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: 905-898-6
CAS number: -
For DEA CAS 111-42-2, an extended One-Generation Reproductive
Toxicity Study (EOGRTS) according to OECD TG 443 has been performed and
finalized on January 29th2018 (Ethanolamine Reach
Consortium). Based on the final decision of the substance evaluation
under CORAP cohorts 2A and B as well as cohort 3 were included for an
investigation of the developmental neurotoxicity module (DNT) and the
developmental immunotoxicity module (DIT), but without the extension to
an F2-generation. Additionally, measurements of the essential nutrient
choline were performed in different tissues of the F0 and F1 pups
(plasma and liver, respectively). Furthermore, additional investigation
of parameters such as platelet-activating factor (PAF) have been
included as another modification to follow-up on a mode-of-action.
2,2’-iminodiethanol was administered to groups of 30 male and 30
female healthy young Wistar rats (F0 parental generation) as a solution
to the drinking water in different concentrations (0, 100, 300 and 1000
ppm). At least 16 days after the beginning of treatment, F0 animals were
mated to produce a litter (F1 generation). Mating pairs were from the
same dose group. Pups of the F1 litter were selected (F1 rearing
animals) and assigned to 5 different cohorts which were continued in
dose groups 10 - 13 in the same fashion as their parents and which were
subjected to specific post weaning examinations. The study terminated
with the terminal sacrifice of the male and female animals of cohort 1B.
Test drinking water containing 2,2’-iminodiethanol were offered
continuously throughout the study.
Intake of test substance: the overall mean dose of
2,2’-iminodiethanol throughout all study phase and across all cohorts
was approx. 12.75 mg/kg body weight/day (mg/kg bw/d) in the 100 ppm
group, approx. 37.68 mg/kg bw/d in the 300 ppm group and approx. 128.35
mg/kg bw/d in the 1000 ppm group.
Under the conditions of the present modified extended 1-generation
reproduction toxicity study the NOAEL (no observed adverse effect level)
for general toxicity is 100 ppm for the F0 parental animals, based on
evidence for distinct kidney toxicity and stomach irritation, as well as
corresponding effects on water consumption, food consumption, body
weights and clinicopathological parameters, which were observed at the
LOAEL (Lowest Observed Adverse Effect Level) of 300 ppm. Similar
toxicity was noted in the adolescent F1 animals, which had no stomach
irritation but liver toxicity in addition.
The NOAEL for fertility and reproductive performance for the F0
and F1 rats is 300 ppm, based on a lower number of implants,
prolonged/irregular estrous cycles as well as pathological changes in
sexual organs, pituitary and mammary glands of both genders at the LOAEL
(Lowest Observed Adverse Effect Level) of 1000 ppm. Most of the reported
effects on reproduction and reproductive organs occurred in the range of
general and systemic toxicity and have been assessed to be secondary in
nature. Please compare with the section “Justification for
classification or non classification” for a detailed discussion.
However, eosinophilic cysts in the pituitary gland were present in the
F1 animals of cohort 1A down to the 100 ppm dose level, but no
assessment on adversity of this finding is possible at present.
Therefore, no NOEL can be established for this particular effect.
The increased mean dose-group T4 values observed in some of the
dose groups were within historical control data and there were no
significant changes of TSH observed in any dose groups.
A dose dependent statistically significant choline depletion was
observed in plasma and liver tissue of both male and female F1B animals
starting already from the lowest dose tested.
The NOAEL for developmental toxicity in the F1 progeny is 100 ppm,
based on impaired pup survival at 1000 ppm as well as reduced pup body
weights in the F1 offspring, which were observed at the LOAEL (Lowest
Observed Adverse Effect Level) of 300 ppm. As these weight reductions
were only observed in the presence of maternal toxicity, including lower
weight gain during pregnancy, they are not regarded as independent
effect of the treatment.
The NOAEL for developmental neurotoxicity for the F1 progeny is
300 ppm, based on adverse clinical observations, impaired auditory
startle response and corresponding neuropathological findings at the
LOAEL (Lowest Observed Adverse Effect Level) of 1000 ppm.
The NOAEL for developmental immunotoxicity for the F1 progeny is
300 ppm, based on effects on the T-helper cells and cytotoxic T-cells in
the spleen in the F1 females at the LOAEL (Lowest Observed Adverse
Effect Level) of 1000 ppm. Lower mean and median anti-SRBC IgM antibody
titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide,
oral) demonstrated that the test system worked properly.
In addition to the EOGRTS / OECD 443 that is available for DEA,
there is a 3-month inhalation study (BASF AG, 2002) in rats reporting an
influence on the male reproductive system at the high concentration. The
NOAEC for male fertility parameters was 0.15 mg/l. When DEA was orally
administered to rats via the drinking water for 13 weeks, decreases in
testis and epididymis weights, testicular degeneration, atrophy of the
seminal vesicles and prostate glands and associated effects on
spermatology were observed. The NOAEL for fertility effects in males was
48 mg/kg bw. In all of these studies no histopathological effects were
observed in female reproductive organs.
Other data on Diethanolamine (DEA)
The US National Toxicology Program conducted a prenatal
developmental toxicity study by gavage administration of DEA to groups
of 12 pregnant CD rats at doses of 0, 50, 125, 200, 250, or
300 mg/kg bw/d during GD 6–19 (Price et al., 2005). The test substance
was administered in water, and the pH of the dosing solutions was
adjusted to 7.4±0.2 with hydrochloric acid. Dams were allowed to litter
and raise their offspring to PND 21.
Dams in the high dose group showed signs of excessive toxicity and
were euthanised by GD 15; the following discussion omits further mention
of this group. The principal toxicity noted in the remaining dams was of
a dose-related reduction in bodyweight (gain) during gestation compared
to the control group. This was most noticeable at 250 mg/kg BW/d, in
which animals had lost weight by GD 12 and didn’t start gaining weight
until GD 15, after which weight gain maintained parity with the control.
Animals in the 200 mg/kg bw/d group lost weight by GD 12, but weight
gain maintained parity with the control thereafter. There were no
differences in bodyweight (gain) between control and the other treatment
There was a statistically significant increase in
post-implantation loss in the groups that were administered 200 or
250 mg/kg bw/d, which in the higher dose group was also manifested as
total loss of four litters in dams that survived until term, one of
which consisted of all dead pups at PND 0 and the other three which
consisted only of implantation sites. It is not clear from the
publication as to whether the implantations were lost at an early or
later stage. Two other dams in this group were either found dead on
GD 15 or euthanised moribund on GD 21. Both had litters. One dam in the
200 mg/kg bw/d dose group was euthanised on GD 22 and had a litter of
dead foetuses. There was a statistically significant increase in
postnatal mortality during PND 0–4 in groups administered ≥125 mg
In general, Ethanolamines inhibit the uptake of [3H]-choline in
cultured CHO cells, with estimated EC50values of ~0.2 mM for DEA and
~1.1 mM for the structural analogue TEA (Lehmann-McKeeman and Gamsky,
1999; Stottet al., 2004).
For DEA various mechanistic in vitro and in vivo studies
identified that choline depletion is the key event in hepatic
carcinogenicity. DEA decreased gap junctional intracellular
communication in primary cultured mouse and rat hepatocytes; induced DNA
hypomethylation in mouse hepatocytes; decreased phosphatidylcholine
synthesis; and increased S-phase DNA synthesis in mouse hepatocytes, but
had no effect on apoptosis. All of these effects were mediated by the
inhibition of choline sequestration, and were prevented with choline
supplementation. No such effects were noted in human hepatocytes in
vitro. Apparent differences in the susceptibility of two different mice
strains (B6C3F1 > C57BL) were noted. B6C3F1 mice are extremely sensitive
to non-genotoxic effects and are susceptible to spontaneous liver
tumors. Moreover, chronic stimulation and compensatory adaptive changes
of hepatocyte hypertrophy and proliferation are able to enhance the
incidence of common spontaneous liver tumors in the mouse by mechanisms
not relevant to humans (adapted from the DEA OECD SIAR, 2009).
Effects of DEA on pre- and post-implantation may be mediated by
effects on choline homeostasis (as described above) rather than through
a direct embryo toxicity. These effects may be inhibition of
choline-uptake in the liver, subsequent perturbation of
choline-homeostasis, with subsequent impairment of C1-metabolism,
DNA-methylation, lipid metabolism, and intercellular
communication. Choline metabolism is connected to Phosphatidylcholine
and Betaine. The latter is reported to be central for the synthesis of
SAM (S-Adenosyl-Methionine), a principle methylating agent for
biosynthetic pathways and maintenance of critical gene methylation
patterns (Stott et al. 2004; Zeisel and Blusztajn, 1994).
It has been accepted for DEA tumorigenicity, that the effects
observed are caused by a non-genotoxic modulation of DNA-methylation.
Such effects may also explain the observed effects on implantation.
Significantly, this is important for the final evaluation of DEA as this
potential mode-of-action may display a species-specific effect with
humans being resistant towards choline-deficiency and its consequences
Recently, it could be demonstrated, that Monoethanolamine (MEA)
reduced implantation success in a two-generation reproduction toxicity
study (ACC and CEFIC, 2009). When administered to pregnant rats during
gestation days (GD) 1–3, 4–5, or 6–7, MEA had no effect upon
implantation success. In a second experiment, MEA was administered
either in the diet or by oral gavage from two weeks prior to mating
through to GD 8. Parallel groups also received a diet supplemented with
choline. In the absence of supplementary choline, MEA induced early
resorptions, statistically significant only when administered in the
diet. A slight reduction in implantation success was ameliorated by
supplementary choline. We conclude that implantation is affected by MEA
only when exposure starts before mating; that dietary administration is
more effective than gavage dosing; and that interference with choline
homeostasis may play a role in the etiology of this lesion (Moore et al
2018, Reprod. Tox. 78, 102 -110).
As supporting evidence, in the EOGRTS / OECD443 which is available
for DEA, a statistically significant decrease in the choline levels at
all doses was observed in F1B animals. The analytical results
demonstrated the presence of choline in all plasma samples from the
animals dosed with the test substance DEA (100 ppm, 300 ppm and 1000 ppm
dosed animals) and in those from control, non-dosed animals. In general,
it can be stated that the administration of 2,2’-iminodiethanol led to a
reduction in the content of choline in the plasma samples analyzed. The
mean plasma choline level decreased dose dependently and statistically
significant through the dose groups. Furthermore, also in the offspring
the analytical results demonstrated the clear presence of choline in all
liver samples from the animals dosed with the test substance DEA (100
ppm,300 ppm and 1000 ppm dosed animals) and in those from control,
non-dosed animals. This was true from all time points investigated
(4-day old pups, 22-day old descendants and ~90-day old adolescents). In
general, it can be stated that the presence of the test substance DEA
led to a reduction in the content of choline in the liver samples
analyzed. This effect appears to be dose-dependent, in that higher dose
levels were associated with greater choline reduction. At higher dosing
levels, no further dramatic liver choline content reduction was
observed, indicating that liver choline levels of all non-control
animals have reached an approximate minimum and distinct choline
depletion can be induced already at diet concentrations as low as 100
Lehman-McKeeman LD, Gamsky EA (1999). Diethanolamine inhibits
choline uptake and phosphatidylcholine synthesis in Chinese hamster
ovary cells. Biochem Biophys Res Commun262:600–604.
OECD SIDS (2009). Diethanolamine.
Smyth et al., (1951).Range-finding Toxicity Data: List
IV.Arch.Hyg. Occup. Med.4: 119 - 122
Stott WT, Kleinert KM (2008). Effect of diisopropanolamine upon
choline uptake and phospholipid synthesis in Chinese hamster ovary
cells. Fd Chem Toxicol46:761-766.
Stott WT, Radtke BJ, Linscombe VA, Mar M-H, Zeisel SH (2004).
Evaluation of the potential of triethanolamine to alter hepatic choline
levels in female B6C3F1 mice.Toxicol Sci79:242-247.
Zeisel SH and Blasztajn JK (1994). Cholin and human nutrition.
Ann. Rev. Nutr.14: 269-296
Based on the available developmental toxicity studies (via the inhalation, dermal and oral route of exposure) with rats and rabbits, DEA caused only developmental toxicity in the presence of clear maternal toxicity and at dose levels considered as high. Therefore no DNEL has to be derived based on developmental toxicity. Furthermore, maternal toxicity was observed at levels higher/comparable to general toxic effects in the repeated dose toxicity studies.
The exposure of
pregnant female Wistar rats to an aerosol of DEA in a head/nose exposure
systems for 6 h/day on day 6 through day 15 post coitum at
concentrations of 0; 0.01; 0.05; 0.2 mg/l (0; 10, 50, 200 mg/m³) led to
signs of maternal toxicity at the highest concentration (0.2 mg/l) (BASF
AG, 1993b, OECD TG 414 study).
Maternal toxicity was substantiated by adverse clinical symptoms
(vaginal haemorrhages) in 8 of the 21 pregnant rats on day 14 p.c. At
this dose level a markedly increased number of fetuses with skeletal
variations (mainly cervical rib(s)) were also recorded but
substance-related teratogenic effects were not detected in any foetus.
Thus, signs of prenatal developmental toxicity did only occur at a
maternal toxic concentration. There were no adverse effects on dams or
foetuses at the low or mid concentrations (0.01 or 0.05 mg/l). The NOAEC
for maternal and prenatal developmental toxicity was 0.05 mg/l (50
mg/m³), the NOAEC for teratogenicity was >0.2 mg/l (200 mg/m³).
DEA was administered
dermally to pregnant CD rats from gestation day 6 through day 15 at
doses of 0, 150, 500 and 1500 mg/kg bw/day (Marty et al, 1999, a
protocol equivalent/similar to OECD TG 414). At 500 and 1500 mg/kg
bw/day moderate and severe skin irritation was caused, respectively.
Maternal body weight gain was decreased in the 1500 mg/kg bw. Absolute
and relative kidney weights were increased at 500 and 1500 mg/kg bw/day.
Haematological effects including anaemia, abnormal red cell morphology
(poikilocytosis, anisocytosis, polychromasia), and decreased platelet
count were observed in all treatment groups. The 1500 mg/kg bw/day group
also had increased lymphocytes and total leukocytes. In the fetuses,
there were no effects of treatment on body weight or on incidence of
external, visceral, or skeletal malformations/abnormalities. Increased
incidences of six skeletal variations involving the axial skeleton and
distal appendages were observed in litters from the 1500 mg/kg bw/day
group. The skeletal variations included poor ossification in the
parietal bones, cervical centrum #5, and thoracic centrum #10, lack of
ossification in all proximal hindlimb phalanges and some forelimb
metacarpals, and callused ribs. Consequently, the LOAEL for maternal
toxicity was 150 mg/kg bw/day, while the NOAEL for prenatal
developmental toxicity was adjusted to 380 mg/kg bw due to dosing
discrepancy at the 500 mg/kg bw/day group. The NOAEL for teratogenicity
was >1500 mg/kg bw/day. Thus, signs of prenatal developmental toxicity
did only occur at clearly maternal toxic dose levels.
DEA was administered
dermally to pregnant New Zealand White rabbits from gestation day 6
through day 18 at doses of 0, 35, 100 and 350 mg/kg bw/day (Marty et al,
1999, a protocol equivalent/similar to OECD TG 414). Rabbit dams at 350
mg/kg bw/day showed several signs of marked skin irritation, reduced
food consumption, and colour changes in the kidneys but no
haematological changes. Body weight gain was reduced at 100 mg/kg
bw/day. There was no impairment of gestational parameters. No evidence
of developmental toxicity was observed at any dose level, especially,
there were no apparent effects of treatment on the incidences of
external, visceral, or skeletal abnormalities. Consequently, the NOAEL
for maternal toxicity was 35 mg/kg bw/day, the NOAEL for prenatal
developmental toxicity including teratogenicity was >350 mg/kg bw/day.
time-mated pregnant rats with DEA dose levels of 0, 50, 125, 200, 250,
300 mg/kg bw/day by oral gavage from gestation day 6-19 (Price, 1999)
led to maternal morbidity or mortality occurred at 200 and 250 mg/kg
bw/day and all females at 300 mg/kg bw/day had to be terminated early
due to excessive toxicity. Maternal water intake was transiently
affected during early gestation (125 and 250 mg/kg bw/day) but was
comparable to controls for all measurement periods after GD 12. Maternal
absolute kidney weight was increased on PND 21 (>= 125 mg/kg bw/day)
indicating persistence of DEA-induced toxicity for up to about 3 weeks
after cessation of exposure. Reduced maternal body weight and weight
change, as well as reduced feed intake, were noted at >= 200 mg/kg
bw/day. Exposure to 50 mg/kg bw/day was not associated with any
significant maternal toxicity during or after the treatment period.
Developmental toxicity was observed specifically in form of an increase
in post implantation mortality at >= 200 mg/kg bw/day on PND 0, and
early postnatal mortality (PND 0-4) was increased at >= 125 mg/kg
bw/day. Pup body weight was reduced at >= 200 mg/kg bw/day, with females
more affected than males. When expressed as a percentage of control
weight, pup body weight reductions were most pronounced during the early
postnatal period. Statistically significant differences were also
evident at the end of the lactation period. Consequently, the NOAEL for
maternal and developmental toxicity was 50 mg/kg bw/day. Signs of
developmental toxicity did only occur at maternal toxic dose levels.
For details cf. "Justification for classification or non-classification"
on the available data for diethanolamine, the reaction mass is
classified and labelled for effects on fertility and developmental
toxicity according to EU Classification, Labelling and Packaging of
Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 (cat. 2;
toxicity was substance- and dose related but occurred in the presence of
distinct general systemic toxicity in the dams and in the offspring.
Administration of DEA in the drinking water resulted in reduced water
and food consumption (particularly marked during lactation), with
accompanying reductions in body weight, body weight gain, and terminal
body weight. These findings were consistent across sexes, generations,
and cohorts. The severity of findings increased with dose. In most
cases, by study termination the body weights of treated groups were
within 10% of the control group mean value, except in the case of HD F1
males and females, where mean body weight values were 80±5% of control.
However, more critical reductions in body weight were seen during the
study. Effects have been reported on kidneys where treatment was
associated with tubular degeneration/regeneration in both F0 and F1
animals of the MD and HD groups. This was accompanied by signs of
impaired renal function (increased urine volume, decreased urine
specific gravity). Liver effects were associated with hypertrophy in
both F0 and F1 animals of the HD group. F1 males also exhibited this
change and fatty change also at the MD level. Additionally, a microcytic
anemia was evident in the HD groups. DEA can be transformed into
phosphoglyceride and sphingomyelin analoga leading to disturbances in
phospholipid metabolism and a multitude of physiological functions,
supporting a secondary mode of action (cf. appendix for further details).
the observed reproductive toxicity cannot be regarded as an isolated
effect and may be attributed to general systemic toxicity.
as a possible mode-of-action (MOA) with quantitative differences in
species specificity, DEA caused a clear reduction of choline levels in
plasma and liver of all dose groups. DEA is already labelled for
specific target organ toxicity (STOT RE cat. 2) for effects on blood,
liver, kidney and nervous system which may be attributed to a disturbed
choline-homeostasis. Choline is an essential nutrient; however, rodents
appear to be more susceptible towards an impaired choline-homeostasis
than humans. Leung et al. (2005) summarized the evidence why humans are
less susceptible for choline-deficiency than rodents in the context of
the carcinogenicity endpoint (further references given within the
original article):“…choline is an essential nutrient in all mammals,
the proposedmechanism of DEA-induced choline deficiency is qualitatively
applicable to humans. However, there are marked species differences in
susceptibility to choline deficiency, with rats and mice being far more
susceptible than other species including humans. These differences are
attributed to quantitative differences in the enzyme kinetics
controlling choline metabolism. Rats and mice rapidly metabolize choline
to betaine in the liver and it is likely that choline oxidase activity
determines choline requirements and controls species sensitivity to
choline deficiency. For example, choline oxidase activity is much lower
in primates than rodents and primates are less sensitive to choline
deficiency. Humans have the lowest choline oxidase activity of all
species and are generally refractory to choline deficiency, with
evidence of choline deficiency observed only after prolonged fasting,
significantly depressed liver function or deficient parenteral feeding.
It is noteworthy that there was no evidence of GJIC inhibition in human
hepatocytes treated with DEA or cultured in choline-deficient media.”
to chapter 18.104.22.168.1. of Regulation EC 1272/2008 (CLP), "Classification
as a reproductive toxicant is intended to be used for substances which
have an intrinsic, specific property to produce an adverse effect on
reproduction ... .".
to chapter 22.214.171.124.3 of Regulation EC 1272/2008 (CLP), "Classification
should not automatically be discounted for chemicals that produce
developmental toxicity only in association with maternal toxicity, even
if a specific maternally-mediated mechanism has been demonstrated. In
such a case, classification in Category 2 may be considered more
appropriate than Category 1.".
to chapter 126.96.36.199.2 and 188.8.131.52.5 of Regulation EC 1272/2008 (CLP), ".
. . when the toxicokinetic differences are so marked that it is certain
that the hazardous property will not be expressed in humans then a
substance which produces an adverse effect on reproduction in
experimental animals should not be classified.".
conclusion, classification with category 2 for reproductive toxicity
(H361) is considered the most appropriate in line with the criteria laid
down in Regulation EC 1272/2008 (CLP).
In most cases, cohorts
1A and 1B are combined. Derived from report summary findings. Detailed
explanation for the annotations (1), (2), (3) and (4) are given below
the table. The critical effects for the endpoint evaluation (marked in
bold in the table) and the interpretation thereof are being discussed in
the text below the table.
↓ food consumption, body weight, body weight gain
↑ liver weight, kidney weight
Microcytic anaemia, ↓ prothrombin time, ↑ platelet count
↑ urea, albumin, AST, ALP
↓ urinary specific gravity, ↑ urine volume
Liver: centrilobular hypertrophy (minimal to slight)
Kidney: tubular degeneration/regeneration (minimal to slight)
↑gestation length (4)
↓ implantation sites, litter size (4)
↓ water and food consumption, body weight, body weight gain
Microcytic anaemia,↓ prothrombin time
↑ urea, albumin, ↓ platelet activating factor
Kidney: tubular degeneration/regeneration (minimal to moderate)
Glandular stomach: erosion/ulcer, oedema, inflammation (minimal to slight)
↑ dead and cannibalised offspring (4)
↓ body weight from PND 4 to weaning
↑ T4 in female weanlings (2)
High-stepping gait, piloerection
Small and immature testes (1)
Microcytic anaemia, ↑ platelet count
↑ urea, AST, ALP
Kidney: tubular degeneration/regeneration (slight to moderate)
Liver: centrilobular hypertrophy (minimal to slight), peripheral hypertrophy (slight to moderate), peripheral fatty change (minimal to slight)
Mammary gland: feminisation (slight to severe), diffuse hyperplasia in one animal (moderate) (1)
Pituitary gland: eosinophilic cysts (minimal to moderate) (2)
Testis: immature epithelium (severe), tubular degeneration (slight to moderate) (1)
Prolonged/irregular oestrus cycle (4)
Microcytic anaemia, ↓ prothrombin time, ↓ monocyte count
↑ urea, AST
↓ T-cell helper count and CD4/CD8 ratio
↑ T-cell count
↑ T4 (2)
Liver: centrilobular hypertrophy (minimal to slight), peripheral fatty change (minimal to slight)
Mammary gland: increased secretion
Ovary: ↓ size, diffuse atrophy, luteal cysts, (1)
↓ follicles (primordial and growing), absent corpora lutea (two females of 1B) (4)
↓ maximum amplitude of auditory startle response, no habituation to test environment
CNS: degeneration of nerve fibres of medulla oblongata and spinal cord (3)
Pituitary gland: eosinophilic cysts (2)
High-stepping gait, piloerection (one animal)
↓ maximum amplitude of auditory startle response, no habituation to test environment and
↓ terminal body weight
Small testes, epididymides, prostate, seminal vesicle (one animal) (1)
↓ body weight, body weight gain
↑ kidney weight
↓ prothrombin time,↑ platelet count
Kidney: tubular degeneration/regeneration (minimal)
↓ water and food consumption (postnatal), body weight, body weight gain
↓ prothrombin time
Glandular stomach: erosion/ulcer, oedema, inflammation (minimal to moderate)
↓ body weight from PND 14 to weaning
↑ platelet count in males
Liver: centrilobular hypertrophy (minimal), peripheral fatty change (minimal to slight)
Pituitary gland: eosinophilic cysts (minimal to slight) (2)
↓ water consumption, body weight, body weight gain
Pituitarygland: eosinophilic cysts (2)
↑ T4 in females PND 4 (2)
Effects to maternal toxicity
Effects marked with (1) can be attributed to be secondary to
reductions in body weight in consequence of general systemic toxicity.
References are given within the final report. Therefore, as already
identified in the summary of effects, the following changes in
reproductive organs can be attributed to the reduced body weight and
probably reduced choline-levels in the F1 generation
Testis: Macroscopically small testes were
identified in three HD males from cohort 1A (476,
478, 479), three from cohort 1B (693,
694, 697), and one from cohort 3 (1240). The size of the
secondary sex organs (epididymis, prostate, seminal vesicle) was also
reduced. Small testis was correlated with immature or degenerative
epithelium. Further evaluation shows that the affected individuals were
overall much smaller than their counterparts, and the effects on the
reproductive tract organs are considered secondary to retarded growth.
The mean terminal body weights of the animals in Cohort 1A and
1B were 24 % and 20 % lower compared to the controls. However, the body
weights of animals for which above mentioned findings and effects in the
reproductive organs have been observed were even more reduced (45 – 70 %
lower compared to controls).
Ovary:Four HD animals in cohort 1B (777,
785, 797, 798) had small, atrophied ovaries with reduced or
absent corpora lutea. These animals were of overall retarded growth, and
the ovary changes compared to control are considered a secondary
Differential ovarian follicle count (both primordial and growing
follicles) was reduced in the cohort 1A and 1B (combined) HD group, but
not in the F0HD group.
Mammary gland:Feminisation of the mammary
gland (grade 2–4) was identified in four HD males of cohort 1A, one of
which also showed diffuse hyperplasia (grade 3). It should be noted that
there was insufficient mammary tissue for another four of the 25 males
in this cohort.
There was an increase in mammary gland secretion in six females of
the HD cohort 1A group.
The above-mentioned findings can be clearly attributed to be a
consequence of general systemic toxicity caused by an impaired choline
homeostasis. The body weights were 16 % lower in Cohort 1B (where
findings occurred) respectively and the animals with above mentioned
findings were 18 - 50 % lower compared to controls.
Additionally, animals showed clinical signs (e.g. high-stepping
gait, piloerection etc.) and mortality occurred in the F1 HD groups (2
males and 1 female in two rearing cohorts from cohorts 1A and 1B were
either sacrificed moribund).
Further literature research revealed (Lindern-Moore et al., 1981)
(cite references) that similar effects can be expected in case of
starvation experiments in rats, especially in weanling and growing rats
which appears not to be surprising assuming a higher energy demand.
Unfortunately, similar reports could not be retrieved for an induced
choline-deficiency, where focus has been laid on organ effects and CNS
effects, but not on reproductive organs and outcome.
In addition, “secondary” evidence shows data from genetic knockout
models can be used demonstrating that effects on implantation loss might
be referred to an impaired choline-homeostasis and/or overall altered
lipid metabolism. This before mentioned evidence being derived from the
reproductive outcome of such transgenic animals with a knockout of an
enzyme being involved in phospholipid-synthesis is only secondary and
indirect. Therefore and as there is non direct evidence from e.g. a
reproductive screening using choline-deficiency, certain effects
reported in the EOGRTS for DEA have been categorized as being “critical
effects” which deserve further discussion and mechanistic investigation
(i.e. to establish the link as a secondary effects on a molecular level).
Moreover, effects on choline-homeostasis have been observed in all
dose groups and levels (i.e. reduction of choline-levels.) Choline is an
essential nutrient in humans and other mammalian species such as the
rat. Wistar rats are assumed to be very sensitive towards
choline-deficiency (Kirsch et al., 2003; Nocianitri and Aoyama, 2001).
Effects, that are covered by the existing “Specific target
organ toxicity” STOT RE cat. 2 already:
Kidney: Treatment was associated with tubular
degeneration/regeneration in both F0 and F1 animals of the MD and HD
groups. This was accompanied by signs of impaired renal function
(increased urine volume, decreased urine specific gravity). Similar
findings have been previously reported in rats (please compare with the
repeated dose toxicity section of the IUCLID6 – chapter 7.5.1.)
Liver: Treatment was associated with
hypertrophy in both F0 and F1 animals of the HD group. F1 males also
exhibited this change and fatty change also at the MD level. Similar
findings have been previously reported in mice2.
A STOT RE cat. 2 has a cut-off according to the CLP of mg/kg
BW/day. Therefore, no additional label is warranted and the hazard is
considered to be already covered by the existing classification and
of lower concern not triggering classification (adversity not clearly
In this section, effects are located which were observed at all
The adversity of effects is however unclear and effects need to be
Pituitary gland: eosinophilic cysts occurred
in the pars distalis of F1 males and females from all dose
levels. Pathologists could not determine the adversity of this finding.
Plasma hormones: The increased mean dose-group
T4 values observed in some of the dose groups were within historical
control data and thus not biologically significant. In addition, there
were no significant changes of TSH observed in any dose groups;
supporting the evidence that administration of DEA has no significant
impact on thyroid hormone homeostasis. The production of thyroid
hormones is primarily regulated by thyroid-stimulating hormone (TSH)
released from the anterior pituitary gland. TSH release is in turn
stimulated by the thyrotropin-releasing hormone (TRH) from the
hypothalamus. The thyroid hormones provide negative feedback to TSH and
TRH: when the thyroid hormones are high, TSH production is suppressed
(ECHA, 2018; Joseph-Bravo et al., 2016).
already covered by the existing C&L - STOT RE cat.2
Central nervous system (cohorts 2A and 2): Degeneration of nerve
fibres in the medulla oblongata and spinal cord was observed in HD males
and females of cohort 2A, but not cohort 2B. Demyelination of the
medulla and spinal cord has been reported at a similar dose in a 90-day
repeated-dose toxicity study in F344 rats (NOAEC=630ppm, LOAEC=1250ppm),
but not in mice. It is clear, therefore, that these findings represent a
response to continuous postnatal exposure rather than an effect upon
development per se.Therefore, these effects are thought to be covered
by a STOT RE cat. 2.
The only functional deficits to be identified were a decrease in
the maximum amplitude of the startle response, although latency was
unaffected, and lack of habituation to the test environment. No other
treatment-related changes were identified.
that may triggering further mechanistic research
Effects marked with (4) are not directly associated with the
general and the systemic toxicity observed. However, as a “secondary”
evidence, data from genetic knockout models can be used demonstrating
that effects on implantation loss might be referred to an impaired
choline-homeostasis and/or overall altered lipid metabolism.
Gestation and outcome: Parturition was
slightly (+2%), but statistically significantly, delayed in the HD
group. The number of implantations sites per dam was decreased, and the
incidence of stillborn pups was increased, resulting in an overall
reduction in litter size in this group. Survival through to weaning was
unaffected in all treatment groups compared to control.
Gonads:Differential ovarian follicle count
(both primordial and growing follicles) was reduced in the cohort 1A and
1B (combined) HD group, but not in the F0 HD group.
Secondary evidence comes from the literature. Vance and Vance
reported (2009) on the physiological consequences of disruption of
mammalian phospholipid biosynthetic genes:
“…several lines of mice with disrupted genes of phospholipid
biosynthesis were generated. From this research, we have learned that
embryonic lethality occurs in mice that lack choline kinase (CK) a,
CTP:phosphocholine cytidylyltransferase a, CTP:phosphoethanolamine
cytidylyltransferase, or phosphatidylserine decarboxylase. Whereas mice
that lack CK b are viable but develop hindlimb muscular dystrophy and
neonatal bone deformity. Mice that lack CTP:phosphocholine
cytidylytransferase b have gonadal dysfunction and defective axon
The disruption of genes and enzymes in consequence involved in
phosphatidyl-choline synthesis is thought to be similar to a state of
choline-depletion or at least impaired choline-homeostasis. Accordingly,
e.g. embryonic death has been reported as physiological consequence for
some knockout models of certain enzymes being involved in phospholipid
synthesis (Vance & Vance; 2009).
Additionally, it has been demonstrated by Jackowski and coworkers
(2004) that disruption of CCTß2 (CTP:phosphocholine
cytidylyltransferase, CCT) expression leads to gonadal dysfunction and
degeneration in mice (i.e. defective ovarian follicle development and
disruption of spermatogenesis).
With respect to the effects observed on DOFC both primordial and
growing follicles (down to 89% and 72% compared to controls,
respectively), similar effects have been described after starvation and
semi-starvation of pre-pubertal Wistar rats (Lintern-Moore et al.,
1981). Herein the authors describe that after starvation of young animal
from PND 21 – 42 leads to a reduction growing follicles on day 42.
Additionally, no corpora lutea were present. However, after re-feeding
(ad libitum) up to day 66, growing follicle numbers and corpora lutea
could be restored. The body weights caused by the starvation were
reduced by starvation but recovered after refeeding up to day 66. This
means, that the effects on DOFC might be a secondary consequence of an
impaired choline-homeostasis (i.e. reduced PC content as reported to be
DEA-induced in vitro and in vivo).
A secondary mode of action is further supported by the fact that
DEA is incorporated into phosphoglyceride and sphingomyelin analoga.
are a class of phospholipids that
incorporate choline as a headgroup, a glycerophosphoric acid, with a
variety of fatty acids. Phosphatidylcholine is formed through the
methylation of phosphatidylethanolamine catalyzed by
phosphatidylethanolamine N-methyltransferase (Li and Vance, 2008).
Sphingomyelins are a group of phospholipids, which consist of
phosphocholine (or phosphoethanolamine) as a
group and a ceramide. The formation typically involves the enzymatic
transfer of a phosphocholine head group from phosphatidylcholine to
ceramide (Tafesse et al., 2006). Due to its structural similarity to
ethanolamine and choline, DEA is metabolized by very similar pathways
undergoing O-phosphorylation and N-methylation making
incorporation into phosphoglyceride and sphingomyelin analogs possible
(Leung et al., 2005). Barbee and Hartung could show in vitro and in
vivo that DEA is incorporated into various phospholipid derivates
and thereby competing with choline and ethanolamine inhibiting their
incorporation. DEA-derived phospholipids are characterized by an
increased biological half life and DEA-derived sphingomyelines are not
being a suitable substrate for sphingomyelinase, which hydrolyzes
sphingomyelines leading to free phosphocholine and ceramide (Barbee
and Hartung, 1979). Thus, incorporation of DEA into phospholipid
derivates is leading to disturbances in phospholipid metabolism and a
multitude of physiological functions.
J., Hartung, R. (1979). The effect of diethanolamine on hepatic and
renal phospholipid metabolism in the rat. Toxicology and Applied
Pharmacology 47(3): 421-430.
ECHA Guidance for the identification of endocrine disruptors in
biocides and pesticides; EFSA Journal 2018; 16 (6); 5311
Leung HW, Kamendulis LM, Stott WT. (2005). Review of the
carcinogenic activity of diethanolamine and evidence of choline
deficiency as a plausible mode of action. Reg Tox and Pharmacol, 43:
Vance, D.E. (2008). Phosphatidylcholine and choline homeostasis. J.
Lipid Res. 49, 1187–1194.
Moore NP et al. (2013). Guidance on classification for
reproductive toxicity under the globally harmonized system of
classification and labelling of chemicals (GHS).Critical
Ternes P. and Holthuis J. (2006). The Multigenic Sphingomyelin Synthase
Family. The Journal of Biological Chemistry. 281, 29421-29425
Vance VE and Vance JE. (2009). Physiological consequences of
disruption of mammalian phospholipid biosynthetic genes. Journal of
Lipid Research, April suppl. S132-137
Nocianitri KA and Aoyama Y. (2001). Note: Different response to
choline deficiency of the serum ornithine carbymoyltransferase activity
in four strains of rats. Biosci. Biotechnol. Biochem. 65(4): 935-38.
Kirsch et al. (2003). Rodent nutritional model of non-alcoholic
steatohepatitis: Species, strain and sex difference studies. Journal of
Gastroenterology and Hepatology 18: 1272–1282
Jackowski S et al (2004). Disruption of CCT2 Expression Leads to
Gonadal Dysfunction. MOLECULAR AND CELLULAR BIOLOGY 24(11): 4720–4733
Joseph-Bravo P, Jaimes-Hoy L and Charli JL, 2016. Advances in TRH
signaling. Reviews in Endocrine and Metabolic Disorders, 17, 545–558.
Lindern-Moore S et al. (1981). The effect of restricted food
intake and refeeding on the ovarian follicle population of the
pre-pubertal Wistar rat. Reprod. Nutr. Develop. 21(5A9): 611-620
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.
За да гарантираме, че се възползвате максимално от функциите на нашия уебсайт, сме въвели „бисквитки“.
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