Dibutyl fumarate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 July 2016 to 10 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

OECD Guidelines for Testing Chemicals, No. 414, Prenatal Developmental Toxicity Study, adopted 22nd January 2001

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl fumarate

EC Number:

203-327-9

EC Name:

Dibutyl fumarate

Cas Number:

105-75-9

Molecular formula:

C12H20O4

IUPAC Name:

dibutyl but-2-enedioate

Test material form:

liquid

Details on test material:

Name: DIBUTYLFUMARATEBatch/Lot Number: LEDB2D3051 / LEDB2D1141CAS Number: 105-75-9Chemical name: 2-Butenedioic acid (E)-, dibutyl esterDescription: Clear colourless liquidMolecular formula: C12H20O4Molecular weight: 228.29Purity: 99.2%Manufacture Date: June 2015Expiry Date: 31 December 2016Storage conditions: Refrigerator (2-8°C), protected from lightSafety Precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) for unknown materials were applied to assure personnel health and safety.Hazards: Sensitising to skin, dangerous for the environment.

Specific details on test material used for the study:

Name: DIBUTYLFUMARATEBatch/Lot number: LEDB2D3051CAS number: 105-75-9Chemical name: 2-Butenedioic acid (E)-, dibutyl ester or Fumaric acid di-n-butyl esterDescription: Clear colourless liquidMolecular formula: C12H20O4Molecular weight: 228.29Purity: 99.2%Manufacture date: June 2015Expiry date: 31 December 2016Storage conditions: Refrigerator (2-8°C), protected from lightSafety precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) for unknown materials were applied to assure personnel health and safety.Possible hazard: Sensitising to skin, dangerous for the environmentNo correction for purity of the test item was applied, in agreement with the Sponsor.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species and strain: Hannover Wistar rats (Crl:WI(Han))Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colonyJustification of strain: The rat is regarded as a suitable rodent species for reproduction studies and the test guideline states it is the preferred rodent species. The Hannover Wistar rat was selected due to the experience of the Test Facility with this strain in teratology studies.Housing condition: Standard laboratory conditions; individual housingNumber of animals: 97 female animals, 24 mated female animals in the Control, Low and Mid dose group and 25 mated female animals in the High dose group; 24, 24, 24 and 25 pregnant and evaluated female animals (with more than 5 implantation sites at necropsy) per Control, Low, Mid and High dose groups, respectively; 60 male animals for mating; no study-procedures were carried out on the male animals; untreated, proven breeders from CiToxLAB Hungary Ltd. spare colony were used.Age of animals: Young adult female rats, nulliparous and non-pregnant, at least 11 weeks old at matingStarting body weight: 193-235 g (the variation did not exceed ± 20% of the mean weight)Acclimatisation period: at least 5 daysHusbandryAnimal health: Only healthy animals were used for the test, as certified by the staff VeterinarianRoom: 524Cage type: Type II and/or III polycarbonateBedding: Lignocel® Hygienic Animal Bedding (Batch number: 03018160120 / 03018160506, Expiry date: 20 January 2019 / 06 May 2019) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) was used in the study.Nesting: ARBOCEL crinklets natural nesting material (Batch number: 05072160121, Expiry date: 21 January 2019) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) was used in the study.Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.Temperature: 19.7-25.0°C (target: 22 ± 3 °C)Relative humidity: 35-75% (target: 30-70%)Ventilation: 15-20 air exchanges/hourHousing/Enrichment: Successfully mated animals were housed individually. Deep wood sawdust was use as bedding to allow digging and other normal rodent activities. Nest building material was also added into the cages.The temperature and relative humidity values were measured continuously during the study, minimum and maximum values were recorded twice daily.Diet and water supplyThe animals were provided with ssniff® SM Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Ssniff Spezialdiäten GmbH, Address: D-59494 Soest, Germany) and tap water (in water bottles) as for human consumption, ad libitum. The diet and drinking water were routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.Animal IdentificationAdult animals were identified by temporary numbers written with indelible ink on the tail during the entire study. During necropsy and Caesarean section procedures each evaluated dam was given an additional number (evaluation number indicating group number), and cross-referenced with the numbers used during the in-life phase of the study.The cages were marked with individual identity cards, with information about study code, sex, dose group, cage number, animal number, date of mating and caesarean section/necropsy date. Cages were arranged to minimise any possible effects due to cage placement.The litters were identified at necropsy with litter numbers. The flasks used for fixation and all sheets used for recording data were identified only by the litter numbers up to the end of foetal examinations (facilitating blind examination of foetuses).The foetuses were identified during the Caesarean section with individual numbers according to their implantation sites. For visceral examination they were identified by digit-clipping; for skeletal examination the foetuses were identified by means of a water-proof plastic ribbon tied around their neck.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item was formulated in the vehicle (corn oil) at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of CiToxLAB Hungary Ltd.Formulations were prepared fresh prior to administration to animals or up to three days before use (in that case formulation were stored refrigerated, protected from light).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

According to stability assessment results of the method validation study (CiToxLAB study code 15/260-316AN [6]) formulations were found to be stable in the 1-250 mg/mL concentration range for 5 days when stored refrigerated or for 1 day when stored at room temperature and protected from light.The analysis of test item formulations for concentration and/or homogeneity was performed by a GC method in the Analytical Laboratory of CiToxLAB Hungary Ltd.To verify the test item concentration in formulations, duplicate sample from the top, middle and bottom of the test item formulations were taken two times during the study (during the first and last week of treatment), one set to analyse (which was collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken on each occasion from the control (vehicle) formulation for concentration measurement.

Details on mating procedure:

Mating ProcedureThe oestrus cycle of female animals was examined shortly before start of pairing.After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2 hours (1 male : 1 female) until at least 24 sperm positive females/group are attained. After the daily mating period (a total of 8 mating blocks were performed in the study), a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD0). Sperm positive females were separated and caged individually.RandomisationThe sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group averages of the body weight were as similar as possible. This process was controlled by an appropriate computer software (SPSS PC+4.0) to verify the homogeneity/variability between/within the groups.

Duration of treatment / exposure:

Treatment from GD 6 to 19.See "Any other information" for further details.

Frequency of treatment:

Daily

Duration of test:

25 + days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

97 female animals, 24 mated female animals in the Control, Low and Mid dose group and 25 mated female animals in the High dose group; 24, 24, 24 and 25 pregnant and evaluated female animals 60 male animals for mating; no study-procedures were carried out on the male animals; untreated, proven breeders from CiToxLAB Hungary Ltd. spare colony were used.

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose-selection and route of administrationThe dose levels were set by the Sponsor in consultation with the Study Director based on available data, including the results of a 14-day Dose Range Finding (DRF) toxicity study in rats (CiToxLAB Hungary Study code: 15/260-101PE,), another Dose Range Finding (DRF) toxicity study in pregnant rats (CiToxLAB Hungary Study code: 15/260-105PE,) and the available data of a 90-day repeated dose toxicity study in rats (CiToxLAB Hungary Study Code: 15/260-101P,), with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.In that 14-day DRF study, diuresis was observed in the High (1000 mg/kg/bw/day) dose group from Day 5 until termination and in the Mid (300 mg/kg bw/day) dose group from Day 10 until termination in both sexes. Symptoms observed in the High and Mid dose groups were considered to be test item related. Enlarged livers and kidneys were also observed in both sexes of the 1000 mg/kg bw/day dose group.Following the specific observation of diuresis in that study, water consumption measurements were added to the standard protocol of this study.In the pregnant DRF study, no signs of maternal toxicity were seen and no sign of embryotoxicity or foetotoxicity was observed in any test item treated dose groups (up to and including 500 mg/kg bw/day).Based on all these information, the doses of 75, 250 and 750 mg/kg bw/day are deemed suitable for the purpose of the study.The oral route was selected as being the most direct and controlled way to expose the animals for detection of potential toxic effects of the test item, it was considered suitable to provide the exposure required for this developmental toxicology study.A constant volume of 5 mL/kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals. The control or test item dose formulations were administered to mated, sperm positive (assumed pregnant) female rats daily by oral gavage on a 7 days/week basis, approximately at similar times, from Gestation Day (GD) 6 to GD19, according to the dosing scheme detailed under "Any other information" below.

Examinations

Maternal examinations:

Clinical observationsAnimals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).Cage-side clinical observations were made at twice daily (at the beginning and end of each working day). Detailed clinical observations were made on all animals at the onset of treatment (GD6) then weekly.The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.On GD13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).Body weight measurementThe body weight of each animal was recorded with precision of ±1 g on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.Food consumption measurementFood was measured with precision of ± 1 g on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.Food consumption was calculated for each interval, including GD0-6, GD6-20 and GD0-20.Water consumption measurementThe water consumption was measured from start of the treatment until termination on each day. The average water consumption was reported on a weekly basis.PATHOLOGYCaesarean section and necropsyBefore expected delivery, on GD20, Caesarean section was performed on each treated dam. Sodium pentobarbital (Euthanimal 40% inj. AUV; Alfasan Nederland BV, Address: Kulpersweg 9, Woerden, Nederland; Batch number: 1409236-06, Expiry date: September 2017) administered by intraperitoneal injection and followed by exsanguination was used for euthanasia.The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. Liver, kidney, adrenal glands were retained in 10% buffered formalin solution (or modified Davidson fixative, in case of the eyes, if any abnormalities noted) for possible future evaluation (organ weights were measured before preservation). Organ weights are not part of the OECD guideline; these additional measurements were taken in case the weight differences were very large and could have influenced the study interpretation. This was not the case, so all organ weights made are tabulated in the appendices, but are not included in the Results and Discussion section as the data is not relevant to the study interpretation.The corrected body weight (body weight on GD20 minus weight of the gravid uterus), corrected body weight gain (total body weight gain minus weight of the gravid uterus) and net body weight gain (body weight gain during the treatment period minus weight of the gravid uterus) was calculated.No histopathology evaluation was performed in the study.

Ovaries and uterine content:

The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses.The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percentage of pre- and postimplantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Fetal examinations:

Each live foetus was weighed individually (accuracy of ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses was determined according to the anogenital distance. Thereafter the foetuses were individually identified; approximately half of each litter was subjected to visceral examination, and the other half was processed for skeletal examination.For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomya mixture then after fixation the body was micro-dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.For the foetuses subjected to skeletal examination, the abdominal region was opened, and the viscera and skin of foetuses were removed and the cadaver was fixed in alcianblue- acetic acid - ethanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting microscope.All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded; additionally photographic records were made when possible.

Statistics:

The statistical evaluation of data was performed with the program package SAS 4.3 in case of Provantis v9.3, or SPSS PC+4.0 in the case of data tabulated in Excel, by an appropriate statistical method.See "Any other information" for further details.

Indices:

Data were collected using the software PROVANTIS v9.3 (maternal data and necropsy), or were recorded on the appropriate forms from the relevant SOPs ofCiToxLAB Hungary Ltd. (Caesarean section and foetal data) then tabulated with PROVANTIS v9.3 or Microsoft Office Excel 2010.See "Any other information" for further details.

Historical control data:

Historical control data was used in this study.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related effects or systemic clinical signs were noted in any dose groups. Thin fur and/or alopecia observed in some test item treated animals (1 animal in the Low dose group of 75 mg/kg bw/day, 4 animals in the Mid dose group of 250 mg/kg bw/day and 1 animal in the High dose group of 750 mg/kg bw/day) as well as scar observed in one Low dose animal were considered as irrelevant findings.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no unscheduled mortality during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant changes were observed in the mean body weights of the Low, Mid and High dose groups (75, 250 and 750 mg/kg bw/day, respectively) when compared to control.Significantly lower body weight gain values compared to the Control group were recorded in the High dose group. For the 2 days following the beginning of the treatment (GD6-8) there was almost no weight gain (p<0.01) during the remaining treatment period the weight gain was generally lower than control (without statistical difference). During the overall treatment period (GD6-20) or in the entire study (GD0-20): the lower body weight gain was below control by approximately 17% (p<0.01) and 11% (p<0.05) respectively. This effect was related to the test item treatment. However, there were no body weight gain effects were observed in the Mid and Low dose groups.Similarly, significantly lower corrected body weight (~4%, p<0.05) corrected body weight gain (~22%, p<0.01) and corrected net body weight gain values (when results were adjusted by the gravid uterus weight) (~53%, p<0.01) were observed in the High dose animals compared to the control group, indicating a test item related effect. No statistically significant changes were seen in the Mid and Low dose groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant (p<0.01) decrease in the mean daily food consumption was noted in the High dose group (750 mg/kg bw/day) in the periods from GD6 to GD14 when compared to the control animals. This effect was considered as treatment related. Furthermore, the daily food consumption calculated for the entire study (GD0-20) and the total amount of consumed food were approximately 5% lower in the High dose group than in the Control, although these differences did not gain statistical significance.No test item related effect was seen in the Mid and Low dose groups (250 and 75 mg/kg bw/day, respectively).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in the mean daily water consumption was noted in the High dose group (750 mg/kg bw/day) particularly in the second week of treatment, GD13-20 (26%, p<0.01). In the first week of treatment water intake was about 12.5% above control, but was not statistically significant. This effect was considered as treatment related.No statistically significant differences were observed in the water consumption of the Mid and Low dose animals (250 and 75 mg/kg bw/day, respectively) compared to the control.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

NECROPSY FINDINGS OF DAMSNo observations were recorded for any animals in the study except of recording fur thin for one Low dose (75 mg/kg bw/day) and one Mid dose (250 mg/kg bw/day) animal or a scar on a thoracic region for one Low dose animal. The liver, kidney and adrenal glands of all animals were carefully examined, but no macroscopic changes were observed in any control or test item treated animals.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all test item treated groups. No test item effect was observed in the preimplantation loss of the test item treated groups when compared to the control.The early and the late embryonic loss did not differ significantly from the control in the test item treated groups. There was no statistically significant difference in the postimplantation loss between the test item treated and control groups.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

The total intrauterine mortality was comparable with the control, no statistically significant differences were observed.There was no statistically significant difference in foetal death in any test item treated groups compared to the control.

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

Description (incidence and severity):

Ninety-seven females (24 animals in the Control, Low and Mid dose groups and 25 animals in the High dose group) were mated in the study. The number of confirmed pregnant, evaluated dams was 24, 24, 24 and 25 in the Control, Low (75 mg/kg bw/day), Mid (250 mg/kg bw/day) and High dose (750 mg/kg bw/day) groups, respectively. Based on these data, test item treatment did not affect the pregnancy rate.

Other effects:

no effects observed

Description (incidence and severity):

Evaluation of placentasNo abnormalities were observed on the placentas of any animals in the Control, Low (75 mg/kg bw/day), Mid (250 mg/kg bw/day) or High (750 mg/kg bw/day) dosegroups. Test item treatment did not induce any abnormalities on the placentas.

Details on maternal toxic effects:

There were no toxicologically significant differences, or test item related-changes in the reproductive parameters in the study.The test item was considered to have had no effects on intrauterine development in this study.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

changes in number of pregnant

changes in pregnancy duration

dead fetuses

early or late resorptions

maternal abnormalities

necropsy findings

number of abortions

pre and post implantation loss

total litter losses by resorption

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The weight of foetuses per litter in the Low and High dose groups (75 and 750 mg/kg bw/day, respectively) did not differ significantly from the control mean value. Significant difference was noted in the Mid dose group (250 mg/kg bw/day), however this increase was not considered to be test item related due to the lack of dose response.With regard to the parameter “mean body weight/foetus” (not required in the OECD guideline) significantly higher values (p<0.01) were observed in the Low (75 mg/kg bw/day), Mid (250 mg/kg bw/day) and High (750 mg/kg bw/day) dose groups when compared to the control; but these slightly higher body weights with a lack of a dose response, were not considered as a test item related effect.The total number of retarded foetuses (runts) as well as the number of affected litters in the test item treated groups was comparable with the control value.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No statistically significant increases in the number of foetuses or litters with malformation / variation were detected when compared to the control.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean number of viable foetuses was comparable with the control mean in all test item treated groups.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

No statistically significant increases in the number of foetuses or litters with malformation / variation were detected when compared to the control.Two type of external malformations were recorded for one foetus in the High dose group (750 mg/kg bw/day), but based on the isolated occurrence it was considered incidental, ascribed to individual variability and not related to treatment. Historical control data also contained these or similar observations. No external malformations were recorded in the Mid and Low dose groups (250 and 75 mg/kg bw/day). No external variations were recorded in the study.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No statistically significant increases in the number of foetuses or litters with malformation / variation were detected when compared to the control.There were no malformations in the Mid or High groups (250 and 750 mg/kg bw/day, respectively). Pelvic girdle was recorded for two foetuses in the Low dose group (75 mg/kg bw/day. However, this malformation was also detected in a current control foetus, as well as it was included in the historical control data. Therefore this finding was considered as unrelated to the test item treatment.There was one skeletal variation (Skull, 3 or more bones, incomplete ossification) where the occurrence was higher in the Low (75 mg/kg bw/day) and High (750 mg/kg bw/day) dose groups than in the Control group. However, the difference was statistically not significant, and this is a common background finding (the observed values were within the historical control range), thus it was not considered to be treatment related. For other recorded skeletal variations, in each case the occurrence in the test item treated group(s) was equal or lower than in the control.

Visceral malformations:

no effects observed

Description (incidence and severity):

No statistically significant increases in the number of foetuses or litters with malformation were detected when compared to the control. The number of foetuses / litters with variation was significantly increased in the Low (75 mg/kg bw/day) and Mid (250 mg/kg bw/day) dose groups (p<0.01 and p<0.05, respectively), however all of the visceral findings were consistent in general nature and the observed frequency was within the existing historical control data, therefore they were considered as incidental findings.Only one malformed foetus was recorded in the test item treated groups: one foetus in the High dose group (750 mg/kg bw/day) showed malpositioned intestines, this finding corresponded to the Umbilicus, hernia abnormality found at external evaluation. Based on the isolated occurrence of this observation and according to the historical control data, this finding was considered incidental, ascribed to individual variability and not related to treatment.The occurrence of small renal papilla variation was significantly (p<0.05) increased in the Low dose group (75 mg/kg bw/day) when compared to the control group (this observation was not present in the current study control); however it corresponded to the historical control database and was not dose dependent, thus it was not considered as treatment related. Other variations observed incidentally in the test item treated groups (one foetus in each) were without dose response and in line with the historical control data, thus not related to the test item treatment.

Details on embryotoxic / teratogenic effects:

All of the foetal findings, in incidence and nature, corresponded to the concurrent study control or current historical control data and/or were not part of a dose response; hence these were considered as incidental, ascribed to individual variability and not related to treatment.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryotoxicity/foetotoxicity/teratogenicity

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Relation to maternal toxicity:

not specified

Dose response relationship:

no

Relevant for humans:

yes

Any other information on results incl. tables

Summary of the dose formulation analysis

Nominal concentration (mg/mL)	Measured concentration (mg/mL)	Percentage of the nominal concentration (%)
Analytical sampling #1
Control	Not detectable	-
15 mg/mL	16.4± 0.48	109
50 mg/mL	50.6± 2.40	101
150 mg/mL	138.9± 6.42	92
Analytical sampling #2
Control	Not detectable	-
15 mg/mL	14.0± 0.23	93
50 mg/mL	48.5± 0.45	97
150 mg/mL	153.9± 0.81	103

Note: Mean values and standard deviation are shown (rounded to one or two decimal places)

 

Summary of pregnancy data

Parameters	Dose (mg/kg bw/day)
	0	75	250	750
Number of mated females	24	24	24	25
Number of non-pregnant females	0	0	0	0
Number of females with≤5 implantation sites	0	0	0	0
Number of evaluated females on GD20 (Caesarean section)	24	24	24	25

 

Summary of the intrauterine evaluation

Parameters	Dose (mg/kg bw/day)
	0	75	250	750
Number of evaluated dams	24	24	24	25
Mean number of corpora lutea	12.08	11.54	11.54	11.24	NS
Mean number of implantations	11.00	11.04	11.13	10.44	NS
Preimplantation loss, mean	1.08	0.50	.042	0.80	NS
Preimplantation loss (%), mean	8.13	3.92	3.54	6.76	NS
Early embryonic loss, mean	0.33	0.13	0.33	0.32	NS
Early embryonic loss (%), mean	3.00	1.33	3.29	3.28	NS
Late embryonic loss, mean	0.08	0.08	0.00	0.04	NS
Late embryonic loss (%), mean	0.96	0.75	0.00	0.40	NS
Dead foetuses, mean	0.04	0.08	0.00	0.00	NS
Dead foetuses (%), mean	0.32	0.81	0.00	0.00	NS
Postimplantation loss, mean	0.46	0.29	0.33	0.36	NS
Postimplantation loss (%), mean	4.29	2.92	3.29	3.68	NS
Total intrauterine mortality, mean	1.54	0.79	0.75	1.16	NS
Total intrauterine mortality (%), mean	11.88	6.75	6.71	10.36	NS
Viable foetuses, mean	10.54	10.75	10.79	10.08	NS

Notes: NS: No statistical significance compared to the control

 

Examination of viable foetuses

Parameters	Dose (mg/kg bw/day)
	0	75	250	750
Number of examined litters	24	24	24	25
Viable foetuses, mean	10.54	01.75	10.79	10.08	NS
Male foetuses, mean	4.96	5.88	5.08	4.44	NS
Female foetuses, mean	5.58	4.88	5.71	5.64	NS
Total number of viable foetuses	253	258	259	252	NS
Total number of male foetuses	119	141	122	111	NS
Total number of female foetuses	134	117	137	141	NS
Sex ratio (males), %	47.13	54.54	47.38	45.32	NS
Mean foetal weight / litter (g)	3.339	3.401	3.505**	3.444	DN
Number of foetuses with retarded body weight	7	6	3	5	NS
Number of affected litters (Runts)	6	5	3	4	NS

Notes:

NS: No statistical significance compared to the control

DN: Duncan’s Multiple Range Test, *:p<0.05, **:p<0.01

 

Summary table of the external abnormalities

Parameter	Dose (mg/kg bw/day)				HC data
	Control	75	250	750
Total number of examined litters	24	24	24	25	485
Total number of examined foetuses	253	258	259	252	4955
No. of intact (normal) foetuses	253	258	259	251	--
No. of foetuses / litters with malformation	0 / 0	0 / 0	0 / 0	1 / 1	--
No. of foetuses / litters with variation	0 / 0	0 / 0	0 / 0	0 / 0	--
External malformations
Oedema, generalised	--	--	--	1 / 1	3 /3
Umbilicus, hernia	--	--	--	1 / 1	2 / 1^#
External variations
No external variations were observed

Notes: Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

The two malformations in the High dose group were noted in the same foetus (#4512/8).

#: Although historical control database did not contain the finding Umbilicus, hernia; but it contained the finding Omphalocele that is similar to the observation of Umbilicus, hernia, its incidence is shown in the table.

 

Summary table of the visceral abnormalities

Parameter	Dose (mg/kg bw/day)				HC data
	Control	75	250	750
Total number of examined litters	24	24	24	25	485
Total number of examined foetuses	126	129	129	126	2484
No. of intact (normal) foetuses	125	122^*CH	125	123	--
No. of foetuses / litters with malformation	1 / 1	0 / 0	0 / 0	1 / 1	--
No. of foetuses / litters with variation	0 / 0	7^**CH/ 5	4^*CH/ 4	2 / 2	--
Visceral malformations
Malformed great vessels	1 / 1	--	--	--	--
Intestines, malpositioned^#	--	--	--	1 / 1	2 / 1
Visceral variations
Renal papilla, small	--	5^*CH/ 3	2 / 2	--	18 / 16
Ureter, convoluted	--	1 / 1	1 / 1	1 / 1	3 / 3
Branchiocephalic trunk, short	--	1 / 1	1 / 1	1 / 1	43 / 35

Notes: Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

CH: Chi²test, *: p<0.05, **: p<0.01

#: The findingIntestine, malpositioned(#4512/8) corresponded to abnormalityUmbilicus, herniafound at external evaluation.

 

Summary table of skeletal abnormalities

Parameter	Dose (mg/kg bw/day)				HC data
	Control	75	250	750
Total number of examined litters	24	24	24	25	484
Total number of examined foetuses	127	129	130	126	2467
No. of intact (normal) foetuses	118	118	128^*CH	115	--
No. of foetuses / litters with malformation	1 / 1	2 / 2	0 / 0	0 / 0	--
No. of foetuses / litters with variation	8 / 7	9 / 7	2^*CH/ 2	11 / 8	--
Skeletal malformations
Pelvic girdle, malpositioned#	1 / 1	2 / 2	--	--	2 / 2
Skeletal variations
Skull, 3 or more bones incomplete ossification	2 / 2	4 / 3	2 / 2	5 / 3	144 / 114
Fontanelle, large	1 / 1	--	--	--	2 / 2
Ossified sternebra (3 or less)	4 / 4	4 / 4	1 / 1	4 / 4	46 / 37
Ribs, wavy marked	1 / 1	--	--	--	243 / 149
Vertebra, dumbbell or asymmetric or unilateral ossification (2 or more)	1 / 1	1 / 1	--	2 / 2	79 / 73
Vertebra, dumbbell shaped	1 / 1	1 / 1	--	--	6 / 6
Vertebra, bipartite ossification	1 / 1	1 / 1	--	1 / 1	24 / 24
Tarsal ossified≤3	4 / 3	1 / 1	1 / 1	0 / 0^*CH	43 / 29

Notes: Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

#: Lumbar VI – Sacral I transverse processes, fused

CH: Chi²test, *: p<0.05; **: p<0.01

Applicant's summary and conclusion

Conclusions:

In this study, from the observations made in the dams and their foetuses, the following no-observed-adverse-effect (NOAEL) levels were derived:NOAEL maternal toxicity: 250 mg/kg bw/dayNOAEL embryotoxicity: ≥750 mg/kg bw/day (highest examined dose)NOAEL foetotoxicity: ≥750 mg/kg bw/day (highest examined dose)NOAEL teratogenecity: ≥750 mg/kg bw/day (highest examined dose)

Executive summary:

This developmental toxicity study was performed to assess the effects of the test item DIBUTYLFUMARATE on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, from gestation day (GD) 6 up to and including GD19 (sperm positive day = 0 day of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

 

The dose levels of 75, 250 and 750 mg/kg bw/day were set by the Sponsor in consultation with the Study Director based on available data, including the results of a 14-day Dose Range Finding toxicity study in rats, another Dose Range Finding toxicity study in pregnant rats and the available data of a 90-day repeated dose toxicity study in rats, with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose. Control dams were treated with the vehicle (corn oil) only.

 

Dose formulations were analysed for test item concentration and homogeneity two times during the treatment period using a validated GC method.

 

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities.

Placentas were examined macroscopically.

 

The number of confirmed pregnant, evaluated dams was 24 in the Control, Low dose group (75 mg/kg bw/day) and Mid dose group (250 mg/kg bw/day), and 25 in the High dose group (750 mg/kg bw/day).

 

Results

All formulations were within the range of 92 to 109% of nominal concentration and were found to be homogenous. No test item was detected in the control samples.

Based on these results, test item formulations were considered suitable for the study purposes.

 

There were no unscheduled mortality or treatment related clinical signs in the study.

 

In the High dose group (750 mg/kg bw/day), there was a clear adverse effect on maternal body weight gain in the first 2 days of treatment, in maternal corrected body weight / corrected body weight gain and on food intake. Water intake was higher than controls during the treatment period. No effects were seen in the Mid (250 mg/ kg bw/day) and Low (75 mg/kg bw/day) dose groups.

 

No test item related adverse effects were observed at necropsy of the dams in any dose groups.

 

There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 750 mg/kg bw/day.

 

The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups.

 

The pre-implantation and the early and late embryonic loss were at control level in all treated groups.

 

There was no toxicologically relevant foetal death in any of the treated groups.

 

The mean number of viable foetuses in all test item treated groups as well as their sex distribution and body weight were comparable with the control mean.

 

The incidence of body weight retarded foetuses was similar in the control and test item treated groups.

 

No remarkable abnormalities were observed on the placentas in any examined groups.

 

There was no foetal external, visceral and skeletal malformation ascribed to the test item administration.

 

In conclusion, DIBUTYLFUMARATE, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19, was associated with the following findings: signs of maternal toxicity (body weight and food intake) was detected in the High dose group (750 mg/kg bw/day), but no effect was observed in the Mid (250 mg/kg bw/day) and Low (75 mg/kg bw/day) dose groups. There were no significant toxicological changes on embryos or foetuses in any dose groups.

 

In this study, from the observations made in the dams and their foetuses, the following no-observed-adverse-effect (NOAEL) levels were derived:

NOAEL maternal toxicity: 250 mg/kg bw/day

NOAEL embryotoxicity: ≥750 mg/kg bw/day (highest examined dose)

NOAEL foetotoxicity: ≥750 mg/kg bw/day (highest examined dose)

NOAEL teratogenecity: ≥750 mg/kg bw/day (highest examined dose)