Dibutyl itaconate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 March 2022 - 28 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study performed according to ECHA Decision CCH-D-2114538624-46-01/F with a deadline of 24 October 2022.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl itaconate

EC Number:

218-451-9

EC Name:

Dibutyl itaconate

Cas Number:

2155-60-4

Molecular formula:

C13H22O4

IUPAC Name:

1,4-dibutyl 2-methylidenebutanedioate

Specific details on test material used for the study:

Identification: Dibutyl itaconate
Chemical name: Di-n-butyl itaconate; itaconic acid di-n-butyl ester, DBI
CAS number: 2155-60-4
Molecular weight: 242.31
Volatile: Yes, vapour pressure: 0.19 Pa @ 25°C
Specific gravity: 0.985 g/mL
Physical description: Clear yellow liquid
Storage conditions: At room temperature
Test item handling: No specific handling conditions required
Stability in vehicle (polyethylene glycol 400): Stability for at least 24 hours at room temperature under normal laboratory light conditions, for at least 8 days in the refrigerator and of 0.5 mL samples for at least 3 weeks in the freezer (≤ - 15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions), Test Facility Study No. 20299095.

Test animals

Species:

rat

Strain:

other: Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 11-15 weeks
- Weight at study initiation: 182-276 g
- Fasting period before study: No
- Housing: Polycarbonate cages containing sterilized wooden fibers as bedding material equipped with water bottles. Animals will be individually housed.
- Diet: ad libitum, pellets (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, municipal tap water.
- Acclimation period: 5-6 days
- Contaminants: It is considered that there are no known contaminants in the feed or water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-20 (actual range)
- Humidity (%): 35-60 (actual range); The values that were outside the targeted mean humidity range (40-70%) occurred for 2 days and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 March 2022 To: 1 April 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing formulations were prepared at least weekly. Dose formulations were divided into aliquots where required to allow to be dispensed on each dosing occasion. Formulations (w/w) were homogenized to visually acceptable levels. Test material dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle was continuously stirred until and during dosing.
An adjustment was made for specific gravity (1.125) of the vehicle and test material. No correction was made for the purity/composition of the test material.

VEHICLE
- Concentration in vehicle: 20, 60, or 200 mg/mL
- Dose volume: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis as indicated in Table 1 under 'Any other information on materials and methods, incl. tables'.

Analyses were performed using a validated analytical procedure in Test Facility Study No. 20299095. Accuracy and homogeneity were determined for formulations prepared for use in Week 1.
Samples and remaining formulation were stored in normal glassware causing the samples stored at room temperature to be exposed to normal laboratory light conditions.
For determination of accuracy, duplicate samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations.

Concentration and Homogeneity Analysis
Storage Conditions: Refrigerator set to maintain 4°C
Acceptance Criteria:
- For concentration, mean sample concentration results within or equal to ± 15% of theoretical concentration.
- For homogeneity, relative standard deviation (RSD) of concentrations of ≤10% for each group.

Stability analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20299095) demonstrated that the test material is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Details on mating procedure:

Untreated females were mated at the supplier and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).

Duration of treatment / exposure:

Day 6 to Day 20 post-coitum

Frequency of treatment:

Once daily

Duration of test:

Day 6-21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels in this study were selected based on the results of the Dose Range Finder (Test Facility Study No. 20299103).
- Rationale for animal assignment: random
- Fasting period before blood sampling for (rat) dam thyroid hormones: No
- Time of day for (rat) dam blood sampling: Sampled between 7:00 and 9:00

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily 0 to 1 hours post-dose, starting on Day 6 post coitum up to and including the day prior to necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 2, 6, 15 and 21 post-coitum

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Time schedule: Over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.

WATER CONSUMPTION: Yes (visual inspection)
- Water consumption was monitored by visual inspection of the water bottles for health monitoring of the animals only and was not reported.

TISSUE COLLECTION AND PRESERVATION:
- The thyroid gland and macroscopic abnormalities will be collected from all animals and preserved in 10% buffered formalin.

MICROSCOPIC EVALUATION:
- Thyroid gland was microscopically examined by a pathologist.

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
- Number of live and dead fetuses: Yes
- Sex of each fetus based on anogenital distance: Yes

In case no macroscopically visible implantation sites are present, nongravid uteri will be stained using the Salewski technique in order to detect any former implantation sites.

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected: 1.0 mL
- Fasting: No
- Thyroid hormone parameters: TSH (IMMULITE 1000 analyser) and T#/T$ (LC-MS).

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
- Anogenital distance of all live rodent pups: Yes (normalized to the cube root of the fetal body weight)

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences will be reported as appropriate by dataset.

All statistical tests will be conducted at the 5% significance level. All pairwise comparisons will be conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

The statistical matrix (table 2) can be found under 'Any other information on materials and methods incl. tables'.

Parametric/Non-parametric
Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons was conducted using Dunnett’s or Dunn’s test, respectively.

Non-Parametric
The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.

Incidence
A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Indices:

- Body weight gains: Calculated for the following intervals: Days 2 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21 and 6 to 21 post-coitum.
- Gravid Uterus Adjusted Body Weight: Body weight on Day 21 post-coitum - body weight on Day 6 post-coitum - gravid uterus weight.
- Overall food consumption: Calculated between each scheduled interval (individual data only) and as specified above for body weight gains. Summarization and statistical analysis intervals reflect the same intervals as the body weight gains.
- Pregnancy rate (%): (No. of pregnant females / No. of mated females) x 100
- Organ weight relative to body weight: Calculated against body weight recorded on Day 21 post-coitum
- Male fetuses (%): (No. of male fetuses / No. of fetuses) x 100
- Female fetuses (%): (No. of female fetuses / No. of fetuses) x 100
- Pre-implantation loss (%): ((No. of corpora lutea - No. of implantations) / No of implantations) x 100
- Post-implantation loss (%): ((No. of implantations - No. of live fetuses) / No. of implantations) x 100
- Litter % of fetuses with abnormalities: (No. of fetuses in litter with a give finding / No. of fetuses in litter examined) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No relevant clinical sings were noted at 100 and 300 mg/kg bw/day.
At 1000 mg/kg bw/day, 2/21 females were noted with hunched posture (on Day 21 post-coitum) and 3/21 females with erected fur (on one to four days between Days 12 and 21 post-coitum). Considering the limited number of affected females and days these observations were noted, these signs were considered non-adverse.
Salivation was seen after dosing in 5/22 and 9/21 females at 300 and 1000 mg/kg bw/day, respectively. Taking into account the nature and minor severity of the effect and its time of occurrence (i.e., after dosing), this sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 1000 mg/kg bw/day, one female was sacrificed in extremis on Day 20 post-coitum, because of a body weight loss of 14% between Days 15 and 20 post-coitum. In addition, food consumption was low over Days 15-18 post-coitum and hunched posture and erected fur were noted on Days 19-20 post-coitum in this animal. Furthermore, the animal was observed with red discolored skin in the nose area and decreased activity on Day 20 post-coitum. At necropsy, enlarged adrenal glands, pale discolored kidneys, and a gelatinous appearance of stomach, small intestines (duodenum, ileum, jejunum) and large intestines (cecum, colon and rectum) were found. The animal was pregnant and had 9 live fetuses, 1 early resorption and 4 late resorptions in utero at necropsy. This death was considered test material-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 100 and 300 mg/kg bw/day, mean body weights, body weight gain and gravid uterus adjusted body weight gain remained in the same range as control over the treatment period.
At 1000 mg/kg bw/day, lower mean body weight gain was noted over Days 6-9 and 18-21 post coitum, resulting in a mean body weight that was 10% lower than control at the end of treatment (Day 21 post-coitum). Mean gravid uterus adjusted body weight gain was also lower at this dose (3.6 g vs. 32.35 g in control).
Correlating lower food consumption was observed throughout the Treatment Period. Given the magnitude of these changes, the effects on food intake and body weight were considered adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 100 and 300 mg/kg bw/day, slightly lower mean food consumption was noted from start of treatment onwards, resulting in a 4% (not statistically significant) and 7% lower overall mean food consumption at 100 and 300 mg/kg bw/day, respectively, when compared to control.
At 1000 mg/kg bw/day, mean food consumption was lower compared to control throughout the Treatment Period. The largest difference was observed over Days 6-9 and 18-21 post-coitum where mean food intake was 27% and 31% lower than control, respectively. Overall mean food intake during the Treatment Period was 19% lower than control.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Serum levels of T3, T4 and TSH were considered to be unaffected by treatment with the test material up to and including 300 mg/kg bw/day.
At 1000 mg/kg bw/day, decreased T3 and T4 levels were noted (0.56x and 0.63x of control). The mean values of both T3 (mean: 0.229 ng/mL) and T4 (mean: 13.52 ng/mL) were outside the historical control data range.
An increased level of TSH was observed at 1000 mg/kg bw/day (2.87x of control, not statistically significant). The high mean value (0.8855 Mu/L) was mainly caused by three females with notably high TSH levels (3.440, 3.300 and 4.820 mU/L, respectively). Although the mean value was within historical control data range, a relation to treatment with the test material could not be excluded. However, no morphologic changes were found the in the thyroid glands of these three females. Although TSH levels of the remaining females at 1000 mg/kg bw/day were within the normal range, this increase was considered test material related. Possible adversity of these effects on T3, T4 and TSH could not be assessed within this type of study.

Historical control data for pregnant Wistar Han rats (period 2020-2022):
T3 (ng/mL): mean (P2.5-P97.5): 0.424 (0.270-0.683) (n=347)
T4 (ng/mL): mean (P2.5-P97.5): 23.40 (15.07-45.67) (n=347)
TSH (mU/L): mean (P2.5-P97.5): 0.3272 (0.0838-0.9033) (n=392)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Thyroid weights and thyroid:body weight ratios were considered to be unaffected by treatment with the test material.
At 1000 mg/kg bw/day, mean absolute thyroid gland weight was lower (-8%, not statistically significant) when compared to the control group. This was considered to be the result of a test material-related effect on final body weight (-10%) since mean relative thyroid gland weight was similar to the control mean.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to be related to treatment with the test material up to and including 300 mg/kg bw/day.
At 1000 mg/kg bw/day, 4/21 females were noted with prominent lobular architecture of the liver. As this was only recorded for a few females and without other liver findings and given the mild nature of the findings, this observation was considered non-adverse.
There were no gross observations in the thyroid glands.
Other findings that were noted among control and/or treated animals were considered to be unrelated to treatment with the test material, as they remained within the range of biological variation for rats of this age and strain.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related microscopic observations in the thyroid glands.
All of the recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain. There was no test material related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The number of pre-implantation loss in the control and test groups were in the range of normal biological variation. At 1000 mg/kg bw/day postimplantation loss was higher at this dose (5.41% vs 1.57% in control, not statistically significant). The mean value of postimplantation loss remained within the historical control data, although the value was within the lower end of the available control data. Therefore, the higher postimplantation loss at 1000 mg/kg bw/day was considered unrelated to treatment with the test material.

Historical control data for pregnant Wistar Han rats (period 2020-2022):
Postimplantation Loss (%/Litter): mean (mean±2SD): 4.15 (0.20-8.10) (n=421)

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, the number of late resorptions was higher (0.2 vs. 0.0 in control), which was considered potentially related to treatment with test material. This increase was considered non-adverse, as the postimplantation value remained within historical control data.

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females, except for three were gravid. Those three females were divided in three different dosing groups (control, 100 mg/kg bw/day, and 1000 mg/kg bw/day).

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

mortality

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no test material-related effects on fetal body weights (both sexes) noted up to and including 300 mg/kg bw/day.
At 1000 mg/kg bw/day, mean fetal body weights (male (4.482), female (4.292) and combined (4.374)) were 14% lower compared to control. Mean values for body weight were below the available historical control data range. Based on the magnitude of the change and as the mean values were below the historical control data range, the lower fetal weight was considered to be adverse.
It should be taken into account, that at 1000 mg/kg bw/day the lower fetal weights may have been the result of the strongly reduced body weight gain of the dams, rather than being a direct effect of the test material.

Historical control data for body weight of Wistar Han fetuses (period 2020-2022):
Combined (g): mean (mean±2SD): 5.2 (4.9 – 5.4) (n= 4,828)
Males (g): mean (mean±2SD): 5.3 (5.0 – 5.6)
Females (g): mean (mean±2SD): 5.0 (4.8 – 5.2)

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was considered to be unaffected by treatment with the test material.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Litter size was considered to be unaffected by treatment with the test material.
There were no test material-related effects on fetal body weights (both sexes) noted up to 300 mg/kg bw/day.
At 1000 mg/kg bw/day, mean fetal body weights (male (4.482), female (4.292) and combined (4.374)) were 14% lower compared to control. Mean values for body weight were below the available historical control data range.

Historical control data for body weight of Wistar Han fetuses (period 2020-2022):
Combined (g): mean (mean±2SD): 5.2 (4.9 – 5.4) (n= 4,828)
Males (g): mean (mean±2SD): 5.3 (5.0 – 5.6)
Females (g): mean (mean±2SD): 5.0 (4.8 – 5.2)

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The mean fetal anogenital distance (both sexes) was considered to be unaffected by treatment with the test material.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related external malformations and variations were recorded.
External malformations were observed in 1 (1), 0 (0), 0 (0) and 3 (2) fetuses (litters) of the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The control fetus had a short tail and syndactyly. In the high dose group (1000 mg/kg bw/day), one fetus presented with polydactyly and another fetus had an absent tail and anus. These malformations were considered chance findings and therefore not related to treatment with test material.
Additionally, in the high dose group generalized subcutaneous edema was found in two dead fetuses and a late resorption. As these similar malformations were discovered in just one single litter amongst dead fetuses and a late resorption, further compounded by the fact edema is a likely consequence of autolysis, these observations were considered not to be related to treatment with the test material.
External variations and incidental findings were not observed in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related skeletal malformations and variations were recorded.
Skeletal malformations were observed in 1 (1), 1 (1), 0 (0) and 2 (2) fetuses (litters) of the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Two fetuses of the high dose group (1000 mg/kg bw/day) presented with external malformations which had underlying skeletal malformations consistent with their external findings. Therefore, this ruled out a relationship to treatment with the test material. The control fetus with syndactyly and a short tail had no underlying skeletal malformation. Furthermore, one control group fetus had a fused cervical arch and one low-dose group fetus had a fused thoracic centrum and an absent thoracic centrum. As these malformations were scored only once, they were considered chance findings and not related to treatment with the test material.
The most noteworthy skeletal variations regarded wavy ribs and bent scapulae.
At 300 and 1000 mg/kg bw/day, statistically significantly less wavy ribs were observed when compared to the control group. In the case of the bent scapulae, all three groups treated with the test material had significantly fewer observations (0) when compared to the control (4). However, comparison with historical control data revealed that both observations in the control group were above or at the higher limit of the normal control range in this study giving the appearance of significance. Hence, this was considered not to be related to treatment with the test material.
All other skeletal variations occurred infrequently, not statistically significant from the control group, and/or only in the control group. Therefore, they were considered not related to treatment with the test material.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related visceral malformations and variations were recorded.
Only one visceral malformation was observed in this study, one fetus of the low-dose group had a retroesophageal right subclavian artery. The single occurrence made it a chance observation and not related to treatment with the test material.
Visceral variations were observed in a diverse range of visceral structures across all groups, including carotid artery, gonad, innominate artery, kidney, liver, spleen and ureter. Each was scored infrequently or in instances comparable to the control group and/or historical control data.
Two incidental findings of discoloration (liver lobe) were observed in this study, which were considered unrelated to treatment with the test material.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

presumably yes

Any other information on results incl. tables

Dose level (mg/kg bw/day)	0	100	300	1000
Pregnant/total dams	21/22	21/22	22/22	21/22
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses	0	0	0	0
Dams with live fetuses	21	21	22	21
Corpora lutea (mean number)	12.3	11.8	12.5	13.1
Implantations (mean number)	11.8	11.4	12.1	12.4
Resorptions (mean number) - Early - Late - Total (early + late)	0.2 0 0.2	0.3 0 0.3	0.3 0.1 0.4	0.4 0.2 0.6
Pre implantation loss (%)	3.95	3.32	2.78	5.09
Post implantation loss (%)	1.57	2.35	2.97	5.41
Dead fetuses (mean number)	0	0	0	0.1
Mean body weight on day 21 (g)	338.7	333.3	341.2	305.0**
Mean body weight gain day 6-21 (g)	112.0	105.6	107.0	75.8**
Gravid uterine weight (g)	79.70	76.90	79.83	72.20
Mean adjusted body weight gain (6-abw)	32.35	28.71	27.13	3.60**
Live offspring (mean number)	11.6	11.1	11.7	11.8
Sex ratio (male%)	49.44	49.62	52.52	46.70
Mean fetal body weight males (g)	5.225	5.243	5.227	4.482**
Mean fetal body weight females (g)	4.975	4.946	4.920	4.292**
Mean fetal body weight combined (g)	5.101	5.101	5.072	4.374**
Mean anogenital distance males (mm)	2.88	2.91	2.85	2.78
Mean anogenital distance females (mm)	1.23	1.19	1.29	1.26
Mean normalized anogenital distance males	1.662	1.679	1.646	1.687
Mean normalized anogenital distance females	0.720	0.697	0.757	0.774
Malformations number and % of fetuses number of litters - External - Visceral - Skeletal	1 (0.4%) 1 0 (0%) 0 1 (1.19%) 1	0 (0%) 0 1 (0.79%) 1 1 (0.79%) 1	0 (0%) 0 0 (0%) 0 0 (0.00%) 0	2 (0.81%) 2 0 (0%) 0 2 (1.34%) 2
Variations number and % of fetuses number of litters - External - Visceral - Skeletal	0 (0. %) 0 8 (5.83%) 6 85 (72.07%) 21	0 (0%) 0 9 (8.17%) 8 79 (67.60%) 21	0 (0%) 0 9 (10.06%) 8 86 (67.67%) 22	0 (0 %) 0 11 (9.67%) 10 85 (70.04%) 20

** = p ≤ 0.01

Applicant's summary and conclusion

Conclusions:

A prenatal developmental toxicity study in rats was performed according to OECD 414 and in accordance with GLP principles. Based on the results of this study, the following NOAELs of the test substance were established:
- Maternal NOAEL: 300 mg/kg bw/day, based on mortality, lower body weight (gain) and food intake at 1000 mg/kg bw/day
- Developmental NOAEL: 300 mg/kg bw/day, based on lower fetal weights at 1000 mg/kg bw/day.

Executive summary:

A prenatal developmental toxicity study was performed according to OECD 414 and in accordance with GLP principles. Time mated female wistar Han rats were dosed orally by gavage from Days 6 to 20 post-coitum. 

The dose levels in this study were selected to be 0, 100, 300 and 1000 mg/kg bw/day, based on the results of the Dose Range Finder (Test Facility Study No. 20299103).

The study design was as follows:

Experimental Design

Group No.	Test Material Identification	Dose Level (mg/kg bw/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Females
1	-	0 (Vehicle)	5	0	22
2	Dibutyl itaconate	100	5	20	22
3		300	5	60	22
4		1000	5	200	22

 

Chemical analyses of formulations were conducted once during week one of the study and confirmed that formulations of the test material in polyethylene glycol 400 were prepared accurately and homogenously.

The following parameters and end points were evaluated in this study for the F₀-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), organ weights (thyroid gland), macroscopic examination, microscopic examination (thyroid gland) and uterine contents (including corpora lutea, implantation sites, pre- and postimplantation loss and number of live and dead fetuses).

In addition, the following parameters were determined for the F₁-generation: fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.

One female at 1000 mg/kg bw/day was sacrificed in extremis on Day 20 post-coitum, based on body weight loss, reduced food intake and general poor condition.

At 100 and 300 mg/kg bw/day, non-adverse lower food consumption was noted throughout the treatment period.

At 1000 mg/kg bw/day, non-adverse hunched posture, erected fur and prominent lobular architecture of the liver were noted for some females. Furthermore, lower mean body weight gain over Days 6-9 and 18-21 post-coitum was noted, resulting in a lower terminal body weight. In addition, lower gravid uterus adjusted body weight gain and lower food consumption (throughout the treatment period) were noted. These effects on body weight (gain) and food consumption were considered adverse.

T3 and T4 serum levels were decreased at 1000 mg/kg bw/day, with mean values below the normal range of variation. In addition, TSH levels were increased at this dose level.

No test material-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e., thyroid gland weights, microscopic evaluation of the thyroid gland and uterine contents including corpora lutea implantation sites and pre- and postimplantation loss).

No developmental toxicity was observed up to and including 300 mg/kg bw/day.

At 1000 mg/kg bw/day, a non-adverse increase in late resorptions and adverse lower fetal body weights were noted.

No test material-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e., litter size, sex ratio, anogenital distance, external, visceral and skeletal malformations, developmental variations and incidental findings).

In conclusion, based on the results of this prenatal developmental toxicity study, the following No Observed Adverse Effect Levels (NOAELs) of the test substance were established:

Maternal NOAEL: 300 mg/kg bw/day, based on mortality, lower body weight (gain) and food intake at 1000 mg/kg bw/day.

Developmental NOAEL: 300 mg/kg bw/day, based on lower fetal weights at 1000 mg/kg bw/day.

Note: In this study, a reduction of T4 and T3 was and increased level of TSH were observed at 1000 mg/kg bw/day which was considered to be test material-related. However, possible adversity of these effects could not be assessed within this type of study and was therefore not taken into account when determining the maternal NOAEL.