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EC number: 947-655-7
CAS number: -
report: Validation of an analytical method for the determination of Butanedioic
acid, 2 (or 3)-sulfo-, 4-[2-[(1-oxo-(C12 -C18 (even numbered) and C18
unsaturated)alkyl))amino]ethyl]esters disodium salts in test item formulations
HPLC-UV detection. LPT Study No. 36867.
aim of this study was to validate a HPLC-UV method for the
quantification of Butanedioic
acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18 (even numbered) and C18unsaturated)alkyl))amino]ethyl]esters,
disodium salts (also referred to as 'act.ingr.')
in tap water formulations.
analytical method applied was validated by LPT with regard to the
linearity of the
calibration curve as well as accuracy, precision, stability, specificity
The validation results are summarised in the table below.
1. Summary of validation results
Method for the determination of Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12- C18 (even numbered) and C18 unsaturated)alkyl))amino]ethyl]esters, disodium salts in test item formulation samples.
R2 = 0.9999 (concentration range: 100 to 1000μg/mL)
Inaccuracy = 0.64% (mean)
Imprecision ≤ 1.04% CV intra-day
Imprecision = 0.68% CV inter-day
No apparent degradation of processed samples after 24h storage in the autosampler.
No apparent degradation in tap water after 42 days of storage at -20°C.
Lower Limit of Quantification (LLOQ) = 24.55μg/mL
Limit of Detection (LOD) = 8.10μg/mL
No peak interferences at the retention time of Butanedioic acid, 2(or 3)-sulfo-, 4-[2 -[(1-oxo(C12-C18 (even numbered) and C18 unsaturated)alkyl))amino]ethyl]esters, disodium salts in formulation samples at the applied concentrations.
results of the validation confirm that the method employed is suitable
for the determination and quantification of Butanedioic acid, 2(or
3)-sulfo-, 4-[2-[(1-oxo(C12-C18 (even numbered) and C18
unsaturated)alkyl))amino]ethyl]esters, disodium salts in tap water
aim of the study was to obtain information on the toxicity of
Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18 (even numbered)
and C18 unsaturated)alkyl)) amino]ethyl]esters, disodium salts (= 'act.
ingr.') at dose levels of 100, 300, or 1000 mg/kg bw given to rats by
daily oral administration via gavage for 90 days, and the reversibility
of any effects after a 4-week treatment-free recovery period.
deaths were noted at any dose level. All animals survived until their
of the male and female rats treated orally with 100, 300, or 1000 mg
act. ingr./kg bw/day revealed any test item-related changes in
behaviour, external appearance, consistency of faeces, body posture, and
movement and coordination capabilities
test item-related effects were noted during the observational screening
and the functional tests.
mean body weight of the male and female animals treated with 1000 mg
act. ingr./kg bw/day was slightly reduced by up to 10.7% in females and
up to 7.4% in males in comparison to the control group.
body weight gain and the body weight at autopsy of both sexes were
reduced accordingly. This effect is considered to be test item-related.
toxicologically relevant test item-related influence was noted on
absolute and relative food consumption
with 300 mg act. ingr./kg bw/day led to an increase of the drinking
water consumption of the female animals by up to 25.9% in comparison to
the control group starting in test week 1. No effect was noted for the
corresponding male animals. At the high dose of 1000 mg act. ingr./kg
bw/day, both the male and the female animals revealed an increase of the
drinking water consumption by up to 64.0% in comparison to the control
group starting in test week 1. The increased drinking water consumption
is considered a test item-related effect.
test item-related changes were noted in haematological and coagulation
test item-related changes were noted in clinical chemistry.
test item-related or toxicologically relevant influence was noted on the
serum levels of triiodothyronine (T3), thyroxine (T4), and
thyroid-stimulating hormone (TSH).
test item-related changes were noted in urinalysis.
test item-related changes were noted in ophtalmological and auditory
necropsy, one of 10 female animals treated with 1000 mg act. ingr./kg
bw/day revealed test item-related changes in form of a thickened mucosa
in the cardia region of the stomach with multiple elevated foci with a
diameter of 1 to 3 mm that were partly crater-like. Histopathological
correlates were noted at microscopic examination.
test item-related changes were noted in organ weights.
examination restricted to control and high dose group, revealed test
item-related local changes in the stomach (non-glandular) of almost all
animals treated with 1000 mg act. ingr./kg bw/day in form of squamous
cell hyperplasia and hyperkeratosis (10 of 10 males, 9 of 10 females)
and submucosal mixed inflammatory cell infiltrate (4 of 10 animals per
stomach examination of low and intermediate dose groups revealed no
local changes in the stomachs at 100 and 300 mg act. ingr./kg bw/day.
of the animals previously treated with 1000 mg act. ingr./kg bw/day
revealed any abnormalities related to the previous treatment during the
4-week recovery period.
body weight of the male and female animals recovered towards the normal
range, no noteworthy difference compared to the control group was noted
at the end of the recovery period.
drinking water intake of all previously high-dosed animals was in the
normal range during the recovery period.
histomorphological examination did not reveal any findings related to
the previous test item-treatment, in particular no abnormalities were
noted in the stomach.
consideration of the observations described above, in particular the
reduced body weight and the substantially increased water consumption,
the experimental NOAEL was 300 mg act. ingr./kg bw by daily oral
of the animals previously treated with 1000 mg/kg bw/day revealed any
abnormalities related to the previous treatment during the recovery
period, thus all toxicological relevant changes observed at the end of
the treatment period were completely reversible within the 4 -week
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