Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 202-987-5
CAS number: 101-90-6
The test item was positive in Salmonella typhimurium strains
TA1535 and TA100 (with and without metabolic activation) in out of 4
tested Salmonella typhimurium strains: TA1535, TA1537, TA98 and
TA100 (Canter and Zeiger, 1984). The test item showed also a positive
result in the Salmonella typhimurium strain TA100 of another
paper (Seiler, 1984), however, there the metabolic activation was not
in vitro chromosomal aberration test
Treatment of cultured Chinese hamster ovary CHO cells with the test item
produced highly significant increases in chromosomal aberrations and
sister chromatid exchanges both with and without S9 (Gulati et al, 1989).
The mutagenic and toxicity potential of the test item has been
demonstrated in Chinese hamster CHO cells. The metabolic activation was
not specified (Seiler, 1984).
A weight of evidence of the analyzed results suggest that the test item
is positive for the genetic toxicity in vitro endpoint.
Table 1: Alkylating Potency of Resorcinol Diglycidyl Ether Measured by
the Formation of Coloured NBP Adducts
Table 2: Salmonella Mutanegenicity Test Data
Table indicates the median numbers of revertant colonies per plate for
TA 100, together with the median deviation in brackets. The number of
spontaneous revertants (116 colonies per plate) has been subtracted.
5.8*10E07 cells were seeded per plate.
The mutagenic potential of resorcinol diglycidyl ether has been
demonstrated in Salmonella TA100 bacterial cells. There was
concentration-related increase over the range tested: 50, 100 and 200
µg/plate. At 500 µg/plate the test item showed already cytotoxicity.
There was no information on used or not used metabolic activation
system. In addition, there are no data on positive or negative control
used. According to the current OECD 471 guideline requirements there
only one (S. typhimurium TA100) out of five minimum required strains of
bacteria was used.
Table 1: In Vitro Chromosomal Aberrations in CHO Cells induced by
Resorcinol Diglycidyl Ether
* Metaphases with multiple aberrations
The mutagenic and toxicity potential of resorcinol diglycidyl ether has
been demonstrated in Chinese hamster CHO cells. The metabolic activation
was not specified.
Short-term tests that detect the induction of chromosomal effects in
cultured mammalian cells are an integral part of most testing schemes
proposed to identify potential chemical carcinogens acting through
genotoxic mechanisms. Cultured Chinese hamster ovary (CHO) cells are
widely used to test chemicals for their ability to induce chromosomal
aberrations (ABS) and sister chromatid exchanges (SCE).
Cell line and Culture Medium: CHO cells, up to 15 passages since
cloning, were used for all testing. Stock cells were obtained from
Litton Bionetics (Kensington, MD) and stored in liquid nitrogen. At
least once per year representative cells were sent to Flow Laboratories
(McLean, VA) for mycoplasma testing using the Hoechst stain test
followed by the Agar and Hyorhinis test. Results from all tests for
mycoplasma contamination were negative. CHO cells were maintained in
McCoy’s 5A medium (modified) supplemented with L-glutamine (2 mM),
antibiotics, and 10% fetal bovine serum (FBS). Premixed culture medium
and FBS were purchased from GIBCO Laboratories (Grand Island, NY). All
stock and experimental cultures were maintained at 37°C in an atmosphere
of 5% CO2 in air and 95% relative humidity.
Metabolic Activation: A liver fraction (S9) prepared from Aroclor
1254-induced male Sprague Dawley rats (Microbiological Associates,
Bethesda, MD) was used to provide exogenous metabolic activation. The
final concentrations of the S9 fraction, NADP, and isocitric acid were
0.02 mL, 2.4 mg and 4.5 mg, respectively, per milliliter culture medium.
Dose Selection: Ten or eleven dose levels, at half-log intervals
beginning at a high dose of 5 mg/ml (or as limited by solubility), were
used for the first trial of the SCE study. The dose levels for ABS
studies were chosen based on the toxicity of the test chemical observed
in the SCE studies.
Protocol for SCE Studies Approximately 24 h prior to cell treatment,
1x10^6 cells were seeded per 75 cm2 flask. A culture was established for
each dose both with and without metabolic activation. For assays without
metabolic activation, the medium was replaced with fresh medium
immediately before chemical treatment. Cells were treated with test or
control substances for 2 h to allow interaction with cells before the
addition of bromodeoxyuridine (BrdUrd). BrdUrd was then added (final
concentration 10 µM), and incubation was continued for an additional 24
h. The medium was removed, and fresh medium containing 10 µM BrdUrd and
colcemid was added and incubation was continued for 2-3 h. For assays
with metabolic activation, the cells were rinsed twice with phosphate
buffered saline (PBS), after which culture medium without FBS was added.
Cells were incubated for 2 h in the presence of the test or control
substances and the S9 reaction mixture. FBS was omitted to avoid the
binding of serum proteins to short-lived, highly reactive
intermediates.After the 2 h exposure period, cells were washed twice
with PBS, and then complete medium containing 10% FBS and 10 µM BrdUrd
was added. Cells were incubated for an additional 26 h, with colcemid
present for the final 2-3 h of incubation.
Two to three hours after addition of colcemid, cells were harvested by
mitotic shake-off. Prior to harvesting, the percent confluency in each
flask was estimated using a widefield microscope. Harvested cells were
treated for about 3 min at room temperature with hypotonic KCI (75 mM),
washed with fixative (3:1 methano1:glacial acetic acid, v/v), dropped
onto slides, and air dried. Staining for the detection of SCE was
accomplished by a modified fluorescence plus Giemsa (FPG) technique
[Goto et al., 19781]. Fifty seconddivision metaphase cells were scored
per dose for the incidence of SCE. The number of chromosomes in each
cell was also recorded. Any cell that had fewer than 19 or more than 23
chromosomes was excluded. All slides except for the high-dose positive
controls were coded.
Protocol for ABS Assay: Approximately 24 h prior to cell treatment, 1.2
x 10^6 cells were seeded per 75 cm2 flask. A culture was established for
each dose both with and without metabolic activation. For assays without
metabolic activation, the testing approach was similar to the
corresponding SCE studies except that cells were treated for about 10 hr
and BrdUrd was omitted. Colcemid was added 2-3 h prior to cell harvest
by mitotic shake-off.
The test protocol for assays with metabolic activation was also similar
to the corresponding SCE studies except that BrdUrd was omitted and
cells were harvested approximately 11 h after removal of the S9
fraction. Colcemid was added 2 h prior to harvest. Slides were stained
in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose
in early studies and 200 cells per dose in later studies. All slides
except high-dose positive controls were coded. Only metaphase cells in
which the chromosome number was between 19 and 23 were scored. The
chromosome number was recorded for each cell and chromosome or chromatid
type aberrations were classified into three categories: simple (breaks,
fragments, double minutes), complex (interchanges, rearrangements), and
other (pulverized, more than ten aberrations/cell).
Repeat Tests: Positive results in initial tests were confirmed by
additional tests. If both -S9 and +S9 studies gave a positive response
and required confirmation, they were done sequentially (-S9 first). If
the -S9 repeat was positive, the repeat +S9 study was not always
Cell Cycle Delay: The standard time for obtaining second-division
metaphase cells in SCE studies was 26 h after adding BrdUrd. For
chemicals that caused cell cycle delay, harvest times were extended,
generally in 5 h increments, with colcemid present for the last 2 h. For
ABS tests, harvest times were similarly extended based on the
observation of cell cycle delay in the SCE trials.
pH During Chemical Treatment: In instances when a change in the pH of
the culture medium was noticed after addition of the test chemical and
the overall response was negative, the test was considered sufficient.
If, however, the overall response was positive, the experiment was
repeated with the pH adjusted to 7.4.
Statistical Analysis: Statistical analyses were conducted on both the
slopes of the dose-response curves and the individual dose points. An
SCE frequency 20% above the concurrent solvent control value was chosen
as a statistically conservative positive response. The probability of
this level of difference occurring by chance at one dose point is cO.01;
the probability for such a chance occurrence at two dose points is
<0.001. Thus a trial with one dose showing an increase of 20% or greater
was considered weak evidence of a positive response, and a trial with
two doses showing an increase of 20% or greater was concluded to be
positive. For the ABS data, the percentage of cells with aberrations was
analyzed. As with SCE, both the dose-response curve and individual dose
points were statistically analyzed. A statistically significant (P <
0.003) trend test or a significantly elevated dose point (P < 0.05) was
sufficient to indicate a chemical effect. A detailed discussion of these
statistical methods is presented in Margolin et al. [ 19861, and their
application in determining test conclusions is further explained in
Galloway et al. .
Results: Treatment of cultured CHO cells with DGRE produced highly
significant increases in ABS and SCE both with and without S9. These
results agree with those presented by Seiler  showing DGRE to be
an extremely effective inducer of ABS in vitro: Treatment of CHO cells
with 25 µg/ml caused chromosome damage in 93% of the cells. In contrast,
oral administration of DGRE to mice in doses up to the LD50 did not
elevate the frequency of micronucleated erythrocytes in the bone marrow
in vivo mouse: cytogenicity / bonne marrow chromosome
Genetic toxicity tests in vivo in mouse were documented in two
publications (Seiler, 1984 and Shelby et al, 1993). The study of Seiler
(1984) has demonstrated that the test item was completely inactive in
vivo micronucleus assay in mice after single oral dosing at 300 and
600 mg/kg. In the paper of Shelby et al (1993) was noted that due to the
test item toxicity characteristics in a designed protocol was not
possible to use of a sufficiently high exposure to induce observable
genetic toxicity (the highest reported intraperitoneal IP dose 270
mg/kg). Since the paper is conducted not exactly according to guideline
(e.g. missing historical control), it is difficult to conclude on
reliability of outcome. However, based on in vivo micronucleus
results, which were inconsistent depending on the testing protocol, the
Dutch Expert Committee on Occupational Safety a Committee of the Health
Council of the Netherlands considered that resorcinol diglycidyl ether
may have genotoxic potential.
A weight of evidence of the analyzed results suggest that the test item
is negative for the genetic toxicity in vivo endpoint.
Table 1 INDUCTION OF MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES 1N THE
BONE MARROW OF MICE TREATED WITH RESORCINOL DIGLYCIDYL ETHER
Frequency (and standard deviation) of micronucleated erythrocytes (per thousand polychromatic erythrocytes) at dose (mg/kg p.o.) of
The spontaneous frequency of micronucleated polychromatic erythrocytes
(3-5 per thousand polychromatic erythrocytes, standard deviation 1.8)
has already been subtracted.
The micronucleus test was performed in male and female mice of the
ICR-strain. The test item resorcinol diglycidyl ether (RDGE) dissolved
in polyethylene-glycol (PEG 400) was given orally in doses up to acutely
toxic level. 24 h after the single dose (300 mg/kg po), the mice were
sacrificed and the bone marrow cells were flushed out into foetal clf
serum. In the case of negative outcome of the test, a second assay was
performed with fixation times of 24, 48 and 72 h, respectively. After
centrifugation at 400 g, the cells were spread onto slides, air-dried
and stained with May-Grünwald/Giemsa. The slides were coded and analysed
by two individuals separately. The polychromatic erythrocytes from the
bone marrow of treated animals were checked for the presence of
micronuclei. RDGE proved to be completely inactive. The inability of
RDGE to induce micronuclei in the bone marrow of mice in vivo could not
be traced to some influence on the length of the cell cycle, since the
compound was also inactive at 48 and 72 h after dosage, nor was the dose
given insufficient, since 1 out of 4 mice died within 48 h.
Groups of 5 male B6C3F1 mice were treated by intraperitoneal (IP)
injection to the 3 doses of test item at a volume of 0.4 mL per mouse on
three consecutive days. Animals were monitored twice daily and 24 h
after the third treatment. The surviving mice were euthanized by CO2
asphyxiation. Bone marrow smears (two slides/tissue/mouse) were prepared
by a direct technique (Tice et al, 1990). Air-dried smears were fixed
using absolute methanol and stained with acridine orange (Tice et al,
1990a). Bone marrow smears from each animal were evaluated at 1000 x
magnification using epi-illuminated fluorescence microscopy (450-490 nm
excitation, 520 nm emission) for determination of the percentage of PCE
among 200 erythrocytes.
For the initial MN test, groups of 5 mice were injected IP on three
consecutive days with either the test item (at 1x, ½ x and 1/ x ,where x
is the maximum dose determined in the dose determination experiments), a
weakly active dose of the positive control chemical (DMBA in corn oil)
or the appropriate solvent. Mice were euthanized with CO2, 24 hr after
the third treatment. Bone marrow smears (two slides mouse) were
prepared, fixed in absolute methanol and stained with acridine orange.
For each animal, slides were evaluated at 1,000 x magnification for the
number of MN-PCE among 2,000 PCE and for the percentage of PCE among 200
Repeat tests were performed for the test item based on results of the
initial micronucleus test since the results suggested a possible effect.
The data were analyzed using the Micronucleus Assay Data Management and
Statistical software package (version 1.3), which was designed
specifically for in vivo micronucleus data.
The level of significance was set at an alpha level of 0.05. To
determine whether a specific treatment resulted in a significant
increase in MN-PCE. The number of MN-PCE were pooled within each dose
and analyzed by a one-tailed trend test. In the software package used,
the trend test incorporates a variance inflation factor to account for
excess animal variability. In the event that the increase in the dose
response curve is nonmonotonic, the software program allows for the data
to be analyzed for a significant positive trend after data at the
highest dose only has been excluded. However, in this event, the alpha
level is adjusted to 0.01 to protect against false positives. The %PCE
data were analyzed by an analysis of variance (ANOVA) test based on
pooled data. Pairwise comparisons between each group and the concurrent
solvent control group was by an unadjusted one-tailed Pearson chi
squared test which incorporated the calculated variance inflation factor
for the study.
The initial test was positive to 60.8 mg/kg with trend P = 0.038. Repeal
tests to 60.8 mg/kg and 91.2 mg/kg were both negative and the overall
result was concluded to be negative. Because authors knew this chemical
was highly effective at inducing chromosomal aberrations in mouse bone
marrow cells following single exposures to doses up to 300 mg/kg (McFee
personal communication; Tice, unpublished), a single-exposure
micronucleus test was conducted. Bone marrow cells were harvested 24 h
after treatment. A significant dose-related increase in MN-PCE was
observed at doses of 90, 180 and 270 mg/kg (Table 2). It appears that
due to the toxicity characteristics of DGRE, a three-exposure protocol
does not permit use of a sufficiently high exposure to induce observable
With the test item, the range of daily doses used in the three-exposure
protocol (15.2 to 91.2 mg/kg) was also lower than the range of doses
used in the single exposure chromosome aberration studies (62.5 to 400
mg/kg). In a single exposure MN test, positive results were obtained
using doses up to 270 mg/kg, indicating that the cumulative toxicity of
multiple exposures restricted the daily doses to a level that did not
induce a detectable increase in MN. The 91.5 mg/kg/day exposure added up
to approximately 270 mg/kg, but there was apparently no substantial
induction of MN, perhaps due to effective detoxification of the low
exposures of efficient repair of induced damage during the intervals
Resorcinol diglycidyl ether showed some mutagenic potential in vivo
and was mutagenic in vitro, therefore the mode of action may be
described by a stochastic genotoxic mechanism.
The conclusion of the results is supported by the report: Health Council
of the Netherlands. Resorcinol diglycidyl ether. Evaluation of the
carcinogenicity and genotoxicity. Subcommittee on the Classification of
Carcinogenic Substances of the Dutch Expert Committee on Occupational
Safety a Committee of the Health Council of the Netherlands (2016). No.
Based on the available data, it is assumed that the substance acts by a
stochastic genotoxic mechanism and may be classified as a germ cell
mutagen category 2, H341 Suspected of causing genetic defects (state
route of exposure if it is conclusively proven that no other routes of
exposure cause the hazard) in accordance with Regulation (EC) No.
1272/2008 and its amendments.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
Welcome to the ECHA website. This site is not fully supported in Internet Explorer 7 (and earlier versions). Please upgrade your Internet Explorer to a newer version.
Do not show this message again