3-acetylthiazolidine-4-carboxylic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-acetylthiazolidine-4-carboxylic acid

EC Number:

225-713-6

EC Name:

3-acetylthiazolidine-4-carboxylic acid

Cas Number:

5025-82-1

Molecular formula:

C6H9NO3S

IUPAC Name:

3-acetylthiazolidine-4-carboxylic acid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat Wistar Rcc Han/Specific Pathogen Free (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The study was carried out in the Experimental Animal House of Slovak Medical University in Bratislava, part Specific Pathogen Free (SPF) on the 3rd floor, in the room No. B2-310, with the
central pressure air-conditioning and under defined laboratory conditions. The temperature range of 20-24 °C and humidity range of 45-65 % were taken as optimal, and ranges of 20-26 °C and 30-70 % as acceptable. Temperature and humidity were constantly monitored automatically by WEATHERHUB-OKO, WH Observer Platform and recorded daily (data logger No.: OE19A10421A7, date of calibration by Accredited Calibration Laboratory - Slovak Legal Metrology n. o.: 12.04.2019, Certificate No. 0584/321.05/191159/321.15/19; date of last control by working standard: 16.08.2021). Minimal and maximal temperatures in room B2-310 was as follows: min 20.0°C and max 24.0°C. Minimal and maximal humidity in room was 40% and 60%, respectively. The artificial lighting was set to a 12-hour light and 12-hour dark photoperiods.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

The test item, 3-Acetyl-Thiazolidine-4-Carboxylic Acid, in the vehicle (Water for injection) with carrier (Methocel A4M), were administered per os by gavage using an appropriately sized stainless steel ball-tipped dosing cannula connected with syringe.

Details on exposure:

The stability and homogeneity of 3-Acetyl-Thiazolidine-4-Carboxylic Acid in the vehicle – 1%
Methocel A4M suspension in Aqua pro injectione were determined by high-performance liquid
chromatography (HPLC) method. Repeated measurements for weekly stability confirmed the
consistency of the test item concentrations of 6 and 60 mg.mL-1 when storing the formulation
mixtures in well-sealed containers, in refrigerator at temperature of 6.5°C ± 1.5°C.
Homogeneity analysis performed for dose concentrations of 6, 20 and 60 mg.mL-1
confirmed uniform dispersion of 3-Acetyl-Thiazolidine-4-Carboxylic Acid in 1% Methocel A4M
suspension in Aqua pro injectione. The content of test item in the vehicle was checked twice
during the study at the beginning and at termination of the study. The test item concentrations
in analysed samples were 88.75-93.75% of the target concentrations within the acceptable limits
(70% - 110% of the target concentrations).

Details on mating procedure:

Mating procedure and gestation：
One male:one female or one male:two females mating (as it is specified in Table 4A, Appendix
3) was used in the study. The female/females was/were placed with the male until evidence of
copulation – presence of sperm in vaginal lavage – was observed each morning during
examination of sperm positivity. Day 0 of pregnancy was described as the day on which mating
evidence was confirmed. Re-mating of females with proven males was not carried out.
The duration of gestation was recorded and calculated since Day 0 of pregnancy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations for homogeneity and concentration during dosing period was
conducted in the analytical laboratory of Test Site 2: the Central Control and Testing Institute
in Agriculture two times per in-life phase of the study using a validated method. For this
purpose, triplicate samples (5 mL of each) were collected from top, middle and bottom strata
of each dosing formulation (6, 20 and 60 mg. mL-1
) prepared at the beginning and at the end of
the in-life phase of the study (Certificate of Delivery No. 6 from October 19, 2021, and No. 17
from November 30, 2021).

Duration of treatment / exposure:

Male rats were treated during:
• 14-day pre-mating period
• 6-day mating period
• and 16-17-day post-mating period
Males were dosed for a total of 36-37 days.
Female rats were treated during:
• 14-day pre-mating period
• 6-day mating period
• 21-24-day gestation period
• and 13-day lactation period
Females received for a total of 54-57 doses.

Frequency of treatment:

The satellite males and females designated for post-treatment observation (5 animals per sex in
Control and High dose satellite groups, respectively) received for a total of 35 and 56 daily
doses, respectively, and susbsequently remained untreated for 14 days.
Non-pregnant females were humanely killed 27 days after the last day of the mating period after
receiving for a total of 47 doses.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 mg/kg bw/day

Remarks:

High-dose group

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Mid-dose group

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day

Remarks:

Low-dose group

No. of animals per sex per dose:

Females received 14 daily doses prior to pairing and were exposed to the test item throughout
mating (1-6 days), gestation (21-24 days) and lactation period until Day 13 post-partum. Female
rats in main groups received totally from 51 to 57 doses (Necropsy Days: 20.-26.11.2021).
Non-pregnant females were dosed for a total of 47 doses (Necropsy Day: 16.11.2021). Males
were administered 14 daily doses prior to mating and throughout the pairing period for a total
of 36-37 doses (Necropsy Days: 4.-5.11.2021). Female rats in satellite groups were exposed to
test item for total of 56 days (Necropsy Day on Day 71 of the study after 14-day recovery
period: 9.12.2021). Male rats in satellite groups received 35 daily doses (Necropsy Day on Day
50 of the study after 14-day recovery period: 18.11.2021).

Control animals:

yes

Details on study design:

The test item 3-Acetyl-Thiazolidine-4-Carboxylic Acid in vehicle - 1% Methocel suspension
in aqua for injection was administered by gavage in graduated doses to three groups of male
and female Wistar RCC Han rats daily for 7 days per week.
Ten reproducibly healthy male rats and 13 healthy virgin female rats were allocated in one
Control (vehicle) group and three dose level groups (Low-, Mid- and High-dose groups).
Moreover, ten male rats and 10 female rats (5 animals per sex) were used in two satellite groups,
Control satellite group and High-dose satellite group as follows:
• Control group received vehicle - 1% Methocel suspension in aqua for injection
• Low-dose group received 30 mg.kg-1
b.w. of test item.
• Mid-dose group received 100 mg.kg-1
b.w. of test item
• High-dose group received 300 mg.kg-1
b.w. of test item
and
• Control Satellite group received vehicle - 1% Methocel suspension in aqua for injection
• High-dose Satellite group received 300 mg.kg-1
b.w. of test item.
The application volume used was 5 mL.kg-1
actual body weight of the rat.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

Parental and Satellite Male and Female Rats:
Based on inconsistent and fortuitous effects described in adult male and female rats exposed to
the concentration of 300 mg.kg-1 b.w. per day, the 3-Acetyl-Thiazolidine-4-Carboxylic Acid
concentration of 300 mg.kg-1 b.w. per day is recommended for the No-Observed-AdverseEffect-Level (NOAEL) for systemic toxicity in adult male and female rats.

Oestrous cyclicity (parental animals):

Oestrous cycle was monitored two weeks before treatment beginning and two weeks during the
pre-mating treatment period until sperm appeared in vaginal lavage during mating period as
evidence of copulation (mating period: 1-6 days). Vaginal smears were also examined before
necropsy (Day 14 post-partum in parental females and after 14-day recovery period in satellite
females) to correlate the stage of oestrous cycle with histopathological findings of reproductive
organs.

Sperm parameters (parental animals):

One male:one female or one male:two females mating (as it is specified in Table 4A, Appendix
3) was used in the study. The female/females was/were placed with the male until evidence of
copulation – presence of sperm in vaginal lavage – was observed each morning during
examination of sperm positivity. Day 0 of pregnancy was described as the day on which mating
evidence was confirmed. Re-mating of females with proven males was not carried out.
The duration of gestation was recorded and calculated since Day 0 of pregnancy.

Litter observations:

Each litter was observed on daily basis to record survival, abnormalities, and deaths of pups

Postmortem examinations (parental animals):

Tail Flick test, Grip Strength test and Open Field test were performed at the end of the study in
male rats of the main test groups and at second week of 2-week recovery period in satellite
animals of both genders. In parental females, functional observations were made during the last
week of lactation, shortly before scheduled sacrifice. Tests were carried out in 5 randomly
selected animals per sex in the Control and three Dose groups and in all satellite animals.

Postmortem examinations (offspring):

Dead pups and pups humanely sacrificed at Day 4 (culling) and Day 13 post-partum (scheduled
termination) were carefully examined externally for gross abnormalities. Particular attention
was paid to the external reproductive genitals that were examined for signs of altered
development. From physical developmental landmarks, eye opening was evaluated in Day 13
pups to assess delayed or accelerated developmental time course for this parameter.

Statistics:

Statistical analyses:
Male Rat Offspring:
Mean body weight of the neonatal males (Day 0) was significantly different among
experimental groups within 24 hours post-partum (P=0.042), but Mann-Whitney test was not
able to discriminate difference between control and test item treated pups. It is worth to note,
that male neonates in the Mid-dose group had the lowest mean/median body weights.
On Day 4 after births, Kruskal-Wallis test recognized statistically significant difference in mean
body weight of male pup groups (P=0.008). Mann-Whitney test showed, clear dose dependent
decrease of mean body weight in the Low-dose group (P=0.009) and the Mid-dose group
(P=0.001) of males when compared to the control counterparts.
In infantile males on Day 13 of lactation exposure, statistically significant reduction of mean
body weight still persisted in the Mid-dose group males against control pups (Kruskal-Wallis
test P<0.001; Mann Whitney test P=0.016).
Female Rat Offspring:
Similar trends as with male neonates and infants were observed in female offspring.
On Day 0, statistically significant difference among groups of females was recorded (KruskalWallis test, P=0.014), however, the lowest mean body weight in the Mid-dose female pups was not shown as statistically significant when compared to the Control group (Mann-Whitney test, P=0.073). On Day 4 post-partum, marked dose dependent reduction of mean body weight of female pups was registered in Low-dose group (P=0.016) and in Mid-dose group (P=0.000) vs. control female pups. Female pups in High-dose group were comparable with Control.
In lactation period, in Day 13 infantile females, statistically significantly lowest mean body
weight was observed in pups exposed to mid dose of test item in comparison with control
counterparts (Mann-Whitney test, P=0.006).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

The test item, 3-Acetyl-Thiazolidine-4-Carboxylic Acid, orally administered to the parental
male and female rats did not induce significant clinical signs or behavioural deviations in the
experimental animals and subsequently in their offspring exposed to three graduated doses:
Low dose - 30 mg.kg-1 b.w., Mid dose -100 mg.kg-1 b.w., High dose - 300 mg.kg-1 b.w (Adult:
Table 1-3, Appendix 1, and Offspring: Individual data, Appendix 3).

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No test item-related mortality was recorded during the study. One male rat ID38 from Highdose group was euthanized for human reasons on 14.10.2021 (the 1st day of mating period).
Dispnoe, body weight loss, piloerection and vocalization were observed after 9-day application
of the test item (Table 1, Appendix 1). Dilatation of small intestine and stomach with gas
content were observed at necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the case adult animals, the High-dose satellite males (300 mg.kg-1 b.w.) shown significantly
lower mean values of the body weight when compared to the Control satellite group from Day
22 of dosing until overnight fasting at termination of the study (including 2-week recovery
period without application). Significantly lower mean body weight was observed also in the
High-dose satellite females but only at day of necropsy after overnight starvation (P=0.028 vs.
the Control satellite group). The significant alterations described in the satellite rats treated with
test item were inconsistent and might be considered as effects without biological significance
(Table 1-4 and Individual data, Appendix 2).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Different character of the food intake alterations was interpreted in the experimental groups of
both genders. In parental and satellite males, significantly reduced dose dependent mean food
consumption was recorded in treated groups when compared to the Control, probably due to
depression of appetite by the test substance. In females during premating period, there was
revealed significantly different food intake in the Low-dose group against the Control. No
marked food intake changes were observed in parental females during gravidity and lactation
periods. In satellite females during treatment and recovery periods, there was obviously lower
mean food intake in the High-dose satellite group against the control counterparts (except for 2
measurements). The significant alterations described in rats treated with test item were
inconsistent, without corresponding body weight change described above and therefore might
be concluded as biologically irrelevant (Table 5-8 and Individual data, Appendix 2).

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significant decrease of Haemoglobin (HBG) and Haematocrit (HTC) in Low-dose and Middose groups and marked increase of Mean Corpuscular Volume (MCV) in Low-dose group
were measured in the female rats (Tables 3 and Individual data in Appendix 5). Observed
changes were inconsistent, minimal in nature and therefore might be regarded as not test item
related (Gracia-Manzano et al., 2001; Okamura et al. 2011).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry analyses (serum chemistry) were performed in five males and five
females - dams randomly selected from each group and in all satellite animals. Blood collection
was done at prior to euthanasia of females - dams on Day 14 post-partum. Dams starved
overnight from day 13 to day 14 of lactation, after the pups were removed, to prevent lactation
disturbing or atypical nursing behaviour. Blood collection in males were made after the end of
mating, after overnight fasting. Blood samples from all satellite animals were collected after
recovery period and fasting. Blood was taken from abdominal aorta (as an integral part of the
necropsy), processed to obtain serum and analysed immediately or in serum samples frozen at
-20°C.
The clinical biochemistry parameters measured in serum of 5 randomly selected male and
female rats showed some statistically significant differences in group of male satellite rats
exposed to high dose of test item (Alanine aminotransferase, Glucose, Triacylglycerols and
Total proteins; Tables 11-12 and Individual data, Appendix 5). The differences were minor
and had no biological or toxicological significance.
Significant impact of test item on T4 concentrations was identified in the Mid- and High-dose
groups of Day 4 female pups (Table 17 and Individual data, Appendix 5). Based on results
in Day 13 female pups in the Mid- and High-dose groups that are comparable with the control
females, this finding might be considered as biologically irrelevant, though effect can be
manifested at other level of thyroid regulation axis.

Endocrine findings:

not specified

Description (incidence and severity):

the endpoints that are the indications of the major toxic effects, target organs of toxicity and
accumulation of the test item as well as potential adverse endocrine disrupting effects were
examined.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis findings recorded in the Control as well as Dose groups of males and in the satellite
males (The Control and the High-dose groups) can be considered as normal or close to normal
according to available historical data (Table 18-19 and Individual data, Appendix 5),
(Historical Control Data of Urinalysis in HsdRccHanTM: WIST, Wistar Hannover Rats).

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

In our study, statistically significant AGD shortening was revealed in the Mid-dose group male
pups. On the other hand, significant increase of AGD was recorded in the High-dose group of
males. In the female pups, there were recognized statistically significant reduction of AGD in
the Mid-dose group as well as in the High-dose group and AGD reduction at limit of statistical
significance in the Low-dose group (P=0.055) probably due to higher variability of individual
measurements. Significant change in sense of decreased values was subsequently identified also
for AGI calculated.
As AGD is the most sensitive marker of masculinization and/or feminization in the
toxicological studies, its statistically significant change might suggest potential
androgenic/antiandrogenic action of test item. Prenatal exposure to test item might alter
circulating hormone levels during pregnancy, which expose developing embryos to an improper
hormonal environment during masculinization programming window that is critical for
development of the reproductive tract. In males, high intrauterine androgen exposure results in
longer AGD. Conversely, exposure to estrogen-like (anti-androgen) chemicals reduces the
AGD (Crestani et al., 2020). Along this line, the AGD reduction may be associated with
increased likelihood of testicular maldescent, small and indistinct scrotum and smaller penile
size in adult male rats. In adult men, cryptorchidism or hypospadias, lower testosterone levels,
poorer semen quality and infertility were described. In the women, longer adult AGD has been
associated with higher testosterone levels and corresponding ovarian dysgenesis syndrome,
whereas shorter AGD has been linked to endometriosis (Priskorn et al., 2018; Crestani et al.,
2020). Moreover, rodent studies have indicated plasticity in AGD and demonstrated the need
of adequate androgen levels not only prenatally but also postnatally to achieve or retain the
AGD programmed in utero (Kita et al., 2016).
Considering toxicological data, our findings could have implications for reproductive health
and fertility in humans, with AGD being described to be more sensitive in humans as in rodents
and quite consistent across species (Hotchkiss et al., 2005; Hotchkiss et al., 2007; MiraEscolano et al., 2014; Swan et al., 2005).
Specifically, observed statistically significant change of AGD/AGI with adverse potential in
male and female pups (in the Mid-dose and the High-dose group vs. Control) probably realized
via non-traditional mechanisms resulting in U-shape dose-response curve might be regarded as
specific developmental effect of prenatal exposure to test item and should be considered for
setting of NOAEL.
In general, a statistically significant change in AGD that cannot be explained by the size of the
animal indicates effects of the exposure and should be used for setting the NOAEL.

Details on results (P0)

Systemic toxicity:
Parental and Satellite Male and Female Rats:
Based on inconsistent and fortuitous effects described in adult male and female rats exposed to
the concentration of 300 mg.kg-1 b.w. per day, the 3-Acetyl-Thiazolidine-4-Carboxylic Acid
concentration of 300 mg.kg-1 b.w. per day is recommended for the No-Observed-AdverseEffect-Level (NOAEL) for systemic toxicity in adult male and female rats

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

clinical biochemistry

urinalysis

other:

Remarks on result:

other: No adverse changes were noted in any of these parameters.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had induced alteration of physical development demonstrated as reduction of pup
body weight on Day 4 and Day 13 of lactation exposure, in significant extent in Day 4 Lowdose and Mid-dose group pups and in Day 13 Mid-dose group pups.
This fact should be considered in relation to thyroid gland function and structure by means of
plasma T4 levels in Day 4- and Day 13-pups, and thyroid gland weight and its microscopic
pattern in Day 13 pups. In Day 4 female pups (but not in male pups), significant decrease of T4
plasma concentrations in the Mid- and High-dose groups was observed (Table 17 and
Individual data, Appendix 5). This T4 level reduction was accompanied with significant
decrease of mean body weight in Mid-dose group females (as well in Mid-dose group males)
measured at this day (Table 1, Appendix 3).
In Day 13 Mid-dose group pups, no significant dose effects were observed in the mean relative
weight of thyroids (after fixation; Table 4 and 6, Individual data in Appendix 6) neither
altered histopathological pattern of pup thyroid glands (Christian and Trenton, 2003;
Individual data, Appendix 6) following exposure to test Mid dose were described. Though no
significant impact of test item was recorded on T4 levels in Day 13 pups (Table 16 and
Individual data, Appendix 5), marked decrease in mean body weight persisted in the Middose group (Table 1, Appendix 3).
On the other hand, the endocrine disrupting chemicals can interfere with thyroid hormone action
in a more complex manner within the scope of the thyroid regulation axis (the hypothalamus,
the pituitary, the thyroid gland, or the thyroid hormone-regulated tissues) what means that
ability chemicals to interfere with the thyroid function cannot rely solely on their levels in
blood. It is not clear to what degree various adaptive responses in the thyroid axis can
compensate for adverse effects and when during development these adaptive responses become
mature (Gore et al., 2015).
In general, statistically significant changes in offspring body weight in the lactation period
might such as be signs of developmental delay and should be considered as adverse.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significant decrease of Haemoglobin (HBG) and Haematocrit (HTC) in Low-dose and Middose groups and marked increase of Mean Corpuscular Volume (MCV) in Low-dose group
were measured in the female rats (Tables 3 and Individual data in Appendix 5). Observed
changes were inconsistent, minimal in nature and therefore might be regarded as not test item
related (Gracia-Manzano et al., 2001; Okamura et al. 2011).

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis findings recorded in the Control as well as Dose groups of males and in the satellite
males (The Control and the High-dose groups) can be considered as normal or close to normal
according to available historical data (Table 18-19 and Individual data, Appendix 5),
(Historical Control Data of Urinalysis in HsdRccHanTM: WIST, Wistar Hannover Rats).

Sexual maturation:

not specified

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Within the frame of reproduction parameters (except for offspring body weight) anogenital
distance (AGD) / anogenital index (AGI) were parameters that shown significant differences
amongst experimental groups in male as well as female offspring (Table 1-3, Appendix 3).
AGD is determined prenatally by the testosterone metabolite, dihydrotestosterone, therefore,
can be used as a marker for a decrease of prenatal testosterone levels (Swan et al. 2005) and
intrauterine androgen exposure (Crestani et al., 2020). The AGD is developmental marker about
twice as long in males as in females. In addition, in litter-bearing mammals, AGD varies with
intrauterine position of foetuses (sex hormone influence of adjacent foetuses), and it is
predictive of number of adult physiological and behavioural characteristics (Hotchkiss et al.,
2005; voom Saal, 1989).
In our study, statistically significant AGD shortening was revealed in the Mid-dose group male
pups. On the other hand, significant increase of AGD was recorded in the High-dose group of
males. In the female pups, there were recognized statistically significant reduction of AGD in
the Mid-dose group as well as in the High-dose group and AGD reduction at limit of statistical
significance in the Low-dose group (P=0.055) probably due to higher variability of individual
measurements. Significant change in sense of decreased values was subsequently identified also
for AGI calculated.
As AGD is the most sensitive marker of masculinization and/or feminization in the
toxicological studies, its statistically significant change might suggest potential
androgenic/antiandrogenic action of test item. Prenatal exposure to test item might alter
circulating hormone levels during pregnancy, which expose developing embryos to an improper
hormonal environment during masculinization programming window that is critical for
development of the reproductive tract. In males, high intrauterine androgen exposure results in
longer AGD. Conversely, exposure to estrogen-like (anti-androgen) chemicals reduces the
AGD (Crestani et al., 2020). Along this line, the AGD reduction may be associated with
increased likelihood of testicular maldescent, small and indistinct scrotum and smaller penile
size in adult male rats. In adult men, cryptorchidism or hypospadias, lower testosterone levels,
poorer semen quality and infertility were described. In the women, longer adult AGD has been
associated with higher testosterone levels and corresponding ovarian dysgenesis syndrome,
whereas shorter AGD has been linked to endometriosis (Priskorn et al., 2018; Crestani et al.,
2020). Moreover, rodent studies have indicated plasticity in AGD and demonstrated the need
of adequate androgen levels not only prenatally but also postnatally to achieve or retain the
AGD programmed in utero (Kita et al., 2016).
Considering toxicological data, our findings could have implications for reproductive health
and fertility in humans, with AGD being described to be more sensitive in humans as in rodents
and quite consistent across species (Hotchkiss et al., 2005; Hotchkiss et al., 2007; MiraEscolano et al., 2014; Swan et al., 2005).
Specifically, observed statistically significant change of AGD/AGI with adverse potential in
male and female pups (in the Mid-dose and the High-dose group vs. Control) probably realized
via non-traditional mechanisms resulting in U-shape dose-response curve might be regarded as
specific developmental effect of prenatal exposure to test item and should be considered for
setting of NOAEL.
In general, a statistically significant change in AGD that cannot be explained by the size of the
animal indicates effects of the exposure and should be used for setting the NOAEL.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Nipple/areole retention
Nipples or areola retention was assessed in the male rat offspring on Day 13, at scheduled
necropsy. No nipples/areolas were visible in male pups exposed to the three graduated doses of
test item during in utero development and lactation.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

Reproductive and Developmental toxicity:
Male and Female Offspring:
• Statistically significant dose-independent changes in the offspring body weight during
lactation period (Day 4 and Day 13 post-partum) most pronounced in the Mid-dose Day 4
offspring and persisting in the Mid-dose Day 13 offspring might such as be signs of
developmental delay and should be considered as adverse effects of test item exposure.
• Statistically significant dose-independent shortening of the important developmental
landmark - anogenital distance (AGD, being under rigorous hormonal control) with adverse
potential in male as well as female pups (most marked in the Mid dose group offspring)
probably realized via non-traditional mechanisms resulting in non-monotonous U-shape
dose-response curve might be regarded as specific developmental effect of prenatal exposure
to test item and should be considered for setting of NOAEL.
Based on significant and persisting effects observed in male and female offspring treated with
100 mg.kg-1 b.w., the 3-Acetyl-Thiazolidine-4-Carboxylic Acid concentration of 30 mg.kg-1
b.w. per day is suggested for the No-Observed-Adverse-Effect-Level (NOAEL) for
reproductive toxicity in male and female rat offspring.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

clinical biochemistry

other:

Remarks on result:

other: No adverse changes were noted in any of these parameters.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

Considering results obtained under defined experimental conditions following conclusions
were made regarding the test item 3-Acetyl-Thiazolidine-4-Carboxylic Acid:
Systemic toxicity:
Parental and Satellite Male and Female Rats:
Based on inconsistent and fortuitous effects described in adult male and female rats exposed to
the concentration of 300 mg.kg-1 b.w. per day, the 3-Acetyl-Thiazolidine-4-Carboxylic Acid
concentration of 300 mg.kg-1 b.w. per day is recommended for the No-Observed-AdverseEffect-Level (NOAEL) for systemic toxicity in adult male and female rats.
Reproductive and Developmental toxicity:
Male and Female Offspring:
• Statistically significant dose-independent changes in the offspring body weight during
lactation period (Day 4 and Day 13 post-partum) most pronounced in the Mid-dose Day 4
offspring and persisting in the Mid-dose Day 13 offspring might such as be signs of
developmental delay and should be considered as adverse effects of test item exposure.
• Statistically significant dose-independent shortening of the important developmental
landmark - anogenital distance (AGD, being under rigorous hormonal control) with adverse
potential in male as well as female pups (most marked in the Mid dose group offspring)
probably realized via non-traditional mechanisms resulting in non-monotonous U-shape
dose-response curve might be regarded as specific developmental effect of prenatal exposure
to test item and should be considered for setting of NOAEL.
Based on significant and persisting effects observed in male and female offspring treated with
100 mg.kg-1 b.w., the 3-Acetyl-Thiazolidine-4-Carboxylic Acid concentration of 30 mg.kg-1
b.w. per day is suggested for the No-Observed-Adverse-Effect-Level (NOAEL) for
reproductive toxicity in male and female rat offspring.

Executive summary:

3-Acetyl-Thiazolidine-4-Carboxylic Acid Combined Repeated Dose Toxicity Study with the
Reproduction/Developmental Toxicity Screening Test was performed by the test strategy
OECD 422 focused on identification of test item potential to induce disturbance of the male
and female reproductive performance such as gonadal function, mating behaviour, conception,
development of the conceptus and parturition, and early postnatal development. Moreover, the
endpoints that are the indications of the major toxic effects, target organs of toxicity and
accumulation of the test item as well as potential adverse endocrine disrupting effects were
examined.