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EC number: 226-394-6
CAS number: 5392-40-5
the range-finding study and dose selection
were selected based on signs of toxicity noted at dose levels of 100,
300, 500 and 1000 mg/kg bw/d in a previously conducted OECD 421
reproduction/developmental screening study (BASF project 80R0410/07R074)
which preceded this definitive extended one generation reproductive
N was administered daily as an aqueous preparation to groups of 10 male
and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300
and 1000 mg/kg body weight/day (mg/kg bw/d). As severe signs of
intoxication were observed at 1000 mg/kg bw/d (particularly females,
details see below) during the first days of administration (premating
phase), the dose was lowered to 500 mg/kg bw/d from premating day 4
until the end of the study. Control animals (10 male and 10 female
Wistar rats) were dosed daily with the vehicle only (0.5% Sodium
carboxymethyl cellulose (CMC) suspension in deionized water (with 5 mg/
100mL Tween 80)).
duration of treatment covered about 4 weeks in-life period (males)
including 2 weeks mating (mating pairs were from the same test group) as
well as a 2 weeks premating period (females), 2 weeks mating period, the
entire gestation, about 3 weeks of lactation period and about 5 weeks
postmating period in females up to one day prior to the day of scheduled
sacrifice of the animals.
1000 mg/kg bw/d dose caused mortality (3 females) and severe clinical
signs of intoxication (i.e. unsteady gait, severely reduced muscle
tonicity and prone-position with abducted limbs) immediately after the
start of treatment. After the high-dose was lowered to 500 mg/kg bw/d
(premating day 4), still adverse clinical observations (hypothermia,
unsteady gait, apathy, abdominal position, salivation, increased water
consumption, decreased food consumption) were recorded, along with
slight anemia, as well as a macroscopically visible thickening of the
forestomach at necropsy. Histopathological examination of the
forestomach in the 1000/500 mg/kg bw/d animals revealed a full thickness
necrosis of the forestomach epithelium in the 3 female decedents, focal
and transmural inflammation in 9 males and all females (most likely
caused by erosion/ulceration that was not always visible on the slide),
erosion/ulcer in 6 males and 6 females, as well as hyperplasia of the
squamous epithelium and hyperkeratosis in 10 males and 7 females.
300 mg/kg bw/d dose caused salivation and marginally decreased food
consumption at the beginning of the study. Histopathological examination
of the forestomach revealed inflammation in 8 males and 9 females,
erosion/ulcer in 2 males and 2 females, hyperplasia of the squamous
epithelium and hyperkeratosis in 10 males and 9 females, as well as
focal degeneration of the squamous epithelium in1 female.
the 100 mg/kg bw/d dose group histopathological examination of the
forestomach revealed inflammation in 1 male, hyperplasia of the squamous
epithelium and hyperkeratosis in 1 male, as well as diffuse degeneration
of the squamous epithelium in1 male.
dose levels of 25, 80 and 250 mg/kg body weight/day were selected as
dose levels for the present study. The high dose was intended to cause a
similar adverse pathology as in the rangefinder while the low dose was
intended to be a NOAEL. This procedure meets the principles of
guidelines OECD 414 (adopted 2018) and OPPTS 870.3700 (US EPA), as well
as ECHA practical guide 10 (“how to avoid unnecessary testing on
animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in
compliance with EU Directive2010/63/EU on animal protection.
Results of analyses
The stability of test substance was
demonstrated for a period of 7 days in the refrigerator.
The homogeneity of the mixtures was
Concentration control analyses
Overall, measured values for Citral N
were in the expected range of the target concentrations (90 - 110%),
demonstrating the correctness of the preparations. Some departures from
the lower specification limit of 90 % were noted for single samples of
Citral N in 0.5 % sodium carboxymethyl cellulose in drinking water with
Tween 80 (0.5 mg/100 mL), which were found to be between 78.0 % - 89.4 %
of their nominal concentrations. As these values were close to the lower
specification limit and the vast majority of all measurements well met
specification, those departures were considered not to have any
influence on the validity of the present study.
Citral N was administered to groups of 25 male and 25 female
healthy young Wistar rats asan aqueous preparation by stomach tube at
different dosages (0, 25, 80 and 250 mg/kg body weight/day [mg/kg
bw/d]). F0 animals were treated at least for 2 weeks prior to mating 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 2 different cohorts (1A and 1B) which were subjected to
specific postweaning examinations. Cohort 1B (=F1 generation parental
animals) were selected to produce F2 pups. F1 animals selected for
breeding were continued in the same dose group as their parents. Groups
of 25 males and 25 females, selected from F1 pups to become F1 parental
generation, were offered an aqueous preparation by stomach tube at
different dosages (0, 25, 80 and 250 mg/kg bw/d) of the test substance
post weaning, and the breeding program was repeated to produce a F2
litter. The study was terminated with the terminal sacrifice of the F2
weanlings and F1 parental animals. Control animals were dosed daily with
the vehicle(0.5% Sodium carboxymethyl cellulose (CMC) suspension in
drinking water (with 5 mg/ 100mL Tween 80)).
The parents' and the pups' state of health was checked each day,
and parental animals were examined for their mating and reproductive
performances. Water, food consumption and body weights of the F0 and F1
parents and F1 rearing animals was determined regularly and a detailed
clinical observation (DCO) was performed in all F0 and F1 parents and F1
animals in cohorts 1A.
Estrous cycle data were evaluated for F0 and F1B parental females.
In all cohort 1A females, vaginal smears were collected after vaginal
opening until the first cornified smear (estrous) was recorded. The
estrous cycle also was evaluated in cohort 1A females for 2 weeks around
PND 75. Moreover, the estrous stage of each female was determined on the
day of scheduled sacrifice.
The F1 pups were sexed on the day of birth (PND 0) and were
weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14
and 21. Their viability was recorded. At necropsy, all pups were
examined macroscopically. Date of sexual maturation, i.e. day of vaginal
opening (females) or balanopreputial separation (males), of all F1 pups
selected to become F1 parental and rearing animals was recorded.
All surviving F1 and F2 pups were examined for the presence or
absence of nipple/areola anlagen on PND 13. If nipple/areola anlagen
were recorded, all surviving male pups were carefully re-examined one
PND 20. The number of nipple/areola anlagen were counted. Anogenital
distance measurements were conducted on all live F1 and F2 male and
female pups on PND 1.
Urine and blood samples for clinical pathological investigations
were withdrawn from 10 selected F0 and cohort 1A animals per sex and
group. Further blood samples were taken from a maximun of 10 surplus
(culled) PND 4 pups per sex and group as well as from 10 surplus PND 22
pups per sex and group.
Various sperm parameters (motility, sperm head count, morphology)
were assessed in the F0 generation males and cohort 1A males at
scheduled sacrifice or after appropriate staining.
All F0 and F1 parental animals were assessed by gross pathology
(including weight determinations of several organs) and subjected to an
extensive histopathological examination; special attention being paid to
the organs of the reproductive system. A quantitative assessment of
primordial and growing follicles in the ovaries was performed for all
control and high-dose F1 rearing females cohort 1A. All F1 rearing
animals were assessed by different pathological and histopathological
No test substance-related mortalities or adverse clinical
observations were observed in any of the groups, indicating systemic
toxicity. In particular, regularly conducted detailed clinical
observations revealed no test substance-related adverse systemic effects.
Transient salivation during a short time period after gavage
dosing was noted for nearly all high-dose and some mid-dose male and
female animals (F0 and F1 animals across all cohorts) during all
sections of the study. It is likely, that this temporary finding was
induced by a bad taste of the test substance or local affection of the
upper digestive tract. It is not considered to be an adverse finding of
systemic toxicity but may, however, had subsequent consequences, namely
increases of food and water consumption. In the high-dose group (F0 and
F1B animals) intermittent increases of food and water consumption were
noted during all study segments. Similar changes, though to a much
lesser extent, were also observed in the mid-dose group. Concurrent with
food and water consumption the mean body weights and body weight gain of
the high-dose group (F0 and F1B animals in particular) showed
intermittent increases during several study segments. All these
food/water consumption and body weight increases in the high-dose group
were rather mild, in the lower dose groups they were even limited to
short episodes in individual animals. Supposedly, the attempt of the
animals to attenuate an unpleasant taste and/or smell after gavage
dosing of the test material led to those phases of increased food/water
consumption and their consequences. However, as an increase of
food/water consumption was observed, the described changes were neither
considered as adverse findings nor as signs of systemic toxicity.
Concerning clinical pathology, no treatment-related, adverse
effects were observed in the parental F0 generation as well as in the
offspring F1A rats up to a dose of the compound of 250 mg/kg bw/d.
Regarding pathology, the forestomach was the target organ in the
F0 generation parental animals. The macroscopically observed thickening
of the forestomach wall in males and thickening of the margo plicatus in
females corresponded to hyperplasia (diffuse or focal) with
hyperkeratosis (diffuse or focal) by light microscopy. Single animals
revealed erosion/ulcer in addition which, in combination with the other
findings, were seen as an aggravation of lesions. Mid and high dose male
and female animals (80 and 250 mg/kg bw/d) were affected, with
increasing number and severity in the high dose group (250 mg/kg bw/d).
The absolute liver weight in mid and high dose males (80 and 250
mg/kg bw/d) and kidney weight in high dose males (250 mg/kg bw/d) were
above historical control data. These weight changes were assumed to be
treatment-related but not adverse for the following reasons: no
histopathologic correlate, no findings in clinical pathology, relative
liver and kidney weights were within historical control data.
Male and female F1 generation rearing animals of cohort 1A
revealed hyperplasia (diffuse or focal) with hyperkeratosis (diffuse or
focal) in the forestomach. Single animals revealed erosion/ulcer in
addition which, in combination with the other findings, were seen as an
aggravation of lesions. Mid and high dose male and female animals (80
and 250 mg/kg bw/d) were affected with increasing number and severity in
the high dose group (250 mg/kg bw/d).
High dose F1 males (250 mg/kg bw/d) revealed a significant
increase of absolute and relative weight of cauda epididymis, kidney and
liver; high dose F1 females (250 mg/kg bw/d) revealed a significant
increase in absolute and relative weight of the liver. The altered
weights were above historical control values. Nevertheless, these
findings were regarded as treatment-related but not as adverse, because
neither in histopathology nor in clinical pathology findings were
observed which could explain the increased organ weights.
All other findings in the investigated internal organs of the F0
and F1A animals occurred either individually or were biologically
equally distributed over control and treatment groups. They were
considered to be incidental or spontaneous in origin and without any
relation to treatment.
In the F1-generation cohort 1B males and females of the high dose
group (250 mg/kg bw/d), the liver weights were significantly increased
(males: absolute and relative, females: absolute only). In concordance
with the discussion for the F0 and F1 cohort 1A animals these changes
were regarded to be treatment-related but not as adverse.
All other findings occurred either individually or were
biologically equally distributed over control and treatment groups. They
were considered to be incidental or spontaneous in origin and without
any relation to treatment.
In the surplus F1-generation pups on PND 22 (F1 weanlings not
selected for cohorts) neither treatment-related organ weight changes nor
gross lesions were detected. Histopathology was not performed.
There were no indications from clinical examinations, that Citral
N adversely affected the fertility or reproductive performance of the F0
and F1B parental animals up to and including the administered high-dose
of 250 mg/kg bw/d. Estrous cycle data, sperm quality of males, mating
behavior, conception, gestation, parturition, lactation and weaning were
comparable between the rats of all groups including control and ranged
within the historical control data of the test facility. The same is
true for sexual organ weights and gross and histopathological findings
of these organs in F0 and F1A females of all dose groups. Specifically,
the results of the differential ovarian follicle count (DOFC) in F1A
females – comprising the numbers of primordial and growing follicles, as
well as the combined incidence of primordial plus growing follicles –
showed no significant differences between the control and the high dose
For all liveborn male and female pups of the F0 and F1B parents,
no test substance-induced signs of developmental toxicity were noted at
dose levels as high as 250 mg/kg bw/d. Postnatal survival, pup body
weight gain as well as post-weaning development of the offspring until
puberty remained unaffected by the test substance. Furthermore, clinical
and/or gross necropsy examinations of the F1 and F2 pups revealed no
Measurement of thyroid hormones revealed no effect caused by the
test substance, neither in the F0 parental animals nor in the liveborn
Neither the anogenital distance/index nor the check for the
presence of nipples/areolas, both very sensitive marker of potential
endocrine-mediated imbalances, revealed any test substance-related
Vaginal opening and preputial separation are commonly used
developmental markers for onset of puberty in laboratory rats. No delays
beyond a normal range of biological variation in rat (multi)generation
studies which might be attributable to the treatment were noted in any
of the test substance-treated groups.
Thus, under the conditions ofthe present extended one-generation
reproduction toxicity studythe NOAEL (no observed adverse effect level)
for general, systemic toxicity is 25 mg/kg bw/d, based on pathological
findings in the gastrointestinal tract at the LOAEL of 80 mg/kg bw/d.
The NOAEL for fertility and reproductive performance for the parental
rats is250 mg/kg bw/d, the highest tested dose. The NOAEL for
developmental toxicity in the F1 and F2 progeny is250 mg/kg bw/d, the
highest tested dose.
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