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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

The NOAEL (No Observed Adverse Effect Level) for the test item 4-oxovaleric acid for TOXICITY in pregnant females was established as 500 mg/kg bw/day.


 


The NOAEL (No Observed Adverse Effect Level) for the test item 4-oxovaleric acid for PRENATAL DEVELOPMENT was established as 1000 mg/kg bw/day.


 

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 24th to December 06th, 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted by the Council on 25th June 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Wistar CRL (SPF quality - guaranteed)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
- Age at study initiation: Dose-range finding experiment: 10 weeks; Main study: 11 weeks
- Weight at study initiation: 262,55 (mean weight)
- Housing: monitored conditions, microbiologically defined background, according to SOP No. 40
- Diet: complete pelleted diet for rats and mice in SPF breeding (Altromin Spezialfutter) was used.
- Water: ad libitum
- Acclimation period: Dose-range finding experiment: 13 days; Main study: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

IN-LIFE DATES: From: 09.08.2021 To: 01.09.2021
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
aqua pro iniectione
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The application form of the test item was administered to the stomach by gavage. The application forms of the test item in Aqua pro iniectione were prepared daily just before administration. The test item was weighted into glass beaker and dissolved in portion of the vehicle. The remaining vehicle was then replenished and the application form was stirred by magnetic stirrer at 300 rpm for 45 minutes and then during the administration.

VEHICLE
Aqua pro iniectione
Manufacturer: Ardeapharma Ševětín, Czech Republic

DRFE: Batch No.: 2102090110
Main study:
Batch No.: 2103160210
Batch No.: 2103190220
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test item application forms were determined by measuring of a peak area of the test item by a high-performance liquid chromatography based on a method developed at the test facility.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male and 2 females
- Length of cohabitation: 10 days
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
15 days
Frequency of treatment:
daily
Duration of test:
20 days
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
21 females in the control group
22 females in the 100 mg/kg bw group
20 females in the 500 mg/kg bw group
22 females in the 1000 mg/kg bw group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: On the basis of the results after evaluation the DRFE the following dose levels – 100, 500 and 1000 mg/kg bw/day were chosen for the main test. These doses were chosen in order to align it with the doses being tested in an OECD 408 test.
- Rationale for animal assignment: randomly assigned to the treatment groups.
- Time of day for (rat) dam blood sampling: 6.00 – 7.00 am on the 20th day of pregnancy before necropsy.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Females were observed in natural conditions in their cages after application, once a day 3 hours after test item application each day.

BODY WEIGHT: Yes
- Time schedule for examinations: First weighing was performed on the 1st day of pregnancy and then on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy.

FOOD CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20th
- Organs examined: Skeleton: cranium, sternebra, vertebrae, ribs
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
- Plasma: No data
- Serum: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
- Anogenital distance of each foetus was measured by digital calliper on the 20th day of pregnancy of females and the corrected AGD was calculated
Statistics:
For statistical evaluation the software Statgraphic® Centurion (version XV, USA) was used. The data from control group were compared with data from treated groups. The results statistically significant on probability level 0.05 are indicated in the summary tables.

The parametric tests were used for statistical evaluation of:
• body weight of females (5th, 8th, 11th, 14th, 17th, 20th day of pregnancy)
• body weight increment
• corrected body weight (subtraction weight of uterus from surgery body weight of females)
• food consumption (per interval)
• mean weight of foetuses (males, females, both sex)
• anogenital distance
• thyroid hormones
• biometry of thyroid gland (absolute and relative weight)
• biometry of uterus (absolute and relative weight )
• preimplantation (IUDE) and postimplantation (IUDL) losses

As the first step, the test for normality (Shapiro-Wilk test) was performed. If the data were not normally distributed the transformation of data was performed (Box-Cox transformation). If the data were not normal distributed after transformation the non-parametric tests (Kruskal-Wallis Test and Mann-Whitney test) for comparison of the medians were performed.
If data were normally distributed after transformation, the Variance check (Levene’s test) to verify standard deviations within each group was used. One-Way ANOVA (probability level 0.05) was used to detect whether there were any significant differences amongst the means and then the post hoc statistical testing (Fisher's least significant difference - LSD test) for only statistical significant differences was performed.

The non-parametric tests were used for statistical evaluation of following parameters:
• number of corpora lutea, number of implantations, number of resorptions
• number of live foetuses (males, females, both sex)
• number of dead foetuses
The two-groups Mann-Whitney test (probability level 0.05) was applied.
Clinical signs:
no effects observed
Description (incidence and severity):
No unscheduled death of females was recorded during the study
Mortality:
no mortality observed
Description (incidence):
No unscheduled death of females was recorded during the study
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Only females who were found pregnant on the 20th day of gravidity (females with live foetuses) were used for calculation of mean body weights. The statistical evaluation was performed from the 5th to 20th day of pregnancy.
The body weights were decreased at the dose level 1000 mg/kg bw/day compared to the control group, on the 14th and 20th day of pregnancy statistically significantly. The body weight increments were statistically significantly decreased at the dose levels 100 and 1000 mg/kg bw/day.
Description (incidence and severity):
Although this is not a feeding study food consumption was assessed and results are reported hereafter.
Only females which were found to be pregnant on the 20th day of gestation (females with live foetuses) were used for calculation of mean food consumption. The statistical evaluation was performed from the 5th to 20th day of pregnancy.
Mean food consumptions were decreased from the 8th to 14th day of pregnancy at the dose level 1000 mg/kg bw/day compared to the control group, on the 8th day of the pregnancy with statistical significance
Endocrine findings:
no effects observed
Description (incidence and severity):
Blood samples from the females which were found to be pregnant on the 20th day of gestation were assessed for serum levels of thyroid hormones (T3, T4, TSH).
No statistically significant differences were recorded in serum levels of thyroid hormones T3, T4 and TSH in females from treated groups against control females.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The behaviour of treated maternal animals were similar compared to the control animals.
Description (incidence and severity):
The uteri of all females were weighed, but only females which were found to be pregnant on the 20th day of gestation were used for calculation of the mean weight of uterus. The absolute weight of uterus was recorded and the relative weight of uterus was computed. The statistical evaluation of absolute and relative weight of uterus was performed.
The absolute weight of uterus was insignificantly decreased at the dose level 1000 mg/kg/day in comparison with control group. The relative weights of uterus in all dose levels was comparable with control.
The thyroid glands of all females were weighed (after fixation), but only females which were found to be pregnant on the 20th day of gestation were used for calculation of mean weight of thyroid gland. The absolute weight of thyroid gland was recorded and the relative weight of thyroid gland was computed. The statistical evaluation of absolute and relative weight of thyroid glands were performed.
Absolute and relative weights of the thyroid glands were similar in the treated and control females. Statistically significant differences of thyroid weights were not detected in females of any dose level.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination was performed in all females (including non-pregnant females). No finding was noted at necropsy in treated females.
Neuropathological findings:
not examined
Description (incidence and severity):
The histopathology of the thyroid glands was performed for all treated and control females. No histopathological changes were found in more than half of the females in each groups. The changes found in the remaining animals are few in number, weak in expression and affect all groups of animals studied. Mild hypertrophy found in two control females and in four females at the dose level 1000 mg/kg bw/day indicated increased reversible thyroid activity. The changes are isolated, weakly expressed and do not deviate from natural variability of the thyroid histological picture of clinically healthy animals.
Histopathological findings: neoplastic:
not specified
Description (incidence and severity):
Body weight - corrected
Corrected body weight of females was calculated, but only females which were found to be pregnant on the 20th day of gestation were used for calculation of the mean corrected body weight. The statistical evaluation of corrected body weight was performed.
Mean value of corrected body weight of females was statistically significantly decreased at the dose level 1000 mg/kg bw/day in comparison with control
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of implantations and corpora lutea were similar in all treated groups with control group. Preimplantation losses (IUDE) and postimplantation losses (IUDL) were comparable or lower in the treated groups than in the control group. Statistically significant differences were not detected in these parameters.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers resorptions were similar in all treated groups with control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead foetus was found in treated groups and control.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Details on maternal toxic effects:
The reduced fetal weights may be related to the maternal toxicity of the test item (decreased maternal body weight and/or food intake) at the highest dose level, but the maternal toxicity was not evident at the dose level 500 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
The mean body weight of foetuses was decreased at the dose levels 500 and 1000 mg/kg/day, statistical significance was not detected. Male foetuses were heavier than females in all groups
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No statistically significant differences with toxicological significance were recorded in number of litters during the statistical evaluation of skeletal findings.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
The AGD and corrected AGD of male and female foetuses at all dose levels were comparable with control group
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
Examination of symmetry of fore and hind limbs, number of fingers, closing or opening of eye fissures and external auditory canal, symmetry of head, integrity of superior palatum, status of umbilicus and genital papilla were performed.
No external changes were recorded in treated groups and control
Description (incidence and severity):
Examination of foetal cranium revealed mainly incomplete ossification of cranial bones. Incompletely ossified were mostly parietal bone, interparietal bone, supraoccipital bone, squamosal part of temporal bone, arcus zygomaticus, less frequently nasal bone, frontal bone, premaxilla, maxila and basisphenoid. High incidence of incomplete ossification of parietal bone, interparietal bone, supraoccipital bone, squamosal part of temporal bone were recorded at all dose levels as well in control group. The proportion of litters with incomplete ossification of premaxilla, maxilla and basisphenoid were comparable or lower at all dose levels in comparison with control group. The increased portion of litters with incomplete ossification of frontal bone was recorded at the dose levels 500 and 1000 mg/kg bw/day (9.52 % – 18.18 % – 45.00 % – 27.27 %), without statistical significance and dose dependence. The portion of litters with incomplete ossification of nasal bone was increased only at the dose level 500 mg/kg bw/day. The statistically insignificantly increased incidence of litters with incomplete ossification of arcus zygomaticus was recorded at the dose levels 500 and 1000 mg/kg bw/day (57.14 % – 50.00 % – 80.00 % – 68.18 %), without dose dependence.
The litters with holes in the supraoccipital bone were detected in high percentage in all treated groups as well in the control group. Other changes of foetal cranium were found only sporadically.

During examination of the foetal skeletons incomplete ossification of ossification sites of sternebra and unossified ossification sites of sternebra were recorded. The incidence of incomplete ossification of ossification sites on sternebra was very common finding in the treated groups and also in the control group. All treated and control litters were affected with incomplete ossification of ossification sites of sternebra. The proportion of litters with unossified ossification sites of sternebra (61.90 % – 72.73 % – 75.00 % – 77.27 %) was comparable in treated groups with control. The incidence of litters with bipartite ossification of ossification sites of sternebra was lower at dose levels in comparison with control (19.05 % – 13.64 % – 5.00 % – 0.00 %).

Examination of vertebrae revealed mainly bipartite and dumbbell ossification of vertebrae thoracic centrum. The proportion of litters with bipartite ossification of vertebrae thoracic centrum was increased at the dose levels 1000 mg/kg bw/day in comparison with control group (23.81 % – 13.64 % – 25.00 % – 40.91 %), without statistical significance. Insignificantly increased incidence of litters with the bipartite ossification of vertebrae thoracic centrum with asymmetric ossification was recorded also at the highest dose level.
The proportions of litters with dumbbell ossification of vertebrae thoracic centrum was high in treated groups as well as in the control group (80.95 % – 81.82 % – 90.00 % – 95.45 %). During the statistical evaluation of findings on the vertebrae the statistically significantly increased number of foetuses with dumbbell ossification of vertebrae thoracic centrum were detected at the dose level 1000 mg/kg bw/day, but in the case of conversion to litter no statistical significance was found. Other changes of foetal vertebrae were found only sporadically.

Anomaly of ribs – supernumerary ribs – lumbar ossification site and wavy ribs were recorded. The proportion of litters with supernumerary ribs – lumbar ossification site and wavy ribs were comparable or lower at all dose levels in comparison with control group.

The incomplete ossification of humerus was recorded in all treated groups and also in the control group. The proportions of litters with incomplete ossification of humerus were 4.76 % – 0.00 % – 10.00 % – 9.09 %, respectively for the four groups 0, 100, 500 and 1000 mg/kg bw/day.
Visceral malformations:
no effects observed
Description (incidence and severity):
Detailed gross dissections of foetuses were performed. Placing and morphology of organs and big vessels were reviewed during examination of internal alterations. No adverse findings were observed in any of the test groups or the control group.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: This NOAEL is based on no altered growth and no significant treatment-related structural abnormalities in foetuses of pregnant rats dosed up to 1000 mg/kg bw/day.
Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal alterations have been observed, however these effects have not been related to the treatment
Key result
Developmental effects observed:
no
Conclusions:
The NOAEL (No Observed Adverse Effect Level) for the test item acid for TOXICITY in pregnant females was established as 500 mg/kg bw/day.
NOAEL value is based on no mortality of females, no altered growth, no changes in health condition status, no alteration to thyroid parameters, no pathological findings in the dams and no dose related changes in reproduction parameters up to 500 mg/kg bw/day.

The NOAEL (No Observed Adverse Effect Level) for the test item for PRENATAL DEVELOPMENT was established as 1000 mg/kg bw/day.
This NOAEL is based on no altered growth and no significant treatment-related structural abnormalities in foetuses of pregnant rats dosed up to 1000 mg/kg bw/day.
Executive summary:

The test item was tested for prenatal developmental toxicity using the OECD Test Guideline No. 414, Prenatal Developmental Toxicity Study, Adopted by the Council on June 25th 2018.


 


There were no deaths of females during the study at any dose level. No adverse changes in health condition and no clinical symptoms of intoxication were observed in maternal animals following administration of the test item at any dose. In this prenatal developmental study the negative effect of the test item on the growth of maternal animals was observed at the highest dose level 1000 mg/kg bw/day. The body weight of treated females was decreased at the dose level 1000 mg/kg bw/day compared to the control group, on the 14th and 20th day of pregnancy statistically significantly. The body weight increments were significantly decreased at the dose levels 100 and 1000 mg/kg bw/day. Also value of corrected body weight of females was statistically significantly decreased compared to the control group at the highest dose level.


The weight reduction correlated with the decrease of food consumption in treated females at the highest dose. The food consumption of treated mothers was decreased from the 8th to 14th day of pregnancy at the dose level 1000 mg/kg bw/day, on the 8th day of the pregnancy with statistical significance.


 


Evaluation of uterus biometry detected decrease of absolute weights at the dose level 1000 mg/kg bw/day. This decrease in the absolute weight of the uterus was related to the lower necropsy body weight of the treated females whereas the relative uterine weight remains unchanged throughout all the treated as well as control groups.


Macroscopic structure of examined organs of pregnant females and reproduction parameters (number of females with live foetuses, number of live and dead foetuses, early and late resorptions) were unaffected by treatment with the test item.


Examination of the thyroid glands (absolute and relative weight of thyroid gland, histological examination of thyroid gland and serum levels of thyroid hormones) did not reveal any changes associated with the application of the test item.


 


Test item-related foetal mortality was not evident at any dose level. Detailed necropsy of foetuses did not reveal an increase of external and visceral variations and malformations at any dose level.


 


The mean body weights of foetuses were decreased at the dose level 500 and 1000 mg/kg bw/day compared to the control, but without statistical significance and dose dependence.


 


The mean AGD and corrected AGD of male and female foetuses in treated groups was not statistically significantly different from the control group.


 


No statistically significant differences with toxicological significance were recorded in number of litters during the statistical evaluation of skeletal findings.


Increased incidence of delayed ossification of some cranial bones (frontal bone, arcus zygomaticus, nasal bone) was observed at the dose levels 500 and 1000 mg/kg bw/day, without statistical significance and without dose relationship. These changes were not considered to be adverse due to their reversibility in postnatal life.


 


The NOAEL (No Observed Adverse Effect Level) for the test item for TOXICITY in pregnant females was established as 500 mg/kg bw/day.


NOAEL value is based on no mortality of females, no altered growth, no changes in health condition status, no alteration to thyroid parameters, no pathological findings in the dams and no dose related changes in reproduction parameters up to 500 mg/kg bw/day.


 


The NOAEL (No Observed Adverse Effect Level) for the test item for PRENATAL DEVELOPMENT was established as 1000 mg/kg bw/day.


This NOAEL is based on no altered growth and no significant treatment-related structural abnormalities in foetuses of pregnant rats dosed up to 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The test item was tested for prenatal developmental toxicity using the OECD Test Guideline No. 414, Prenatal Developmental Toxicity Study, Adopted by the Council on June 25th 2018.


The NOAEL (No Observed Adverse Effect Level) for the test item for TOXICITY in pregnant females was established as 500 mg/kg bw/day.


NOAEL value is based on no mortality of females, no altered growth, no changes in health condition status, no alteration to thyroid parameters, no pathological findings in the dams and no dose related changes in reproduction parameters up to 500 mg/kg bw/day.


 


The NOAEL (No Observed Adverse Effect Level) for the test item for PRENATAL DEVELOPMENT was established as 1000 mg/kg bw/day.


This NOAEL is based on no altered growth and no significant treatment-related structural abnormalities in foetuses of pregnant rats dosed up to 1000 mg/kg bw/day.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), "Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification.


Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects."


 


The available experimental data are adequate for classification and labelling and the substance is not classified for reproductive and developmental toxicity according to the CLP Regulation (EC 1272/2008).

Additional information