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EC number: 267-956-0
CAS number: 67953-76-8
There were no data available for HEDP potassium salts. Therefore, data
were read-across from HEDP sodium salts and HEDP acid. For HEDP sodium
salts, information is available from in vitro (two Ames tests (Henkel,
1979; P&G, 1976), one mouse lymphoma assay (Wollny, 2010), one
micronucleus test (Envigo, 2015)) and in vivo tests (micronucleus test
(Henkel, 1979), dominant-lethal-assay (P&G 1974)). None of the studies
gave any indication of genotoxic potential. A combined chronic toxicity
/ carcinogenicity study performed with the disodium salt of HEDP gave no
rise to concern regarding genotoxic / carcinogenic effects.
Gene mutation (Bacterial reverse mutation assay / Ames test): negative
with and without activation in all strains tested (similar to OECD TG
Cytogenicity in mammalian cells: not required: in vivo cytogenicity
result is available
Mutagenicity in mammalian cells: negative with and without activation in
L5178Y mouse lymphoma cells (OECD TG 476).
The test item SAT 140001 was assessed for
its potential to induce gene mutations according to the plate
incorporation test (experiment I) and the pre-incubation test
(experiment II) using Salmonella lyphimurium strain TA 102. The assay
was performed in two independent experiments both with and without liver
microsomal activation. Each concentration and the controls, were tested
The test item was tested at the following
concentrations in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and
No precipitation of the test item occurred
up to the highest investigated dose.
The plates incubated with the test item
showed normal background growth up to 5000 µg/plate with and without S9
No toxic effects, evident as a reduction in
the number of revertants (below the indication factor of 0.5), occurred
in the test groups with and without metabolic activation.
No substantial increase in revertant colony
numbers was observed following treatment with SAT 140001 in strain TA
102 at any concentration level, neither in the presence nor absence of
metabolic activation (S9 mix). There was also no tendency of higher
mutation rates with increasing concentrations in the range below the
generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as
positive controls. They showed a distinct increase in induced revertant
SUMMARY EXPERIMENT I
SUMMARY EXPERIMENT II
This study was performed to investigate the
potential of SAT140001 to induce gene mutations according to the plate
incorporation test (experiment I) and the pre-incubation test
(experiment II) using the Salmonella typhimurium strain TA 102. The
assay was performed with and without liver microsomal activation.
Each concentration, including the controls,
was tested in triplicate. The test item was tested at the following
concentrations in both experiments:
3; 10; 33; 100; 333; 1000; 2500; and 5000
No substantial increase in revertant colony
numbers was observed following treatment with SAT140001 in strain TA 102
at any dose level, neither in the presence nor absence of metabolic
activation (S9 mix). There was also no tendency of higher mutation rates
with increasing concentrations in the range below the generally
acknowledged border of biological relevance.
Appropriate reference mutagens were used as
positive controls and showed a distinct increase of induced revertant
In both experiments no precipitation of the
test item in the culture medium was observed.
No relevant influence on osmolarity was
observed. The pH was adjusted to physiological values.
In Experiment I in the absence and presence
of S9 mix and in Experiment II in the absence of S9 mix, no cytotoxicity
was observed up to the highest applied concentration.
No relevant increase in micronucleated cells
was observed after treatment with the test item. However, in Experiment
I in the presence of S9 mix, at a concentration of 691.1 μg/mL a
statistically significant increase in the number of micronucleated cells
(0.75 %) was observed after treatment with the test item. The value lies
within the range of the laboratory historical control data (0.15 – 1.70
% micronucleated cells) and can be estimated as biologically irrelevant.
In both experiments, either Demecolcin
(100.0 ng/mL), MMC (2.0 μg/mL) or CPA (17.5 μg/mL) were used as positive
controls and showed distinct increases in cells with micronuclei.
SC = Solvent control
PC = Positive control
CBPI = Proliferation index
nc = not calculated (CBPI equal or higher
than solvent control value)
The test item SAT 140001, dissolved in
deionised water, was assessed for its potential to induce micronuclei
in human lymphocytes in vitro in two independent experiments. The
following study design was performed:
Cytochalasin B exposure
Total culture period
In each experimental group two parallel
cultures were analysed. Per culture 1000 binucleated cells were
evaluated for cytogenetic damage.
The highest applied concentration in this
study (2116.4 μg/mL of the test item) was chosen with regard to the
purity (94.5%) of the test item and with respect to the current OECD
Guideline 487. Dose selection of the cytogenetic experiment was
performed considering the toxicity data in accordance with OECD
In Experiment I in the absence and
presence of S9 mix and in Experiment II in the absence of S9 mix, no
cytotoxicity was observed up to the highest applied concentration.
No relevant increase in micronucleated
cells was observed after treatment with the test item. However, in
Experiment I in the presence of S9 mix, at a concentration of 691.1
μg/mL a statistically significant increase in the number of
micronucleated cells (0.75 %) was observed after treatment with the
test item. The value lies within the range of the laboratory
historical control data (0.15 – 1.70 % micronucleated cells) and can
be estimated as biologically irrelevant.
Appropriate mutagens were used as positive
controls. They induced statistically significant increases in cells
In conclusion, it can be stated that under
the experimental conditions reported, the test item did not induce
micronuclei as determined by the in vitro micronucleus test in human
lymphocytes. Therefore, SAT 140001 is considered to be non-mutagenic
in this in vitro micronucleus test, when tested up to the highest
required or evaluable concentrations.
Table 1a Plate incorporation assay. Number of revertants per plate (mean
of 3 plates)
* Negative control - exact nature of this control not specified in study
Table 1b Plate incorporation assay. Number of revertants per plate (mean
of 3 plates)
The positive control for TA1538 in the presence of metabolic activation
only produced 2x the number of colonies as the negative control, and
therefore was not considered mutagenic based on it not producing the
The test substance with TA98 in the presence and absence of metabolic
activation gave uneven results at 10 mg: e.g., 75, 29, and 24 colonies
per plate. The complete assay with TA98 was repeated.
Etidronic acid disodium salt was tested for mutagenic potential in the
Salmonella/microsomal assay in the presence and absence of metabolic
activation using tester strains TA1535, TA1537, TA1538, TA98, and TA100.
Cultures were treated for 48 hours using the plate incorporation method
at dose levels ranging from 1.0 -20 mg/plate. Negative controls were
included for all strains, while positive controls were included for
TA1535, TA100 and TA1537 in the absence of metabolic activation and for
TA100, TA1538 and TA98 in the presence of metabolic activation.
All negative control values were within historical range. The positive
control for TA1538 in the presence of metabolic activation did not
induce a mutagenic response. The test substance was negative for
mutagenicity in both the presence and absence of metabolic activation.
number of mutant colonies per 106cells of each solvent
control plus 126
was not continued since a minimum of four concentrations is required by
The study was performed to investigate
the potential of HEDP x-sodium salt (neutral pH) to induce mutations at
the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The assay was performed in two
independent experiments, using two parallel cultures each. The first
main experiment was performed with and without liver microsomal
activation and a treatment period of 4 h. The second experiment was
performed with a treatment period of 24 hours in the absence of
metabolic activation and 4 hours in the presence of metabolic
activation. An additional experimental part was added to the second
experiment with 4h treatment without metabolic activation to adopt the
experimental part of 4 hour treatment without metabolic activation to
the modified molecular weight of 228 g/mol. Only one concentration
(21000 µg/ml equal to about 10 mM) was tested in the additional
The maximum concentration level of the
test item should be 10 mM (corresponding to 18.4 (based on a molecular
weight of 206 g/mol) or 21.0 mg/ml (based on a molecular weight of 263.2
g/mol) of the active ingredient) unless limited by the cytotoxicity of
the test item. Thus the maximum concentration in the pre-experiment and
in the first experiment was 18.4 mg/ml of the test item as supplied
based on a purity of 12.4% and a molecular weight of 228 g/mol. On
request of the sponsor the molecular weight of the test item was
corrected to263.2 g/mol to reflect the true conditions at neutral
pH-values. The maximum concentration in the second experiment was
consequently increased to 21.0 mg/ml in the experimental parts using 4
hours treatment with and without metabolic activation. Following 24
hours treatment without metabolic activation the concentration range was
limited by cytotoxic effects.
No relevant toxic effects indicated by
a relative total growth of less than 50 % of survival in both parallel
cultures were observed up to the maximum concentration with and without
metabolic activation, following 4 hours of treatment. In experiment II
following 24 hours treatment without metabolic activation cytotoxic
effects as described above occurred at 3450 and 4600 µg/mL. Based on the
steep cytotoxic gradient of the test item the relative total growth
(RTG) of both parallel cultures fell short of the lower limit of
approximately 10%. However, according to the current IWGT
recommendations data at RTG levels between 1 and 10% can be used to
support a non mutagenic result provided that the dose spacing was 2.0 or
lower and no relevant increase of the mutation frequency is observed at
RTG levels below 10%.
substantial and reproducible dose dependent increase of the mutation
frequency was observed in both main experiments up to the maximum
concentration with and without metabolic activation. The threshold of
126 plus each solvent control count and the historical range of solvent
controls were exceeded in culture I of the first experiment at the
lowest concentration of 1150 µg/mL without metabolic activation and in
the second culture of the second experiment at 21000 µg/mL, 4 hours
treatment without metabolic activation. However, no comparable increase
was noted in the parallel cultures under identical conditions.
Therefore, the isolated increases described above are judged as
irrelevant fluctuations. In experiment I the range of the historical
solvent control data was exceeded at 4600 µg/mL at culture I without
metabolic activation. Again, this effect was not reproduced in the
parallel culture performed under identical experimental conditions and
therefore, considered irrelevant.
A linear regression analysis (least
squares) was performed to assess a possible dose dependent increase of
mutant frequencies using SYSTATâ11statistics
software. A significant dose dependent trend of the mutation frequency
indicated by a probability value of <0.05 was detected in the first
culture of the second experiment without metabolic activation and in the
second culture of the second experiment with metabolic activation. The
trend noted in the second experiment without metabolic activation was
judged as irrelevant since it actually was reciprocal, going down versus
increasing concentrations. Even the trend noted in the second experiment
with metabolic activation was judged as irrelevant since the actual
levels of the mutation frequency remained within the historical range of
solvent controls and the threshold was not exceeded.
In this study the range of the solvent
controls was from 95 up to 189 mutant colonies per 106cells;
the range of the groups treated with the test item was from 92 up to 298
mutant colonies per 106cells. The highest solvent control
values (189 and 173 colonies per 106cells) exceeded the
recommended 50 – 170 x 106control
range as stated under paragraph 8.12, acceptability of the
assay, of this report. The mean values of both parallel cultures
however, (157 and 156.5 colonies per 106cells) are fully
acceptable. The cloning efficiency exceeded the upper limit of the
acceptance criteria somewhat in the second culture of the second
experiment without metabolic activation. The data are acceptable
however, since the cloning efficiency of the parallel culture remained
within the acceptable range.
MMS (19.5 µg/ml following 4 hours
treatment and 13.0 µg/mL following 24 hours treatment) and CPA (3.0 and
4.5 µg/mL) were used as positive controls and showed a distinct increase
in induced total mutant colonies at acceptable levels of toxicity with
at least one of the concentrations of the controls.
Micronucleus assay in mouse (intraperitoneal administration): negative
(similar to OECD TG 474)
Total litter losses occurred very rarely throughout the study and they showed no relationship to dosage of the test substance. Only two litters approached being totally lost in all test groups. One incident occurred in the untreated controls in which two to three implants in one female were dead and another occurred in the high dose group in which only one of five implants was alive at necropsy. Both litter losses originated during the third week of mating and were attributed to a malfunction in the individual animals and therefore were not considered test-related.
The number of deaths in females mated with males given the test substance did not differ significantly from those in concurrent controls for any of the mating periods.
Mating performance of all test groups compared favorably with the controls. As in the examination of individual female data, individual poor performance of the sire which was not test related could easily be detected. For example one male in the mid test group failed to impregnate a female until the fifth week of mating. In the final two weeks of the study, three of the four females mated with this male mouse conceived but only carried an average of six implants per pregnancy. The average number of implants for the rest of the group was 8.9 for the entire study and 9.0 for all other sires combined during the same two weeks of mating. The fact that eighteen implants sired by the male mouse mentioned above produced no fetal deaths provides additional evidence supporting the decision that the performance of this male was not test related.
When it came to conception rates, implants and fetal mortality, little variation occurred between test groups and controls for any of the parameters measured.
The mutagenic index was measured. It was a calculation based on two independent variables, fetal deaths and total implants, used as a convenient comparison of group to group performance. No significant differences (P= 0.05. D.F=1) were found when test group mutagenic indices were compared to either the vehicle or untreated controls.
Phosphonic acid, P,'-(1 -hydroxyethylidene)bis-,sodium salt was administered orally (0.25 ml volumes) to male mice of the C3D2F1/J strain at three dose levels (1000, 200 and 20 mg/kg) for five consecutive days. The study also included a vehicle control (water) and an untreated group. 20 mice were assigned to each group.
Immediately following treatment, each male was caged with two untreated females for a period seven days and with two fresh females the following week. This procedure was continued for a total of six weeks, thereby encompassing the entire spermatogenic cycle of the mouse, which was purported to be 35 days.
Consequently, each male was mated to twelve females over a six-week period: A total of 240 matings per dose group
On day 13 or 14 of gestation (as measured from the mid-week of presumptive mating), the females were sacrificed. Total implants, resorptions and dead embryos were enumerated and recorded.
Various parameters such as average number of implants per female, average number of fetal deaths/female, fetal deaths per sire, and percent pregnancy per group were subjected to statistical analysis.
The test substance produced data that were not significantly different from control values in conception rates, total implant averages, fetal death averages, resorption percentage and mutagenic indices.
Therefore, the test substance was considered to be non-mutagenic when administered orally at maximum tolerated doses in the test system employed.
1: Mean Results of in vivo
micronucleus test with mouse bone marrow
Positive Control (Endoxan)
Sampling time (h)
Number of cells analysed
Micronucleated cells per animal %
For the induction of genotoxicity, two principle mechanistic actions can
be distinguished: the potential to induce either mainly gene- or
chromosome mutations. For the conclusion on the mutagenic potential of a
substance, both modes of action have to be investigated with suitable
Concerning the induction of gene mutations, two Ames tests (Klimisch 2)
(for HEDP-3Na salt and HEDP-H acid) (Harlan 2015; Henkel 1979) and one
mouse lymphoma study (Klimisch 1) (for HEDP-2Na salt) (Wollny, 2010) are
available. The Ames tests followed the principles of the OECD TG 471,
but were conducted in the 1970s, where GLP principles have not yet been
established. Furthermore, they include neither the Salmonella
typhimurium tester strain TA 102 nor an equivalent Escherichia coli
strain to evaluate the induction of DNA crosslinking. Therefore, another
Ames test according to OECD TG 471 and GLP principles was conducted with
the strain TA 102 to address this specific question. No indication for a
genotoxic potential was observed in this test either. The hypothesis
that HEDP salts or acid do not induce gene mutations is not only
supported by the two already available Ames tests, but also by a fully
OECD- and GLP-compliant mouse lymphoma assay conducted in 2010, that
gave no indication for mutagenicity.
With regard to the induction of chromosome aberrations, information from
two in vitro studies (mouse lymphoma assay, Klimisch 1 (Wollny, 2010);
micronucleus test, Klimisch 1 (Envigo, 2015)) and two in vivo tests
(dominant-lethal-assay, Klimisch 2 (P&G, 1974); micronucleus test,
Klimisch 4 (Henkel, 1979)) are available.
Though the main focus of the mouse lymphoma assay lies on the
identification of gene mutations, it is also suited to detect chromosome
aberrations. According to the OECD TG 476, these could be differentiated
from the gene mutations by their smaller colony size. As no
dose-related, reproducible increase in the mutant frequency occurred at
all in the mouse lymphoma test with HEDP-2Na, it can be concluded that
the substance did neither induce gene-, nor chromosome mutations under
the conditions of the test (Wollny, 2010).
The potential to induce micronuclei was investigated in an in vitro
study with human lymphocytes following OECD TG 487 (Envigo, 2015). In
two independent experiments, the substance did not induce a relevant
increase of micronucleated cells in the absence (exposure period: 4 and
20 h) and presence of S9 mix (exposure period: 4 h). The only observed
significant induction of micronuclei occurred at the lowest
concentration in experiment I with S9 mix, but the value was within the
historical control and thus considered as biologically irrelevant.
Neither precipitation nor cytotoxicity were detected up to the highest
The in vivo micronucleus test included several deviations from current
OECD standards (2 instead of 3 doses; highest dose tested lower than
MTD; 4 instead of 5 animals per dose; bone marrow samples collected
after 6 h, not after 18-24 h). Though the sampling time was shorter than
recommended in the guideline, the positive control induced a clear
effect (Henkel, 1979).
Another relevant in vivo study for the evaluation of chromosome
mutations is the dominant-lethal-assay (P&G, 1974). The study reviews
the chromosomal aberration potential in germ cells of male mice by
observation of pre-and post-implantation losses. The formation of
chromosomal alterations in germ cells is basically the same as that for
somatic cells (e.g., deletions, inversions, translocations). As stated
in the OECD TG 478, dominant lethal are generally accepted to be the
result of chromosomal aberrations (structural and numerical anomalies),
although intrauterine deaths may also be caused by gene mutations and
reproduction toxic effects. Though the study was conducted prior to the
adoption of the respective OECD and GLP guideline, it met the principles
laid down in the current OECD TG 478. However, a concurrent positive
control was not included in the assay, but might according to the OECD
guideline be omitted if positive control data are available from another
dominant-lethal-assay in the same laboratory within the last 12 months.
In the dominant-lethal assay, mice were dosed by gavage up to 1000 mg of
HEDP-2Na/kg bw/d for 5 days. No statistically significant chromosomal
aberrations were observed in any of the treatments. Under the conditions
of the dominant-lethal-assay, HEDP-2Na did not induce cytogenic effects.
Taking into consideration the lack of effects seen in the two
micronucleus assays and the in vitro mouse lymphoma test, it can be
concluded that sodium salts or acid of HEDP are not suspected to induce
cytogenetic effects in vitro or in vivo. This is supported by negative
results in the carcinogenicity study and the dominant lethal assay,
In conclusion, no potential to induce gene or chromosome mutations was
observed in any of the in vitro- and in vivo genetic toxicity assays or
the carcinogenicity study.
Justification for grouping:
See CSR Annex I or IUCLID section 13.
The available information on read across substances indicates that, when
tested in vitro, HEDP potassium salt does not cause mutagenicity in
mammalian cells; this is supported by evidence for lack of bacterial
mutagenicity from disodium HEDP. In addition, no evidence for
cytogenicity was shown when disodium HEDP was tested in vitro or in
vivo. Therefore it is concluded that classification for mutagenicity is
not required for potassium salts of HEDP according to Regulation (EC) No
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