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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 09, 2020 to November 18, 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted: 25th June 2018
Deviations:
yes
Remarks:
All deviations will be documented in the study records with an assessment of impact on the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Maleic acid
EC Number:
203-742-5
EC Name:
Maleic acid
Cas Number:
110-16-7
Molecular formula:
C4H4O4
IUPAC Name:
but-2-enedioic acid
Test material form:
solid: crystalline
Remarks:
A white, crystalline powder

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: VELAZ, Czech Republic – Origin Crl:WI(Han)
- Age at study initiation: At least 11–12 weeks; female animals were non-pregnant and nulliparous.
- Number and Sex of Animals: 100 females and 30 males
- Housing: up to 5 animals per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: The animals were acclimated to the condition identical to the condition during the experiment at least 5 days prior to the start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%): 55±10%
- Air changes (per hr): central airconditioning
- Photoperiod (hrs dark / hrs light): 12-hour light /12-hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Aqua pro Injectione
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Calculations for the doses preparation will be based on the dose (20, 60 and 140 mg/kg bw), administered volume (2 ml/kg body weight),
documented in spreadsheets and maintained in the study file as printouts. The test item was dissolved in the required volume of vehicle (Aqua pro Injectione) in order to achieve the concentrations of 10, 30 and 70 mg/mL and then homogenized using a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency): Once daily
- Mixing appropriate amounts with (Type of food): The test item, the vehicle (Aqua pro Injectione)
- Storage temperature of food: Room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): Ultra pure water such as API is a common vehicle for water soluble items in toxicity studies like OECD TG 423
- Concentration in vehicle: 10, 30 and 70 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg bw
- Lot/batch no. (if required): 19L1001
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosage levels 20, 60, and 140 mg/kg bw/day with ~3-fold optimal interval were selected for the current study based on dose range-finding study (DRF) (DRF 02-2020, UEFT CEM SAS). In the DRF study, repeated administration of Maleic Acid at the doses of 150, 300 and 500 mg/kg bw/day over the next 14 days caused toxicity signs presented as excess vocalization, piloerection and mortality. Two out of three animals died after administration of Maleic Acid at the doses of 300 and two out of three animals died after administration of Maleic Acid at the doses of 500 mg/kg bw/day. One animal died in the group of animals dosed with 150 mg/kg bw/day of Maleic Acid. Aqua pro injection was used as a solvent/vehicle.

Analysis of formulations for homogeneity and concentration during dosing period will be conducted in the test facility approximately three times for the in-life phase using a validated method.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 3:1
- Length of cohabitation: cohoused until mating
- Proof of pregnancy: sperm-positive in vaginal smear referred to as day 0 of pregnancy.
Duration of treatment / exposure:
from day 5 to day 19 (including) of post mating (GD5-GD19)
Frequency of treatment:
Once daily, at approximately the same time each day at the first half of the day.
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
140 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
No. of animal: 100 females
Group 1 - vehicle (Aqua pro Injectione) 2 ml - control sample
Group 2 - 20 mg/kg bw/day
Group 3 - 60 mg/kg bw/day
Grup 4 - 140 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Chosen with the goal of observing mild maternal toxicity at highest dose level and a NOEL at the lowest dose level
- Fasting period before blood sampling for (rat) dam thyroid hormones: Animals were not fasted prior to blood collection.
- Time of day for (rat) dam blood sampling: On gestation day 20, all females were euthanized by anesthesia followed by terminal blood sampling. Blood samplings should be made within two hours on the first part of the day of necropsy in randomized order to avoid bias.

Examinations

Maternal examinations:
CAGE SIDE and CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats were observed for morbidity and mortality. Each female was also observed for signs of toxicity approximately 15-45 minutes following dose administration. In addition, the presence of findings at the time of dose administration was recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual female body weights were recorded during animal identification, at day of confirmed mating and group assignment (gestation day 0), on the first day of dose administration (GD5), and at three-day intervals thereafter (gestation days 8, 11, 14, 17, and 20 as the day of euthanasia). Body weight value on gestation day 20 was corrected for gravid uterine weight to calculate maternal body weight change.

FOOD CONSUMPTION (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was assessed for each female quantitatively as g/kg of body weight/day by weighing of feeder (cage lid) at the beginning of the day and 24 hours after. Food consumption was recorded prior to the initiation of dose administration (gestation days 0-1), and at three-day intervals thereafter (gestation days 4-5, 7-8, 10-11, 13-14, 16-17 and 19-20).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Uterine Content; Thyroid Gland; Soft Tissues Examination

OTHER: Blood Sample Collection for Hormones Assay; T4, T3 and TSH Assay; Examination of Foetuses; External Examination, Body Weight and AGD; Skeletal Examination
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
- Live / dead fetuses: yes
- Fetal weight: Yes
- Fetal sex: Yes
Blood sampling:
- Plasma: No data
- Serum: Yes
- Volume collected: At least 3.0 mL
- Other: The blood will be allowed to clot for 50 min and centrifuged (1600 x g, 4 °C, 15 min) for serum separation. Serum from each animal will be divided into 2 aliquots and immediately frozen at –70 °C until assayed.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [the organs and structures of the head: all per litter ; the bone structure of the head: half per litter] /
- Anogenital distance of all live rodent pups: Yes
Statistics:
All statistical tests will be performed using Microsoft Excel (descriptive statistics) and statistical software Statistica for Windows v.7.1 to compare the treated groups to the control group. Descriptive statistics (mean, standard deviation (S.D.), and N) will be presented for all measurement data and shown in the summary tables. The litter will be the experimental unit for statistical analysis.

Continuous data variables (mean body weights and food consumption data) will be analyzed by multi-factor analysis of variance ANOVA-2, followed by the Duncan test, to determine inter-group differences. Former implantation sites, number of corpora lutea, implantation loss indices, hormones concentration value, uterine, and thyroid weights will be analyzed by parametric one-way analysis of variance (ANOVA). If the results of the ANOVA are significant (p<0.05), Dunnett's test will be applied to the data to compare the treated groups to the control group. The t-test will be e used additionally to compare each dose group with the control value.
Offspring sex ratio data and AGD value will be subjected to the Kruskal-Wallis non-parametric ANOVA test to determine intergroup difference. If the results of the ANOVA are significant (<0.05), Dunn's test will be applied to the data to compare the treated groups to the control group.
The fetal body weight will be analyzed by sex as well as for both sex combined using one-way analysis of variance (ANOVA) as described above. Additionally, statistical analysis for fetal body weight will be done using analysis of covariant with litter size as a covariant.
Descriptive data, percentage values, and pathomorphological data will be analyzed by Fisher's Exact Test.
Indices:
Anogenital distance index

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
Not observe signs of toxicity during dosing period except slight diarrhea in high dose group (900 mg/kg) appearing approximately from 16th day of gestation. No animals died during study.

BODY WEIGHT AND WEIGHT GAIN
The final body weight of pregnant females was slightly decreased in the 20 and 140 mg/kg dose groups (by 3.57% and 2.02% compared to the control group). However these changes were not statistically significant.
However, body weight gain in the 20 mg/kg group was statistically reduced compared to controls. The final body weight of pregnant females was not influenced by administration of tested substance and similarly the weight gain did not differ compared to control females except in the low dose group, where the body weight gain was reduced at the end of treatment (p<0.01).
However, the netto weight of females (without uterus) or uterine weight was not changed in any treatment group compared to control group.

FOOD CONSUMPTION
Food consumption was not affected by administration of tested substance.

Pre- and post-implantation loss
Pre-implantation and post-implantation loss was similar in all groups with no statistical significance among groups.

Thyroid Gland Weight
The weight of thyroid gland was not influenced by the treatment of tested substance

T4, T3 and TSH Hormone Assay
Significant differences among groups were seen in thyroidal hormones (T3, T4 and TSH).

Histological of Thyroid Gland
Histological examination of thyroid gland did not reveal obvious histomorphological changes.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
140 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical signs
dead fetuses
early or late resorptions
food consumption and compound intake
gross pathology
maternal abnormalities
mortality
necropsy findings
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption

Results (fetuses)

Details on embryotoxic / teratogenic effects:
External of Foetuses, Body Weight and AGD
There were no test item related external malformations in foetuses.
The mean total body weight of fetuses was slightly decreased in low and high dose group (5.9% and 4.8%) compared to the control value without statistical significance. Similar findings were observed in the mean placental weight (ranging from 5.4% to 7.1% reduction), again without statistical significance.
No changes in anogenital distance was noted in both sexes. The percentage of male fetuses was not changed compared to control group).

Skeletal
Analysis of skeletal malformations revealed delayed ossification of the cervical vertebrae in all dose groups, less than 4 metacarpal ossification centers present in all dose groups and delayed ossification metatarsal centers, pelvic bone, caudal vertebrae and sternebrae in low treatment group. These anomalies might be connected to slightly lower body weight of foetuses in treatment groups.


Soft Tissues
Increased incidence of visceral anomalies was not observed in either treatment group.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
<= 20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The study of Prenatal Developmental Toxicity Study according OECD TG 414 on Maleic Acid was conducted. Based on the results, the NOAEL for maternal toxicity was determined to be 140 mg/kg body weight/day (actual dose received). Although we did not find any malformations and abnormalities of skeletal and visceral development, but rather transient changes and anomalies in development, the NOAEL for developmental toxicity was determined to be ≤20 mg/kg body weight/day (actual dose received) due to presence of delayed ossification and unsignificant growth delay observed in low and high treatment groups.
Executive summary:

The objective of this prenatal developmental toxicity study was to evaluate the potential effects of the test item, Maleic Acid, on pregnancy and on embryo-foetal development in rats following daily oral (gavage) administration at doses 20, 60 and 140 mg/kg body weight/day from implantation to the day prior to scheduled caesarean section (day 5 to day 19 post-mating inclusive).
There were slight adverse clinical manifestations observed in highest dose group (140 mg/kg), such as vocalization and piloerection at the beginning of treatment. All females were schedule sacrificed on gestation day 20. The final body weight of pregnant females was slightly decreased in the 20 and 140 mg/kg dose groups (by 3.57% and 2.02% compared to the control group). However these changes were not statistically significant.
However, body weight gain in the 20 mg/kg group was statistically reduced compared to controls. The final body weight of pregnant females was not influenced by administration of tested substance and similarly the weight gain did not differ compared to control females except in the low dose group, where the body weight gain was reduced at the end of treatment (p<0.01). However, the netto weight of females (without uterus) or uterine weight was not changed in any treatment group compared to control group.
Pre-implantation and post-implantation loss was similar in all groups with no statistical significance among groups. The weight of thyroid gland was not influenced by the treatment of tested substance and significant differences among groups were seen in thyroidal hormones (T3, T4 and TSH). Histological examination of thyroid gland did not reveal obvious histomorphological changes.
The mean total body weight of fetuses was slightly decreased in low and high dose group (5.9% and 4.8%) compared to the control value without statistical significance. Similar findings were observed in the mean placental weight (ranging from 5.4% to 7.1% reduction), again without statistical significance. No changes in anogenital distance was noted in both sexes. The percentage of male fetuses was not changed compared to control group).
There were no test item related external malformations in foetuses. Analysis of skeletal malformations revealed delayed ossification of the cervical vertebrae in all dose groups, less than 4 metacarpal ossification centers present in all dose groups and delayed ossification metatarsal centers, pelvic bone, caudal vertebrae and sternebrae in low treatment group. These anomalies might be connected to slightly lower body weight of foetuses in treatment groups. Increased incidence of visceral anomalies was not observed in either treatment group.
Based on the results, the NOAEL for maternal toxicity was determined to be 140 mg/kg body weight/day (actual dose received). Although we did not find any malformations and abnormalities of skeletal and visceral development, but rather transient changes and anomalies in development, the NOAEL for developmental toxicity was determined to be ≤20 mg/kg body weight/day (actual dose received) due to presence of delayed ossification and unsignificant growth delay observed in low and high treatment groups.

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