Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl lactate
EC Number:
202-598-0
EC Name:
Ethyl lactate
Cas Number:
97-64-3
Molecular formula:
C5H10O3
IUPAC Name:
ethyl 2-hydroxypropanoate
Test material form:
liquid
Remarks:
colorless transparent liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Test Guideline OECD 422 is designed for use with the rat. The
rats are the standard experimental rodent of choice and
recommended by OECD Guideline and in the international
validation program for the detection of endocrine disrupters the rat
was the only species used.
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animals were acclimated to the condition identical to the
condition during the experiment 5 days prior to the start of treatment.
The acclimation was according to standard operation procedures.
The animals were acclimated during 5 days to the animal room
conditions identical with the conditions defined for the experimental
part of the study. On the arrival, they were placed into the cages, 3
rats per cage and were weighted after 48-hours.
The animals were housed in TECNIPLAST cages 1500U from the
Tecniplast Company, Italy. The cages have high density
polypropylene body, measuring 480 x 375 x 210 mm - floor area
1500 cm2. The cages, cage racks and other equipment were sanitized
according to standard operation procedures.
On arrival, animals were placed into the cages, 5 rats per cage.
48-hours after arrival animals were weighted and kept in their cages
until the start of the study to allow for acclimation to the animal
room conditions, which are identical as defined for the experimental
part of the study. After randomisation were placed three and two rats
of the same sex.
The study was carried out in the experimental animal house of the
Test Site with the pressure air-condition system. The animals were
housed in one room under the defined laboratory conditions. The
temperature and relative humidity in the animal room were recorded
daily (dataloger No.: TH 464); mean values of temperature were
23.1±0.6°C at mean relative humidity 54.0±4.6% (mean±SD). The
artificial lighting was setting in a 12-hour light and 12-hour dark
photoperiod.
The sanitation was performed according to standard operation
procedures.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Duration of treatment / exposure:
The animals were dosed daily for 7 days a week.
Frequency of treatment:
Once per day.
Females were treated during:
• 14-day pre-mating,
• 14-day mating (maximum)
• 22-day gestation (approximately)
• 13-day lactation
Females with no evidence of copulation and that failed to deliver
were dosed for a total of 51 doses (from 27.8.2018 to 17.10.2018).
Males were treated during:
• 14-day pre-mating,
• 14-day mating (maximum)
The animals designated for post-treatment observation (5
animals per sex in control and high dose groups, respectively)
were remain untreated for subsequent 14 days.
The animals in all dose groups were treated during declared
periods excluding 4.9.2018 because of change of test doses. The
control animals received doses of vehicle without interruption on
this day, it means 1 day more as it is stated for dose groups.
Control animals:
yes, concurrent vehicle

Examinations

Statistics:
Individual results (the body weight, food consumption, haematology, clinical chemistry, relative
weights of organs and reproduction parameters) obtained during the study were statistically
evaluated by statistical programme Statgraphics. Statistical evaluation was done separately for
males and females. Basic statistic (mean, sd) was made and Kruskal – Wallis test was applied. In
the case of statistically significant result the Kruskal Wallis test was followed by Mann Whitney W
test to determine which medians are significantly different from the one in control group.
Significance level of 0.05 was considered to make relevant statistical conclusions.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical observations throughout the first two weeks of test item application indicated presence of
signs of systemic toxicity in the high dose group of female rats (800 mg/kg b.w.). There were three
test item-related deaths of animals (High dose) during the study. Parental female mortality at this
dose overreached 10%.
Mortality:
no mortality observed
Description (incidence):
After reduction of test item doses tested, since Day 10 of exposure, no test item-related mortality,
no significant behavioural changes (except the female rat No.: 81), no clinical signs or any deviation
from normal findings were recorded during the study neither in adult exposed animals nor in
offspring.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences in body weight amongst the treated groups of adult male and
female rats including the animals in the both satellite groups were observed.
Unlike adult animals, the test item induced alteration of physical development being demonstrated
as increase of pup body weight on Day 13 of lactation exposure, in significant extent in High dose
group male pups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not significantly different amongst the experimental groups. Influence of
treatment associated stress and/or decreased appetite was observed at the start of treatment and
increased physiological demands in pregnant and lactating female rats was evident. The decrease in
food consumption in the adult male rats exposed to high dose of test item observed on a week 3 was
not statistically significant and not accompanied with statistically significant changes in the body
weight; it is not considered to be of toxicological relevance.
Haematological findings:
no effects observed
Description (incidence and severity):
No significant differences in haematological parameters among groups exposed to the test item and
controls were found in all evaluated parameter with exception of PT and APTT values. The
differences were minor had no biological or toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The clinical chemistry parameters measured in serum of male and female rats showed some
statistically significant differences. These differences were minimal in nature and had no biological
or toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
In the urine no significant changes against normal physiological conditions were detected.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological lesions found in the treated animals were either of spontaneous character or not
in direct relation with the test item administered. Macroscopically described lesions in the stomach
or jejunum as change of colour (diffuse or focal), were histologically detected as hyperaemia of
capillaries in the mucosa and submucosa. Sporadic findings as defect in renal pelvis histologically
evaluated as dilatation of the renal pelvis, mucification found in the cervix uteri, dilatation of acinus
in prostate, gold brown pigment particles in ovary, cystic dilatation in the vagina wall and uterus
lumen dilatation were classified as lesions not related with application of test item.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
In summary none of used doses significantly influenced length of pregnancy, number of implants, survival of pups, anogenital distance, or other variables.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
ca. 600 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
< 75 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical signs

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
75 mg/kg bw/day (nominal)
Treatment related:
no

Any other information on results incl. tables

Clinical observations throughout the first two weeks of test item application indicated presence of

signs of systemic toxicity in the high dose group of female rats (800 mg/kg b.w.). There were three

test item-related deaths of animals (High dose) during the study. Parental female mortality at this

dose overreached 10%.  

After reduction of test item doses tested, since Day 10 of exposure, no test item-related mortality,

no significant behavioural changes (except the female rat No.: 81), no clinical signs or any deviation

from normal findings were recorded during the study neither in adult exposed animals nor in

offspring.

No statistically significant differences in body weight amongst the treated groups of adult male and

female rats including the animals in the both satellite groups were observed.  

Unlike adult animals, the test item induced alteration of physical development being demonstrated

as increase of pup body weight on Day 13 of lactation exposure, in significant extent in High dose

group male pups.  

Food consumption was not significantly different amongst the experimental groups. Influence of

treatment associated stress and/or decreased appetite was observed at the start of treatment and

increased physiological demands in pregnant and lactating female rats was evident. The decrease in

food consumption in the adult male rats exposed to high dose of test item observed on a week 3 was

not statistically significant and not accompanied with statistically significant changes in the body

weight; it is not considered to be of toxicological relevance.

Within the scope of all observed reproductive parameters (except of body weight), anogenital

distance/anogenital index (AGD/AGI) were only parameters that showed significant differences

amongst experimental groups in male as well as in female pups. Dose dependent statistically

significant reduction of AGI in male offspring (from the Low- and Mid dose group) and in female

offspring (from the Low dose, the Mid dose at the limit of significance and the High dose group)

unexplainable by significant change of animal size at birth was observed.  

Records of pre-implantation and early post-implantation loss might suggest potential effect of Low-

and Medium-dose of the test item during a very short critical developmental window.  

Changes of number live pups per dam on postnatal day 0 and 4 and changes in litter weight at birth

described in Mid dose offspring and analysed as statistically nonsignificant should not be omitted

due to high differences in litter characteristics.  

Nonsignificant post-natal loss (higher incidence of records of clinical observations, cases of

morbidity and mortality) described in the Mid dose group could point out on potential to induce

non-monotonous dose response effects by the action of the test item.

The clinical chemistry parameters measured in serum of male and female rats showed some

statistically significant differences. These differences were minimal in nature and had no biological

or toxicological significance.  

No significant differences in haematological parameters among groups exposed to the test item and

controls were found in all evaluated parameter with exception of PT and APTT values. The

differences were minor had no biological or toxicological significance.  

The observed significant changes in T4 levels in the male and female offspring rats (significant

decrease) not accompanied with significant alterations in changes in the mean relative weight of

thyroids (after fixation) as well as with the histological examinations revealed no serious

morphological alterations were considered to be findings without biological/toxicological

significance.

In the urine no significant changes against normal physiological conditions were detected.

Functional battery observation tests (tail flick test and grip strength test) conducted at the end of the

dosing period and at the end of recovery period revealed no abnormalities.

During the gross necropsy, there were found some abnormal tissues in the treated male and female

rats. Described macroscopic findings (being sporadic incidence and without test item dose

dependence) were supplemented/elucidated with histopathological evaluations and considered not

to be test item related alterations in the sense of the prevalence, severity and character.

Significant changes of relative organ weights, being sensitive indicator of the adverse effect of the

test item, were observed in the Cowper´s glands, kidneys (males), spleen (males) and Levartor ani

& bulbocavernosus muscles. Yet, the histological examinations revealed no serious morphological

alterations and mentioned statistically significant differences (when compared to the control

animals) were considered to be without biological significance.

Histopathological lesions found in the treated animals were either of spontaneous character or not

in direct relation with the test item administered. Macroscopically described lesions in the stomach

or jejunum as change of colour (diffuse or focal), were histologically detected as hyperaemia of

capillaries in the mucosa and submucosa. Sporadic findings as defect in renal pelvis histologically  

evaluated as dilatation of the renal pelvis, mucification found in the cervix uteri, dilatation of acinus

in prostate, gold brown pigment particles in ovary, cystic dilatation in the vagina wall   and uterus

lumen dilatation were classified as lesions not related with application of test item.

Applicant's summary and conclusion

Conclusions:
The study was designed to evaluate the potential toxic effects of the Ethyl lactate when administered
to rats for a minimum of 28 days and to evaluate the potential of the test substance to affect male
and female reproductive performance such as gonadal function, mating behavior, conception,
parturition, and early postnatal development. Moreover, the study might comment on system
general toxicity due to evaluations of haematological parameters, clinical biochemistry analyses,
urinalysis and histopathological examinations.
• Clinical observations throughout the first two weeks of test item application indicated presence
of signs of systemic toxicity in the high dose group of female rats (800 mg/kg b.w.).
• No test item-related mortality in significant extent was recorded during the study. No moribund
animals were isolated due to exposure to three abovementioned doses of test substance.
• No statistically significant differences in body weight amongst the treated groups of adult male
and female rats were revealed. The animals grew over time in all experimental groups or
stagnation of body weight was recorded.
• Food consumption was not significantly different amongst the experimental groups.
• During the gross necropsy, macroscopic findings were described in the all treated groups but
incidence was sporadic without clear dose dependency. From microscopic point of view no
significant item related alterations in the sense of the prevalence, severity and character were
described.
• Significant changes of mean relative organ weights in male rats, being sensitive indicator of the
adverse effect of the test substance, were observed in Cowper`s glands, levator ani plus musculus
bulbocavernosus, kidneys and spleen. The histological examinations revealed no serious
morphological alterations and statistically significant differences (when compared to the control
animals) were considered to be findings without biological significance.
• No significant differences in haematological parameters among groups exposed to the test item
and controls were found in all evaluated parameters with exception of PT and APTT values. The
differences were minor, had no biological or toxicological significance.
• The clinical chemistry parameters measured in serum of male and female rats showed some
statistically significant differences. These differences were minimal in nature and had no
biological or toxicological significance.
• In the urine no significant changes against normal physiological conditions were detected.
Based on these results, the no-observed-adverse-effect-level (NOAEL) was 600 mg/kg/day for
parental systemic toxicity.
• No test item-related effects on parental reproductive performance, gestation length, parturition,
or reproductive organs were noted at any dosage level.
• Significant alteration of physical development was demonstrated as increase of body weight in
High dose group male pups on Day 13 post partum.
• The observed significant reduction in T4 levels in the male and female offspring not
accompanied with significant alterations in the mean relative weight of thyroids (after fixation)
as well as with no serious morphological alterations were considered to be findings without
biological/toxicological significance.
• Dose-dependent statistically significant reduction of AGI with adverse potential in male pups
(feminization in Low and Mid dose group male pups) and probably realized via non-traditional
mechanisms resulting in inverted-U-shape dose-response curve (because of mechanism deletion
in High dose group) might be regarded as specific developmental effect of prenatal exposure to
test item and should be considered for setting of NOAEL.
• Records of pre-implantation and early post-implantation loss might suggest potential effect of
Low- and Medium-dose of the test item during a very short critical developmental window.
• Changes of number live pups per dam on postnatal day 0 and 4 and changes in litter weight at
birth described in Mid dose offspring and analysed as statistically nonsignificant should not be
omitted due to high differences in litter characteristics
• Nonsignificant post-natal loss (higher incidence of records of clinical observations, cases of
morbidity and mortality) described in the Mid dose group could point out on potential to induce
non-monotonous dose response effects by the action of the test item.
In summary, the potential of Low and Mid dose of test item to induce statistically significant change
of important developmental landmark (AGI) being under rigorous hormonal control supported with
nonsignificant potential the Low- and the Mid dose to raise pre-implantation and early post-
implantation loss to a greater extent in the Low dose (four non-pregnant females and two females
that failed to deliver vs. two non-pregnant females and two females that failed to deliver in the Mid
dose), which rejected females from next evaluation of reproductive variables and suggested
potential activity also during very short period of uterine receptivity and placentation along with
more variable litter characteristics and higher number (but nonsignificant) of post-natal loss
recorded in the Mid dose group should be considered for setting of no-observable-adverse-effect-
level (NOAEL).
Based on these results, the NOAEL for reproductive toxicity was considered to be less than 75
mg/kg/day.