensitivity of the assay, and the efficacy of the S9-mix.
In conclusion, C.I Pigment Red 4 did not induce gene mutations in the
HPRT-test with V79 Chinese hamster cells, both in the presence as well
as in the absence of a metabolic activation system, under the
experimental conditions described.
This report describes the results of an in vitro study for the detection
of structural chromosomal aberrations in cultured mammalian cells. It
supplements microbial systems insofar as it identifies potential
mutagens that produce chromosomal aberrations rather than gene mutations
(Scott et al, 1990). The method used was designed to be compatible with
that described in the OECD Guidelines for Testing of Chemicals (1997)
No. 473 "Genetic Toxicology: Chromosome Aberration Test", Method B10 of
Commission Regulation (EC) No. 44012008 of 30 May 2008 and the USA EPA
OPPTS guideline 40 CFR 799.9537. The study design also meets the
requirements of the UK Department of Health Guidelines for Testing of
Chemicals for Mutagenicity.
Duplicate cultures of human lymphocytes, treated with the test item,
were evaluated for chromosome aberrations at up to four dose levels,
together with vehicle and positive controls. Four treatment conditions
were used for the study; i.e. In Experiment 1, 4 hours in the presence
of an induced rat liver homogenate metabolising system (S9), at a 2%
final concentration with cell harvest after a 20-hour expression period
and a 4 hours exposure in the absence of metabolic activation (S9) with
a 20-hour expression period. In Experiment 2, the 4 hours exposure with
addition of S9 was repeated (using a 1% final S9 concentration), whilst
in the absence of metabolic activation the exposure time was increased
to 24 hours.
The dose levels used in the main experiments were selected using data
from the preliminary toxicity test and were as follows:
Test item (ug/ml) 25, 50, 100, 200, 400, 800
All vehicle (solvent) controls had frequencies of cells with aberrations
within the range expected for normal human lymphocytes. All the positive
control items induced statistically significant increases in the
frequency of cells with aberrations indicating the satisfactory
performance of the test and of the activity of the metabolising system.
The test item did not induce any statistically significant increases in
the frequency of cells with aberrations, in either the absence or
presence of a liver enzyme metabolising system in either of two separate
experiments. The maximum dose tested was limited by formulation
difficulties and was also limited by precipitate of the test item
occurring on the slides at the maximum dose and restricting the ability
to accurately Score metaphases.
The test item was considered to be non-clastogenic to human
lymphocytes in vitro.
with data on study results is attached (PigmentRed4_CometAssay_result
RED 2GC was assayed in an in vivo alkaline comet (single cell gel
electrophoresis) assay for the detection of DNA strand breaks in cells
or nuclei isolated from liver, glandular stomach, duodenum and testis
tissue of male CD rats, as well as ovary tissue from female CD rats. The
test item was administered orally to rats once daily for three days at
0, 24 and 45 hours, and the tissues were sampled at 48 hours (3 hours
after the last dose).
RED 2GC was suspended in 0.8% aqueous hydroxypropylmethylcellulose. The
dose levels for the main study had been selected based on the result of
a preliminary Limit test of 2000 mg/kg b.w./day, p.o employing two
animals per dose. No signs of systemic toxicity were noted.
ascending dose levels of 400, 1000 or 2000 mg VERSAL RED 2GC/kg
b.w./day, p.o. and the vehicle (0.8% aqueous
hydroxypropylmethylcellulose) were administered three times at 0, 24 and
45 hours. The positive reference item (125 mg ethyl methane sulfonate/kg
b.w./day, p.o.) was administered two times at 24 and 45 hours. Each
group consisted of 5 male and 5 female rats. The administration volume
was 20 mL/kg b.w./day. The test was divided in 4 different preparation
groups containing one or two animals of each group which were tested in
parallel at each stage of the experiment.
signs of systemic toxicity were noted up to the highest dose level of
2000 mg VERSAL RED 2GC/kg b.w./day, p.o.
cells of liver, glandular stomach, duodenum and testis (males) or
ovaries (females) per animal and tissue collected 48 hours post 1st
administration were evaluated. The grade of DNA fragmentation is
expressed in percent tail DNA (% tail intensity).
with VERSAL RED 2GC did not cause any test item-related increase in the
tail intensity for the cells of liver, glandular stomach, duodenum and
testis (all males) or ovaries (females) at any of the three tested dose
levels of 400, 1000 and 2000 mg test item/kg b.w./day compared to the
vehicle control data.
group mean values of the median tail intensities of the test item
treatment groups were between 6.325% and 7.83%, 9.58% and 11.806% or
11.554% and 12.856% for liver, stomach and duodenum cells, respectively.
The corresponding values for vehicle control were 4.726%, 8.152% or
12.554% for liver, stomach and duodenum cells, respectively. The mean
values of the median tail intensity were between 5.341% and 6.220% or
1.082% and 1.681% for the cells of testis and ovaries, respectively. The
corresponding values for vehicle control were 5.326% or 1.782% for the
cells of testis and ovaries, respectively.
values obtained were within the upper limits of the historical control
data, except for the vehicle control group of stomach cells and test
item treatment groups of liver and stomach cells which were slightly
above the background data. As the slightly higher data are no extreme
outliers, were within published historical control data and older rats
at an age of 9 – 12 weeks were used, to assure maternity of the animals
for evaluation of testis and ovary cells, the results are considered
acceptable for the inclusion into the historical control data and the
test is considered valid.
of ethyl methane sulfonate (positive reference) significantly increased
the % tail intensity for the cells of liver, glandular stomach, duodenum
and testis (all males) or ovaries (females).
values were well within the historical background data, except the
positive control for the stomach and duodenum cells which were above the
historical background data. As the positive control is significantly
increased, the data are considered acceptable for the inclusion into the
historical control data and the test is considered valid.
conclusion, under the present test conditions VERSAL RED 2GC tested up
to the highest reasonable dose level of 2000 mg/kg b.w. per day by
repeated oral administration to male and female rats for three days
showed no genotoxic properties in the in vivo alkaline comet (single
cell gel electrophoresis) assay employing cells of the liver, glandular
stomach, duodenum and testis (males) or ovaries (females).
the same system, ethyl methane sulfonate (positive reference item)
induced significant damage.
Additional information from genetic
toxicity in vitro:
adverse effects due to the test item C.I. Pigment
Red 4 have
been seen in vitro in OECD 473 and OECD 476 studies while ambiguous,
positive and negative test results have been seen in OECD
with and without metabolic activation.
Additional information from genetic
toxicity in vivo:
test item C.I. Pigment Red 4 tested up to the highest reasonable dose
level of 2000 mg/kg b.w. per day by repeated oral administration to male
and female rats for three days showed no genotoxic properties in the in
vivo alkaline comet (single cell gel electrophoresis) assay (OECD
cells of the liver, glandular stomach, duodenum and testis (males) or
conclusion: the test item C.I.
Pigment Red 4 is
not considered to be mutagenic based on overall information from
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