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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
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 W
EATHERHUB-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
Details on route of administration:
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. A separate cannula for
treatment groups and control groups were used. The dose of test formulation was given once
daily at similar time for 7 days per week in single dose volume of 5 mL.kg-1
body weight. Dose
volume was adjusted according to the weigh development of the animals. A concurrent control
group received the vehicle with carrier only (Water for injection with Methocel A4M; 1%
solution) on a comparable regime. Satellite animals treated on the same regime were not mated
and subsequently used for 14-day post-treatment observation (after scheduled human kill of the
males and females in main groups) for the potential reversibility or persistence, or delayed
occurence of any toxic effects.
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.
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.
Vehicle:
water
Remarks:
Water for Injection (Aqua pro injectione), 500 mL
Details on oral 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-1confirmed 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).
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. 17from 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 / concentrationsopen allclose all
Dose / conc.:
30 other: mg.kg-1 b.w. of test item
Remarks:
Low-dose group
Dose / conc.:
100 other: mg.kg-1 b.w. of test item.
Remarks:
Mid-dose group
Dose / conc.:
300 other: mg.kg-1 b.w. of test item.
Remarks:
High-dose group
No. of animals per sex per dose:
Thirteen females were used per group for pre-treatment estrous cycle examination, treatment,
mating and rearing of the pups. Ten males per group were treated and paired with females in
ratio 1:1 or 1:2 (one male : one female or one male : two females). Ten males and 10 females
treated in satellite groups were not used in reproduction part of the study, they were designated
for post-treatment observation during 14 subsequent days without test item exposure.
Summarily, 50 males and 62 females were treated in the study.
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

Observations and examinations performed and frequency:
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-AdverseEffectLevel (NOAEL) for systemic toxicity in adult male and female rats.
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 register
ed 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 of examinations

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).
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
Description (incidence and severity):
Regarding relative organ weights, statistically significant decrease in mean relative weight of
kidneys in High-dose group males, and increase in right epididymidis, left epididymidis,
coagulating glands-seminal vesicles complex, adrenal glands, spleen, and heart were observed
in High-dose satellite males in comparison with control counterparts treated with 1% Methocel
suspension in Water for injection (Tables 4, 5 and Individual data, Appendix 6).
When absolute organ weights were analysed, the same distribution of mean organ weights
across compared experimental groups was recorded. With respect to statistically significantly
higher mean body weight of Control satellite males when compared to High-dose satellite males and small number of the animals (5 per group) evaluated by means of non-parametric tests,
observed statistically significant differences (especially in reproductive organs) might be
considered as biologically irrelevant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Some abnormal tissues were found in the treated male and female rats during the gross
necropsy. Described macroscopic findings in the Control and the High-dose groups (being sporadic
incidence and without test item dose dependence) were correlated with histopathology
evaluations and considered not test item related alterations. Microscopic changes were
considered as incidental findings or results of experimental manipulation other than
administration of the substance. Summarily, there were no test item related alterations in the
sense of prevalence, severity, or histological character of incidentally found lesions.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
urinalysis
other:
Remarks on result:
not determinable because of methodological limitations

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Systemic toxicity:
Adult 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.