Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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)
Limit test:
yes

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
Details on route of administration:
Dose volume was 5.0 mL/kg of body weight and was adjusted according to the weight development of the animals.
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.
Vehicle:
olive oil
Details on oral exposure:
Thirteen females were used per group for pre-treatment estrous
cycle examination, treatment and mating. Ten males per group were
treated and paired with females in ratio 1:1 (one male:one female).
In summary, 40 males and 52 females were treated within the
reproductive part of study. Five males and five females per group
were orally exposed to test item in the two satellite groups (Control
and High dose group). Totally, 50 male rats and 62 female rats were
used in the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The tested item was administered orally by gavage in a single dose daily. Test item Ethyl lactate was available as a suspension in olive oil. The dose volume was 0.5 ml/100 g body weight of rat.
The dose of thetest substance was given at similar times each day.
• Animals of Group 1 received 100 mg/kg b.w.
• Animals of Group 2 received 200 mg/kgb.w.
• Animals of Group 3 received 500 mg/kg b.w.
• Animals of Group 4 received 800 mg/ kg b.w.
• Animals of Group 4 received 1200 mg/ kg b.w.
Duration of treatment / exposure:
The animals were dosed daily for 7 days a week.
Frequency of treatment:
daily
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
One day before the treatment, all animals were randomized. Within the frame of treatment groups, each rat was marked by code on the tail root to identify animal individually. Each cage was provided with a cage card.
Rats were inspected twice daily for evidence of reaction to treatment or ill-health. The observation focused on changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity as lacrimation, piloerection, pupil size, and unusual respiratory pattern. Moreover, possible changes in gait, posture and response to handling as well as the presence of clonic or tonic movements or bizarre behaviour (self-mutilation, walking backwards) were observed.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Rats were inspected twice daily for sings of toxicity. No deviations from normal findings in terms of: clinical signs, sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli), changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity as lacrimation, piloerection, pupil size, and unusual respiratory pattern were observed. No changes in gait, posture and response to handling as well as the presence of clonic or tonic movements or bizarre behaviour (self-mutilation, walking backwards) were observed.
Mortality:
no mortality observed
Description (incidence):
Three mortality cases were recorded during the study. Animals No 15 (♂) and No 29 (♀) died one week after administration of the test substance. Animal No 8 (♂) died in second week of administration of test substance. All three animals died probably during the night hours, because cadavers were in state of autolysis.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
During the study, body weight of animals increased in time in all group of rats. The test substance was an unpleasant taste and smell for the animals which resulted in lack of appetite of animals. Body weight of male rats in groups G4 800mg/kg and G5 1200mg/kg was little lower.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the end of the study, the animals were subjected to gross necropsy. During gross pathology examination, organs were examined macroscopically and any abnormal tissue were collected and stored for possible future analysis. Tissues were added into fixative solution (10 % buffered formalin).
During the necropsy there were observed diffuse discolorations (red) on thymus in male rats No (♂5), (♂6) from Group 2 –200mg/ kg, (♂7), (♂9) from Group 3 – 500mg/kg and No (♂11) from Group 4 – 800mg/kg.
These findings are not considered to be caused by the test substance.
No macroscopic findings were observed during the necropsy in female rats.

Effect levels

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

Target system / organ toxicity

Critical effects observed:
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:
After consideration of the study results following conclusions were made regarding the test item.
• The High dose of Ethyl lactate 800 mg/kg b.w. induced systemic parental toxicity which
overreached 10% mortality, therefore test doses were reduced since 10th day of application.
• Test item did not induce significant clinical findings or behavioural deviations in the
experimental animals exposed to doses of Low dose - 75 mg/kg b.w., Mid dose -300 mg/kg
b.w. and High dose - 600 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 item.
• 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,
no significant reduction or stagnation of body weight was recorded.
• The test item caused statistically significant increase of body weight in offspring males at
the high dose (600 mg/kg) at Day 13 post-partum.
• Test item did not affect the food consumption. Food consumption was not significantly
different amongst the experimental groups.
• 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 parameters with exception of PT and APTT
values. The differences were minor, had no biological or toxicological significance.
• In the urine no significant changes against normal physiological conditions were detected.
• 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.
• 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.
• Macroscopic findings described during the gross necropsy, were observed 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.
• Test item did not cause histopathological changes on examined reproduction organs
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, 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.
• 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.
Based on these results,
- the no-observed-adverse-effect-level (NOAEL) was 600 mg/kg/day for parental systemic
toxicity.
- the NOAEL for reproductive toxicity was considered to be less than 75 mg/kg/day.