diethyl pyridine-2,4-dicarboxylate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed under GLP condition and according to guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

MEXORYL SBU

IUPAC Name:

MEXORYL SBU

Constituent 2

Reference substance name:

diethyl pyridine-2,4-dicarboxylate

EC Number:

680-341-5

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

diethyl pyridine-2,4-dicarboxylate

Test material form:

other: yellow lumps/liquid

Details on test material:

- Name of test material: MEXORYL SBU

Test animals

Species:

rat

Strain:

other: Wistar Hannover

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: BIOAGRI Laboratórios Ltda - DF- Age at study initiation: 8 week old when supplied, 11 weeks old at mating (0 day of gestation)- Weight at study initiation: mean 214 g- Fasting period before study: no- Housing: 4 animals/cage (male acclimation period), 2 animals/cage (female acclimation period), 1 male in female cage (mating), individually (females post-mating period)- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: 20 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 19.0 - 23.5- Humidity (%): 40.7 - 70.0- Air changes (per hr): 10 - 20- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % methylcellulose (MC) aqueous solution in purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For each dosage group an appropriate amount of MEXORYL SBU was weighed into a precalibrated beaker. In presence of solid or partly solid particles, the test item was first liquefied before being sampled: the container was placed a few minutes in a water-bath at +40°C until complete liquefaction. After sampled under magnetic stirring, the temperature of test item has returned to ambient temperature before added the vehicle. The vehicle, 0.5% methylcellulose (MC) aqueous solution in purified water was added in sufficient quantity until achieved the desired concentration. Each suspension was stirred and dispensed into individual containers properly identified. A sufficient quantity of the vehicle was similarly dispensed for administration to control animals. Test suspensions were prepared at the Testing Facility, stored at room temperature under inert gas, protected from light and humidity.VEHICLE- Justification for use and choice of vehicle (if other than water): no data- Concentration in vehicle: 0, 25, 75, 250 mg/mL- Amount of vehicle (if gavage): 4 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability analyses of the test item in prepared suspensions were performed during the treatment. The samples were analyzed by BlOAGRl Laboratorios Ltda-DF after validation of the analytical method by HPLC (High Performance Liquid Chromatography) method.

Details on mating procedure:

- Impregnation procedure: cohoused- If cohoused:- M/F ratio per cage: 1/2- Length of cohabitation: from about 4:30 pm to about 8:00 am of the following day- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from day 6 through day 19 of gestation

Frequency of treatment:

daily

Duration of test:

20 days

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage levels (mg/kg bw/day) were selected based on the results of a preliminary prenatal developmental toxicity study by oral rout in rats where the minimal maternal toxicity consisted of enlarge spleen as well as colored contents associated with dilatation in both uterine horns, observed on few females at 1000 or I00 mg/kg bw/day, and 1000 or 300 mg/kg bw/day respectively. None of the pregnancy parameters were clearly affected by the test item. At 1000 mg/kg bw/day, a malformation was observed in one fetus. Accordingly, the following doses were chosenfor this study. 100 mg/kg bw/day: as the expected dose which causes no signs of toxicity; 300 mg/kg bw/day: as the intermediate dose level; .1000 mg/kg bw/day: as the expected dose which causes signs of maternal toxicity.- Rationale for animal assignment (if not random): only animals within ±20 % from mean body weight were used

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: twice a day on working days or once a day on weekends or public holidaysDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: weeklyBODY WEIGHT: Yes- Time schedule for examinations: on days 0, 3, 6, 9, 12, 15, 18 and 20 of gestationFOOD CONSUMPTION AND COMPOUND INTAKE : Yes - Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: YesPOST-MORTEM EXAMINATIONS: Yes- Sacrifice on gestation day #20- Organs examined: not specified

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: YesExaminations included:- Gravid uterus weight: Yes- Number of corpora lutea: Yes- Number of implantations: Yes- Number of early resorptions: Yes- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter- Soft tissue examinations: Yes: half per litter- Skeletal examinations: Yes: half per litter- Head examinations: No data

Statistics:

One Way Analyses of Variance (ANOVA), followed by Dunnett's Test, was used for statistical evaluation of fetal and maternal body weights, maternal body weight changes, weight of the uterus, placental weights, food consumption, number of implantation, fetuses, corpora lutea, resorption, and pre and postimplantation losses. Wilcoxon Test (non parametric test) was used for data that did not present a normal distribution. The Chi-square Test was used to evaluate the incidence of lesions. The litter was used as the experimental unit for the purpose of statistical evaluation. The level of significance was set at 5%, and the statistical program used was SAS Software (SAS Institute lnc., Cary, NC)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day, dam No. 32 presented slight Hypotrichosis in the forelimbs. At 300 mg/kg bw/day, dam No. 70 exhibited forelimb, hind limb and abdominal alopecia. At 1000 mg/kg bw/day, dam No. 85 had abdominal and hind limb alopecia. These isolated findings have no dose-trend relation and are considered incidental

Mortality:

no mortality observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effectsDetails on maternal toxic effects:Few clinical signs were observed during the study at 100 mg/kg bw/day, dam No. 32 presented slight Hypotrichosis in the forelimbs. At 300 mg/kg bw/day, dam No. 70 exhibited forelimb, hind limb and abdominal alopecia. At 1000 mg/kg bw/day, dam No. 85 had abdominal and hind limb alopecia. These isolated findings have no dose-trend relation and are considered incidental.There were no test-item related effects on gestational parameters.No treatment-related findings were observed at the necropsy of the dams.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other:

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Total placental and male placental mean weights were statistically significant higher in animals from 1,000 mg/kg bw/day (+ 11% and 10.3% respectively) when compared to the control. This finding has no toxicological significance because no abnormalities were found in the tissue and no dose-trend response was present. Fetal mean weights of both male and female fetuses was unaffected at the 100 and 300 mg/kg bw/day dose level; at 1000 mg/kg bw/day, fetal weights were slightly higher for both male and female fetuses (+8.4% and +9.9% respectively when compared to control) but this difference did not reach statistical significance.

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal MalformationsMinor skeletal malformations (absent rib) were observed in 1 fetus of group 2, group 3 and group 4. This is considered as spontaneous findings and without toxicological significance.Skeletal VariationsThe incidence of fetuses and litter affected by wavy ribs was statistically lower at the 300 mg/kg bw/day dose-level when compared to the control group. Such finding was also observed at the highest dose but did not reach statistical significance. These lower incidences of wavy ribs did not show clear dose-relationship and were considered to be not test-item related. Skeletal RetardationFetal incidence of sternebrae with incomplete ossification wasstatistically significant lower in fetuses from 100 mg/kg bw/day in comparison to the control group while it remained unaffected by treatment with the test-item at dose-level of 300 and 1000 mg/kg bw/day. The fetal incidence of sternebrae not ossified was significantly higher in 100 mg/kg bw/day, but lower at 1000 mg/kg bw/day when compared to the control group. Litter incidence of unossified sternebrae was similar between the 100 mg/kg bw/day and the control group, but was lower (without reaching statistical significance) for the 1000 mg/kg bw/day dose-level (27.3% versus 59.1% for the control).Fetal incidence of interparietal bone with incomplete ossification was statistically significant higher in animals from 100 mg/kg bw/day group when compared to the control, but remained within our historical control range (4.8-51.6). Moreover, litter incidence was similar between these two groups. The aforementioned three findings occurred spontaneously and since their fetal incidence was either not dose-related, or not confirmed at the litter level, or lower in some of the test-item treated group compared to control, they were not attributed to the test-item. Fetal incidence of supraoccipital bone incomplete ossification was statistically significant higher at doses of 300 and 1000 mg/kg bw/day when compared to the control group, occurred with dose-effect relationship (29.3% and 39.8% versus 17%), but at 300 mg/kg bw/day this value was inside our historical control range (6.22- 30.62). Litter incidence was also higher but without reaching statistical difference (65 and 77% respectively at 300 and 1000 mg/kg bw/day versus 50% for the control group. This effect was attributed to treatment with the test-item but was regarded as non adverse since this retardation was an isolated finding (in the absence of similar findings in other skull bones) that could have resumed to normal after birth.A statistically significant higher sternebrae ossification centers was observed in fetuses in the 1000 mg/kg bw/day group. This finding was considered to be without toxicological significance because of its low magnitude (6%) when compared to the control group. Data from groups 2 (-2.40%) and 3 (+2.9%) did not differ from the control group.

Visceral malformations:

no effects observed

Description (incidence and severity):

Although not reaching statistical significance, a higher fetal and litter incidence of enlarged nasal cavity was observed with dose-response relationship. This finding was considered to bear no toxicological significance. A statistically significant higher fetal and litter incidence of unilateral kidney dilatation was observed at 300 mg/kg bw/day dose-level. This finding was regarded as unrelated to theadministration of the test item since no dose-response was observed and since the fetal and litter incidences of bilateral kidney dilatation was comparable between groups

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effectsDetails on embryotoxic / teratogenic effects:Total placental and male placental mean weights were statistically significant higher in animals from 1,000 mg/kg bw/day (+ 11% and 10.3% respectively) when compared to the control. This finding has no toxicological significance because no abnormalities were found in the tissue and no dose-trend response was present. Fetal mean weights of both male and female fetuses was unaffected at the 100 and 300 mg/kg bw/day dose level; at 1000 mg/kg bw/day, fetal weights were slightly higher for both male and female fetuses (+8.4% and +9.9% respectively when compared to control) but this difference did not reach statistical significance.The external examination of the fetuses revealed no variations or malformations in any of the experimental groups.No malformations in soft tissue were observed in the fetuses of all groups. Although not reaching statistical significance, a higher fetal and litter incidence of enlarged nasal cavity was observed with dose-response relationship. This finding was considered to bear no toxicological significance. A statistically significant higher fetal and litter incidence of unilateral kidney dilatation was observed at 300 mg/kg bw/day dose-level. This finding was regarded as unrelated to theadministration of the test item since no dose-response was observed and since the fetal and litter incidences of bilateral kidney dilatation was comparable between groups.Skeletal MalformationsMinor skeletal malformations (absent rib) were observed in 1 fetus of group 2, group 3 and group 4. This is considered as spontaneous findings and without toxicological significance.Skeletal VariationsThe incidence of fetuses and litter affected by wavy ribs was statistically lower at the 300 mg/kg bw/day dose-level when compared to the control group. Such finding was also observed at the highest dose but did not reach statistical significance. These lower incidences of wavy ribs did not show clear dose-relationship and were considered to be not test-item related. Skeletal RetardationFetal incidence of sternebrae with incomplete ossification wasstatistically significant lower in fetuses from 100 mg/kg bw/day in comparison to the control group while it remained unaffected by treatment with the test-item at dose-level of 300 and 1000 mg/kg bw/day. The fetal incidence of sternebrae not ossified was significantly higher in 100 mg/kg bw/day, but lower at 1000 mg/kg bw/day when compared to the control group. Litter incidence of unossified sternebrae was similar between the 100 mg/kg bw/day and the control group, but was lower (without reaching statistical significance) for the 1000 mg/kg bw/day dose-level (27.3% versus 59.1% for the control).Fetal incidence of interparietal bone with incomplete ossification was statistically significant higher in animals from 100 mg/kg bw/day group when compared to the control, but remained within our historical control range (4.8-51.6). Moreover, litter incidence was similar between these two groups. The aforementioned three findings occurred spontaneously and since their fetal incidence was either not dose-related, or not confirmed at the litter level, or lower in some of the test-item treated group compared to control, they were not attributed to the test-item. Fetal incidence of supraoccipital bone incomplete ossification was statistically significant higher at doses of 300 and 1000 mg/kg bw/day when compared to the control group, occurred with dose-effect relationship (29.3% and 39.8% versus 17%), but at 300 mg/kg bw/day this value was inside our historical control range (6.22- 30.62). Litter incidence was also higher but without reaching statistical difference (65 and 77% respectively at 300 and 1000 mg/kg bw/day versus 50% for the control group. This effect was attributed to treatment with the test-item but was regarded as non adverse since this retardation was an isolated finding (in the absence of similar findings in other skull bones) that could have resumed to normal after birth.A statistically significant higher sternebrae ossification centers was observed in fetuses in the 1000 mg/kg bw/day group. This finding was considered to be without toxicological significance because of its low magnitude (6%) when compared to the control group. Data from groups 2 (-2.40%) and 3 (+2.9%) did not differ from the control group.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other:

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Treatment related:

no

Dose response relationship:

no

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

At doses of 100, 300 and 1000 mg/kg bw/day test item to pregnant Wistar rats by gavage from day 6 through 19 of gestation did not produce toxicological effects on the dams nor had an adverse effects on gestational parameters. The NOAEL for maternal toxicity and for developmental toxicity is 1000 mg/kg bw/day.

Executive summary:

MEXORYL SBU was tested for prenatal developmental oral toxicity study in Wistar rats according to OECD TG 414 and EU Method B.31. The test item was suspended in a 0.5% aqueous solution of methylcellulose and administered daily by gavage to 22, 20, 21, 23 pregnant rats per groups at doses of 0 (control), 100, 300 and 1000 mg/kg bw/day respectively from gestation day 6 to 19. The control group received the vehicle alone. Mortality and clinical signs were checked daily. Body weights and food consumption of the dams were recorded every 3 days during the gestation period. On day 20 of gestation all females were euthanized and assessed for gross lesions. The ovaries were removed and the number of corpora lutea was recorded. The unopened uteri were removed, weighed and their contents assessed (implantations, early and late resorptions live and dead fetuses were recorded). Placentae and fetuses were removed and weighed. The fetuses were sexed and observed for any external, soft tissue or skeletal abnormality (malformations, variations and retardations).

The results of this prenatal developmental toxicity study indicate that the oral administration of MEXORYL SBU at doses of 100, 300 and 1,000 mg/kg bw/day to pregnant Wistar rats by gavage from day 6 through 19 of gestation did not produce toxicological effects on the dams or have adverse effects on gestational parameters. MEXORYL SBU was not teratogenic and produced the following test-item related

fetal findings:

In group 4 (1,000 mg/kg bw/day) statistically significant higher fetal incidence of supraoccipital bone with incomplete ossification. This test-item related finding was regarded as non adverse since this retardation was an isolated finding (in the absence of similar findings in other skull bones) that could have resumed to normal after birth.

Under the conditions of this study, the oral administration of MEXORYL SBU to pregnant Wistar rats after the pre-implantation period, the NOAEL (No-Observed-Adverse-Effect-Level) for maternal toxicity and for developmental toxicity is established at 1000 mg/kg bw/day.