Dihexyl fumarate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 14 May 2018 and 02 October 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

These deviations were considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dihexyl fumarate

EC Number:

242-833-4

EC Name:

Dihexyl fumarate

Cas Number:

19139-31-2

Molecular formula:

C16H28O4

IUPAC Name:

dihexyl fumarate

Specific details on test material used for the study:

Identification: Dihexyl fumarate (EC 242-833-4)
CAS: 19139-31-2
Physical form/color: Clear colourless liquid
Batch number: 800317920
Purity/concentration: 98.6%; no correction factor for purity was applied
Expiry date: 19 December 2018
Storage conditions: At room temperature (20 ± 5 ºC) in the dark.
Safety precautions: Safety precautions were taken according to the test item hazard class and the
results of the risk assessment

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hannover Wistar Rat (HsdHan®:WIST)

Details on species / strain selection:

Recognized by international guidelines as a recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
Species: Hannover Wistar Rat (HsdHan®:WIST)
Supplier: Envigo RMS, S.L.
Breeder: Envigo RMS B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
Total number of animals ordered: 44 males and 48 females
Total number of animals in the study: 40 males and 40 females (80 in total).
Each group started with 10 males and 10 females per group.
Age (at treatment start) Males: 10 weeks
Females: 10 weeks
Body-weight range (at start of treatment) Males: 283-349 g
Females: 190-239 g

Identification
-Acclimatization: F0 => Cage card and tail mark (later ear tattoo)
-Treatment: F0 => Cage card and individual ear tattoo
F1 offspring – Back mark (animals were observed daily together with pup status and the back mark was checked. It was not considered necessary to tattoo them).

Animal Care and Husbandry
Animal Care
Acclimatization: Five days after arrival and before the start of pre-test. After acclimatization period, the animals were subjected to a 15-day pre test period.
Veterinary examination: During acclimatization (at arrival and prior to treatment start), the animals were examined by a veterinary surgeon. Only animals without any visible signs of illness were used for treatment.
Environmental enrichment program: Different types of material specific for this species were supplied to reduce stress, enhance wellbeing and improve behavior.

Husbandry
Room number: 4
Conditions: Optimum hygienic conditions behind a barrier system: air-conditioned with a minimum of 15-20 air changes per hour, and continuously monitored environment with ranges for temperature of 20-24 ºC and for relative humidity between 30 and 70%. 12 hours fluorescent light/12 hours dark.
Accommodation: Cages with standard, granulated, S8-15 sawdust bedding (J. Rettenmaier & Söhne)
Premating period (maximum 5 animals/cage) Makrolon type-IV cages
Mating period (one male and one female/cage) Makrolon type-III cages
Postmating, gestation and lactation periods (individual) Makrolon type-III
Diet: Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum (supplied by Envigo RMS, S.L.). Results of analyses for composition and contaminant analyses are included in the raw data.
Pelleted standard Teklad 2018C rat/mouse maintenance diet (supplied by Envigo RMS, S.L.) ad libitum, for lactating females and pups (until sacrifice).
Water: Tap water in bottles ad libitum. Results of bacteriological, chemical and contaminant analyses are included in the raw data.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% CMCNa (medium viscosity) + 0.5 % Tween 80 in water

Details on exposure:

Formulation
Treatment Formulated concentration*
Group 1 Vehicle control
Group 2 Dihexyl fumarate; 10 mg/mL
Group 3 Dihexyl fumarate; 30 mg/mL
Group 4 Dihexyl fumarate; 100 mg/mL
* As supplied
Dose volume :10 mL/kg/day
Validated concentration range: 5 mg/mL to 150 mg/mL
Frequency of dose formulation preparation: No more than 7 days between preparation and administration based on the stability data.
Storage of dose formulations: At room temperature (20 ± 5 ºC) and in the absence of light when they were prepared on the day of administration, or refrigerated (5 ± 3 ºC) and in the absence of light when they were prepared for subsequent days.
Stability of dose formulations: At room temperature (20 ± 5 ºC) in the absence of light for 24h. Refrigerated (5 ± 3 ºC) and in the absence of light for 8 days
Safety precautions: Routine hygienic procedures (gloves, goggles, face mask)

Method of Preparation
The formulations were prepared ahead of time when they were covered by the proven stability period.
Formulation for Group 1 (Vehicle)
The necessary volume of the vehicle was added into a suitable container (stock solution) and samples for analysis / aliquots were taken for each day of analysis / administration, when required.
Formulation for Groups 2, 3 and 4
1. The required quantity of test item was weighed in a single-use container.
2. The test item was transferred to a suitable container.
3. Small amounts of vehicle were added (approximately 60-70% of the total volume) and the single use container was rinsed completely with the vehicle to ensure that there were no remnants of the test item and mixing the formulation until the emulsion was homogenous.
4. The emulsion was transferred to a volumetric flask or graduated measuring cylinder that was previously moistened with vehicle.
5. The container was completely rinsed with vehicle to ensure that there were no remnants of the formulation. This vehicle was added to the volumetric container and made up to the mark.
6. The formulation was transferred to a final container and mixed.
7. When it was necessary to adjust volume, the volume was first added into volumetric vessel and after the remainder was passed at final container.
After mixing the formulation with an agitator for five minutes and checking that the formulation was homogenous, samples for analysis were taken and the formulation was aliquoted for each day of administration, when required.
NOTE: The density was taken the first day each concentration was prepared.

Details on mating procedure:

Mating
Paired for mating After 2 weeks of treatment.
Male/female ratio 1:1
Duration of pairing 1-14 days (all animals mated in this period)
Daily checks for evidence of mating Ejected copulation plugs. Sperm within vaginal smear.
Day 0 of gestation When positive evidence of mating detected.
Male/female separation Day when mating evidence detected.
Pre-coital interval Calculated for each female as time between first pairing and evidence of mating.
Females showing no evidence of pregnancy (female 55 at 0 mg/kg/day, female 73 at 300 mg/kg/day and female 92 at 1000 mg/kg/day) were sacrificed 26 days after the last day of the mating period.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

Analytical method
Before commencement of treatment, an analytical method (M0361_HPLC_DFA_Dihexyl fumarate_Formulation_Vehicle_ISV) was validated in the present study. Formulations at two concentration levels (target concentrations: 5 and 150 mg/mL) were prepared and the following parameters were determined:
• System suitability (SST)
• Linearity
• Accuracy/repeatability
• Homogeneity
• Specificity
• Limit of Quantification (LOQ) and Limit of Detection (LOD)
• Stability [autosampler, formulations ( at 20 ± 5 ºC for 20-48 hours and at 5 ± 3 ºC for 8-15 days; in both cases in the absence of light)]
This method also includes the procedure followed for the analysis of administered formulations.
Analysis of the administered formulations: The formulations prepared at three different concentrations were analyzed twice over the course of the study to verify its correct preparation (see table below).
Control (vehicle) formulations were analyzed to confirm the absence of test item or other substances at the retention time of the test item.
The test item was used as analytical standard.
Results were expected to be within ±15% of nominal value and the coefficient of variation (CV) was ≤10%.
Other parameters were also tested, meeting the following acceptance criteria:
• System Suitability Test (SST) --- CV ≤ 2%.
Calibration curve --- R2 ≥ 0.99; deviation of the calibration standards ≤ 10% (75% complies).
Sampling: 12-mL aliquots (in duplicate) were taken from each analyzed formulation (see the table below). Each aliquot was taken from the formulation freshly prepared and poured into an amber glass vial. The second aliquot was considered as a contingency sample.
Labeling: Each aliquot was labeled with the study code, test item, concentration, date of preparation, storage conditions and aliquot number.
Sample shipment: Aliquot number 1 was transferred refrigerated at 5±3ºC in the absence of light to the Analytical and Bioanalytical Sciences Department at Envigo Barcelona for analysis.
Sample storage: Aliquot number 2 was stored (in the Pharmacy facilities at the Santiga site) at 5 ± 3 ºC and in the absence of light for a maximum of the days when stability has been demonstrated in the previous validation study.
Sample disposal: As samples for the formulation analysis were taken in duplicate, the first sample was used; any amount of this sample not used during analysis was disposed of without consultation with the Study Director. The second (contingency) sample was be disposed of once the stability period is accomplished.

Dose Formulation Analysis
Occasion Test item concentrations (mg/mL) Sample volume (mL) Samples taken
1st 0 mg/mL
10 mg/mL
30 mg/mL
100 mg/mL 12 2 (middle)
2nd 0 mg/mL
10 mg/mL
30 mg/mL
100 mg/mL 12 2 (middle)
Calculation: The formulation analysis results (including validation and DFA) were calculated using the Empower (version 2.0, Waters, USA). Other parameters such as accuracy, mean, SD and CV were calculated using an Excel spreadsheet.

Duration of treatment / exposure:

5-8 weeks
Males: 2 weeks before pairing up to necropsy after 5 weeks of treatment
Females: 2 weeks before pairing, then throughout pairing and gestation until days 13/15 of lactation (until the day before sacrifice)

Frequency of treatment:

Once daily
-F0 males: Two weeks prior to mating start until the day before sacrifice (after 5 weeks of treatment). They were then killed.
-F0 females: Two weeks prior to mating start until day 13/15 of lactation, including the day before sacrifice.
-F1: Potential indirect exposure in utero and through the milk during lactation

Details on study schedule:

See "Details on Mating Procedure"

Parturition Observations and Gestation Length
Duration of gestation Time elapsing between mating and commencement of parturition.
Parturition observations From day 20 after mating, animals were checked twice daily for evidence of parturition.
Number of live and dead offspring was recorded.

Records Made During Littering Phase
Clinical observations Observed 24 hours after the considered birth and then daily for evidence of ill-health or reaction to maternal treatment
Litter size Daily from Day 1-13 of age
Sex ratio Days 1, 4, 7 and 13 of age
Individual offspring body weights Days 1, 4, 7 and 13 of age
Ano-genital distance Day 1 - all F1 offspring
Nipple/areolae count Day 13 of age - male offspring.

Necropsy
F0 Males : After final investigations completed (after 5 weeks of treatment)
F0 Females failing to produce viable litter (not pregnant): Day 26 after mating
Females killed at termination: Day 14/16 of lactation
F1 Offspring : Selected offspring for thyroid hormone analysis – Day 4 of age (two females per litter where possible)
Scheduled sacrifice - Day 13/15 of age

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

Each group started with 10 males and 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels
The doses were chosen based on the preliminary results obtained in non GLP Study JJ43CF 14-day Oral (Gavage) Dose-Range Toxicity Study for OECD 422 conducted at Envigo CRS, S.A.U.
As no toxicity was obtained at the administered doses in JJ43CF, then:
- The high dose was selected as no toxicity was observed in the preliminary study at 1000 mg/kg/day and considering it as a limit dose to be tested.
- Intermediate and low dose levels were selected considering approximately a 3-fold increment between doses.

Randomization
Method based on the similarity of mean body weights among groups. Before treatment start, four females were exchanged after estrous cycle observations done. Rejected females, took no part of the study as well as the rest of spare animals.

Allocation
The animals were allocated at random to four treatment groups. Spare animals were used and/or animals were exchanged. The rejected animals took no further part in the study after commencement of treatment. The group identification and number of animals per group assigned for treatment are stated below:
Dose Group Dose Level(mg/kg/day) Animal Numbers
Males Females
1 0 1-3, 5-7, 9, 10, 42, 44 52-59, 61, 95
2 100 11-15, 17-20, 41 60, 62-67, 69, 91, 96
3 300 21-30 70-73, 75-79, 97
4 1000 31-40 80, 81, 83-85, 87-89, 90, 92

Method of Sacrifice
All F0 animals By intraperitoneal injection of sodium pentobarbital. Each animal was subsequently exsanguinated.
Offspring Selected for thyroid hormone sampling: Cardiac puncture. Death was assured with sodium pentobarbital injection.
All other offspring: injection of sodium pentobarbital.

Examinations

Parental animals: Observations and examinations:

Viability / Mortality / Cage-side Observations
Animals and their cages: Visually inspected twice daily for evidence of reaction to treatment or ill-health.

Body Weight
-See "Any Other Information on methods and materials"
The animals were weighed before sacrifice.

Food Consumption
-See "Any Other Information on methods and materials"

Clinical Signs / Detailed Signs
-See "Any Other Information on methods and materials"

Sensory Reactivity and Grip Strength
-See "Any Other Information on methods and materials"

Motor Activity
-See "Any Other Information on methods and materials

In-life Sampling and Analysis
Hematology, Peripheral Blood
Conditions: Males and females were deprived of food from 6-8 hours before blood extraction. Pups were removed from the dam the day before this procedure. Samples collected under light general anesthesia.
Anesthetic: Isoflurane.
Sample site: Retro-orbital sinus
Anticoagulant/Sample volume EDTA/0.5 mL
Citrate/0.5 mL
Routine hematology parameters were measured by the Advia 120 (Siemens Healthcare), version 3.1.8. Coagulation parameters were measured using a STA Compact analyzer (Roche), version 108.08.02.
All samples were examined for the following characteristics:
Using EDTA as anticoagulant
Hematocrit (Hct)
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)
Mean cell hemoglobin concentration (MCHC)
Mean cell volume (MCV)
Total leucocyte count (WBC)
Differential leucocyte count (N: neutrophils, L: lymphocytes, E: eosinophils, B: basophils, M: monocytes, LUC: large unstained cells)
Platelet count (Plt)
In addition, the following parameters were analyzed as considered necessary based on the analysis system alarm.
Grade of anisocytosis
Hypochromic Cells
Hyperchromic Cells
Using citrate as anticoagulant
Prothrombin time (SPT)
Activated partial thromboplastin time (SAPT)

Blood Chemistry
Conditions: Males and females were deprived of food from 6-8 hours before blood extraction. Pups were removed from dam the day before this procedure. Samples collected under light general anesthesia.
Anesthetic: Isoflurane
Sampling site: Retro-orbital sinus
Anticoagulant/Sample volume: Lithium heparin/0.8 mL
Routine biochemistry parameters were measured using the Cobas 6000 analyzer (Roche), version Cobas Link.
Electrophoretic parameters were performed by HYDRASIS LC from Sebia, version 5.1.8.
The albumin/globulin ratio was derived from the total protein value from the Cobas 6000 and the albumin value generated by the HYDRASYS LC from Sebia.
All samples were examined for the following characteristics:
Using lithium heparin as anticoagulant
Alkaline phosphatase (ALP)
Alanine amino-transferase (ALT)
Aspartate amino-transferase (AST)
Creatine kinase (CK)
Bilirubin – total (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Cholesterol – total (Chol)
HDL
LDL
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio)
Protein electrophoretogram

Oestrous cyclicity (parental animals):

Dry smears
Using inoculation loops during the following phases:
• For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
• Daily from the beginning of treatment period until evidence of mating.
• On the day of necropsy*
A cotton swab impregnated in physiological saline was used in order to check the evidence of mating during the mating period.
*No vaginal smear was taken at termination for females 59 (0 mg/kg/day), 75 (300 mg/kg/day) and 88 (1000 mg/kg/day).

Sperm parameters (parental animals):

Testes: detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

Clinical observations: Observed 24 hours after the considered birth and then daily for evidence of ill-health or reaction to maternal treatment
Litter size: Daily from Day 1-13 of age
Sex ratio: Days 1, 4, 7 and 13 of age
Individual offspring body weights: Days 1, 4, 7 and 13 of age
Ano-genital distance: Day 1 - all F1 offspring
Nipple/areolae count: Day 13 of age - male offspring.

Postmortem examinations (parental animals):

Organ Weights
Adult animals See "Any Other Information on methods and materials"
Data collection For bilateral organs, left and right organs were weighed together.

Macroscopic Pathology
Blood sampling required: specific details are included in this section as regards animals from which samples were required for the analysis of thyroid hormone levels. Blood samples were also required at termination from specific adult animals for the evaluation of hematology and blood chemistry parameters and details relating to these examinations are included.
Scheduled termination
F0 animals (six males and five lactating females with a surviving litter per group) Blood sampling required from all surviving males and females.
Complete necropsy as per Pathology procedures for the five males and the five lactating F0 females with a surviving litter per group at scheduled termination, and all F0 adult decedents: all animals.
Checks: retained tissues.
Number of uterine implantation sites counted and checked.
Remaining F0 males and females / Females not pregnant Limited list as per Pathology procedures for remaining F0 males and females per group.
Checks: retained tissues.
Number of uterine implantation sites counted and checked.

Fixation
Fixatives Standard – 10% Neutral Buffered Formalin.
Testes and epididymides: Initially in modified Davidson’s fixative.
Eyes: Davidson’s fixative.

Histology

Processing – Full list The five males and five lactating females with a surviving litter selected in Groups 1 and 4 at scheduled termination.
Processing – Abnormalities only All adult animals
Routine staining 4-5 µm sections stained with hematoxylin and eosin, except testes which are also stained with periodic-acid Schiff

Pathology
Data Recording
Ovaries: qualitative evaluation of one section from each ovary.

Postmortem examinations (offspring):

Macroscopic Pathology
F1 offspring on Day 4 of age Blood sampling required from two females per litter where possible.
F1 offspring on Day 13 of age Blood sampling required from two males and two females per litter, where possible.
Thyroid gland to be retained from one male and one female in each litter where possible.
All animals were subjected to an external macroscopic examination; particular attention was paid to the external genitalia.

Statistics:

See "Any Other Information on methods and materials"

Reproductive indices:

Percentage mating = (Number animals mating/Animals paired) x 100
Conception rate = (Number animals achieving pregnancy/Animals mated) x 100
Fertility index = (Number animals achieving pregnancy/Animals paired) x 100
Gestation index: Calculated for each group as: (Number of live litter born/Number pregnant) x 100

Offspring viability indices:

Post-implantation survival index: (Total number offspring born/Total number uterine implantation sites)x 100
Live birth index: (Number live offspring on Day 1 after littering/Total number of offspring born) x 100
Viability index: (Number live offspring on Day 4/Number live offspring on Day 1 after littering) x 100
Lactation index: (Number live offspring on Day 13 after littering/Number live offspring on Day 4(after
selection for thyroid hormone bleed)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation was recorded occasionally at 1000 and 300 mg/kg/day in males and females from day 8 of treatment until the end of the study. It tended to occur approximately within ½ hour of dosing. Incidence was higher at 1000 mg/kg/day. Post-dosing salivation has been related with taste aversion (Ronald, 2006).
No other signs considered related to the administration of Dihexyl fumarate were observed during the study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was recorded during the course of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with Dihexyl fumarate had no relevant effect on body weight. From treatment day 29 to the end of the study, males showed slightly lower and statistically significant mean body weights compared to the Control group (always within 7% with respect to Control); however it cannot be considered adverse due to the magnitude and the fact that the same profile was not observed in females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Administration of Dihexyl fumarate had no effects on food consumption measurements during the whole study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of treatment period, lower mean values with statistically significant differences with respect to Control were observed at 1000 mg/kg/day in Mean Cell Hemoglobin (MCH) in males and Platelet count (Plt) in females, as well as higher than Control mean values at 300 and 1000 mg/kg/day in Mean cell hemoglobin concentration (MCHC) in females and lower than Control at these doses and 100 mg/kg/day in Mean Corpuscular Volume (MCV) in males. The significant differences observed were considered not relevant, given the magnitude of the differences between means and taking into account that these values were closed to those recorded in the Control, there was no dose relation and as they were within the Historical Control Data.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry after 5 weeks of treatment in males revealed higher mean values than Control for cholesterol, HDL and triglycerides mean values in the test item administered groups with statistically significant differences at 300 and 1000 mg/kg/day. Mean triglyceride values in females were also higher in the test item administered groups when compared to Control, but no dose relation was observed (statistically significant differences were only observed at 300 mg/kg/day). Statistical significance observed at 1000 mg/kg/day in males was considered occasional as there was no dose response and given the magnitude.
Creatinine mean values in the test item administered groups in males and females were always lower than those recorded in the Control, following a dose relationship. Differences were also statistically significant at 300 and 1000 mg/kg/day.
Statistically significant differences were observed in urea mean values at 300 and 1000 mg/kg/day being the effect different between both sexes (higher in males and lower in females with respect to Control).
In males, a dose related decrease in glucose mean values was observed, being it statistically significant at 1000 mg/kg/day. However, all values were within those recorded in males under the same conditions, and consequently this fact is not considered test item related.
There were statistically significant differences in mean calcium values in females at 1000 mg/kg/day with respect to Control. It is considered of no toxicological relevance as there is no dose relation and considering the differences within the normal individual values recorded.
In males at 300 and 1000 mg/kg/day, there was an increase in albumin and, consequently, in total proteins. The difference was statistically significant at 1000 mg/kg/day for total proteins and albumin, and only for albumin at 300 mg/kg/day. Based on the magnitude of the change, it was not considered an adverse effect. Consequently, and in the absence of any relevant effect in the total proteins or albumin, the significant differences observed in protein electrophoresis were considered of no relevance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Sensory reactivity conducted during week 5 in males and between days 7-9 of lactation in females revealed no findings considered treatment-related. An isolated male (no. 23, administered at 300 mg/kg/day) did not show tail pinch response. It was considered not test item related and occasional as it wasobserved at the intermediate dose.
Grip strength recordings revealed significantly lower mean forelimb mean value among males at 1000 mg/kg/day with respect to Control. However there was no evidence of this effect in females and there were no clinical signs that could corroborate this finding in males. This value was not taken into consideration given the high variability and as values observed were within those recorded in rats of thisstrain and age.
Motor activity assessment revealed significantly higher mean values at 10, 20, 30 minutes and, consequently, in total mean values in females at 1000 mg/kg/day with respect to Control. However, there was no evidence of this effect in males nor were there clinical signs that could corroborate thisfinding. In addition, the significant values observed were similar to those recorded in the historicalcontrol data in females under the same conditions.
Statistically significant differences observed in males at 50 minutes were considered related to the low mean values recorded in the Control group and those recorded at 100 mg/kg/day at 60 minutes wereconsidered occasional (as they were only observed at the low dose).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The microscopic examination performed 5-8 weeks after treatment at 1000 mg/kg/day revealed test item-related changes in the stomach (minimal to slight, diffuse epithelial hyperplasia of the nonglandular region (forestomach)) of both males and females accompanied by a minimal degree of hyperkeratosis in all males and few females. Minimal to slight hypertrophy of centrilobular hepatocytes was observed in the liver of most of the males administered at 1000 mg/kg/day.
All other histological findings were considered incidental and unrelated to the test item.
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted in males given 1000 mg/kg/day.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Analyses of samples for thyroxine (T4) obtained from main study males and F1 offspring on Day 13 of age did not reveal any differences attributable to treatment. The statistically significant differences observed in males at 300 and 1000 mg/kg/day are considered devoid of any toxicological effect and could be attributed to the normal biological variability, taking into account that no effects have been observed in the thyroid histopathology and considering that mean values in Control group are slightly lower than expected based on the Historical Control Data.
Statistically significant differences observed in offspring females at 100 mg/kg/day are not considered of any toxicological effect and could also be attributed to the normal biological variability as there is no dose-effect relationship and they were not observed in male offspring.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to the study showed regular 4-day estrous cycles prior to treatment start and the frequency of regular cycles during treatment was the same or higher than that observed in the Control group.
At termination, all surviving reproductive phase females showed diestrus with the exception of three females in the Control group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted in males given 1000 mg/kg/day.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on the pre-coital interval; all animals (with the exception of female no. 75 at 300 mg/kg/day) mated within four days of mating at the first estrus.
There was no effect of treatment on gestation length or gestation index.
Mating performance was not affected by treatment (100% for all groups) and fertility was 100% (10/10) at 100 mg/kg/day, 90% (9/10) at 0, 300 and 1000 mg/kg/day.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs observed among the F1 litters that could be clearly attributable to parental treatment with Dihexyl fumarate.
Although little or no milk was observed in the stomach and occasionally abnormally cold to touch body temperature were recorded in some pups during the first days of lactation, the majority of the offspring survived the first days of the phase.
One pup at 300 mg/kg/day (97-7F) showed a deformity in tail (partially absent). Taking into account that it was occasional, it is considered not to be treatment related.

Dermal irritation (if dermal study):

no effects observed

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no effect of treatment on offspring survival, litter size or sex ratios.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on mean body weights of male or female offspring on day 1 of age or on subsequent body weight measurements until day 13 of age.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings in the offspring that died prior to the scheduled termination or among those offspring killed on days 4 or 13-15 of age that were attributable to maternal treatment with Dihexyl fumarate.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There was no effect observed in ano-genital distance or male nipple observations.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There was no treatement-related adverse effect found on any F1 effect level parameters.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Any other information on results incl. tables

Sensory Reactivity and Grip Strength

Group	Forelimb Mean (g) Males
Statistical test	Wi
0 mg/kg/day	817.54
100 mg/kg/day	704.55
300 mg/kg/day	662.22
1000 mg/kg/day	583.68*
Historical Control Data[1]	Mean 796.7 Range 433.3 to 3017.9

[1] In six studies conducted between 2017 and 2018, control rats of the same strain housed in similar environmental conditions.

Motor Activity

Group	Motor Activity / Total (min) Females
Statistical test	Wi
0 mg/kg/day	1003.6
100 mg/kg/day	1040.8
300 mg/kg/day	1133.8
1000 mg/kg/day	1600.0*
Historical Control Data[1]	Mean 1188 Range 586 to 2179

[1]In six studies conducted between 2017 and 2018, control rats of the same strain housed in similar environmental conditions.

Hematology and Coagulation

Group	MCH (pg) Males	MCV (fL) Males	MCHC (g/dL) Females	Plt (x10^9/L)
Statistical test	Wi	Wi	Wi	Wi
0 mg/kg/day	18.7	57.3	32.5	877
100 mg/kg/day	18.2	55.5*	32.8	912
300 mg/kg/day	17.8	54.9**	33.4*	809
1000 mg/kg/day	17.7*	55.0**	33.4*	698*
Historical Control Data[1]	Mean 17.83 Range 16.1 to 19.8	Mean 55.94 Range 52.9 to 60.3	Mean 32.44 Range 29.9 to 35.1	Mean 774.1 Range 214 to 1012

[1]In six studies conducted between 2017 and 2018, control rats of the same strain housed in similar environmental conditions.

Blood Chemistry

Group	Chol (mmol/L) Males	HDL (mmol/L) Males	Trig (mmol/L) Males	Trig (mmol/L) Females	Creat (µmol/L) Males	Creat (µmol/L) Females
Statistical test	Wi	Wi	Wi	Du	Wi	Wi
0 mg/kg/day	2.01	1.63	0.83	1.56	25	31
100 mg/kg/day	2.24	1.82	1.13	1.95	22	29
300 mg/kg/day	2.69**	2.14**	1.58*	4.21**	21*	26*
1000 mg/kg/day	2.88**	2.22**	2.15**	2.33	20**	21**
Historical Control Data[1]	Mean 2.096 Range 1.27 to 2.66	Mean 1.715 Range 1.20 to 2.14	Mean 1.043 Range 0.50 to 1.88	Mean 2.397 Range 0.54 to 6.44	Mean 23.3 Range 19 to 31	Mean 31.3 Range 24 to 42

[1]   In six studies conducted between 2017 and 2018, control rats of the same strain housed in similar environmental conditions.

Group	Gluc (mmol/L) Males	Urea (mmol/L) Males	Urea (mmol/L) Females	Ca (mmol/L) Females	Total Prot (g/L) Males	Alb (g/L) Males
Statistical test	Wi	Wi	Wi	Wi	Wi	Wi
0 mg/kg/day	10.32	4.04	10.58	2.52	62	33
100 mg/kg/day	9.67	4.31	8.29	2.35	62	32
300 mg/kg/day	9.05	5.02*	8.11*	2.47	66	35*
1000 mg/kg/day	7.94*	4.99*	6.87**	2.18*	69**	38**
Historical Control Data^1	Mean 10.779 Range 7.75 to 15.60	Mean 4.163 Range 2.51 to 5.71	Mean 8.720 Range 5.74 to 14.90	Mean 2.696 Range 2.34 to 3.00	Mean 62.6 Range 50 to 69	Mean 34.1 Range 31 to 37

[1]   In six studies conducted between 2017 and 2018, control rats of the same strain housed in similar environmental conditions.

Thyroid Hormones

Mean serum T4 concentrations (pg/mL)

Dose		F0	Day 13 Offspring	Day 13 Offspring
(mg/kg/day)		Males	Males	Females
Statistical test		Wi	Wi	Du
0	Mean SD N	48298.76 9079.610 9	50408.04 6268.237 9	52255.90 6382.421 9
100	Mean SD N	53409.43 5863.183 10	48896.15 4600.132 10	45047.76* 5402.492 10
300	Mean SD N	60255.10** 6944.427 10	54383.90 7480.805 10	55902.44 7477.535 9
1000	Mean SD N	60996.09** 10519.452 10	45600.07 7580.444 9	49923.75 5778.930 9
Historical Control Data[1]		Mean 54308.0 Range 35963.81 to 80106.20

[1]      In seven studies conducted between 2017 and 2018, control rats of the same strain housed in similar environmental conditions.

Organ Weights

Dose	Absolute weight	Kidneys	Kidneys	Liver	Liver
(mg/kg/day)	(g)	Males	Females	Males	Females
Statistical test		Wi	Wi	Wi	Wi
0	Mean	2.334	1.662	10.544	11.364
100	Mean	2.138	1.731	10.932	12.348
300	Mean	2.464	1.770	12.202*	12.422
1000	Mean	2.563*	2.002**	14.185**	13.234*
Historical Control Data[1]		Mean 2.3580 Range 1.811 to 3.161	Mean 1.6898 Range 1.259 to 2.105	Mean 12.0232 Range 9.288 to 16.601	Mean 11.5380 Range 8.015 to 13.891

[1]      In six studies conducted between 2017 and 2018, control rats of the same strain housed in similar environmental conditions

Applicant's summary and conclusion

Conclusions:

In conclusion, the effects of oral (gavage) administration of Dihexyl fumarate to Wistar rats receiving 100, 300 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:
The No Adverse Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on estrous cycle, pre-coital interval, mating performance, fertility and gestation length or in the offspring, on litter size, survival, sex ratio, clinical signs, body weights, ano genital distances or macropathology.

Executive summary:

Introduction

The purpose of this study was an assessment of the general systemic toxic potential ofDihexyl fumaratein Hannover Wistar rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, after administration of the test item by gavage for 5-8 weeks.

Three groups of 10 male and 10 female rats receivedDihexyl fumarateat the doses of 100, 300 and 1000 mg/kg/day. Males were treated continuously for two weeks before pairing up to necropsy, after a minimum of 36 consecutive days. Females were treated continuously for two weeks before pairing, throughout pairing and gestation, and until Day 13-15 of lactation (the day before sacrifice). Females were allowed to litter and rear their offspring, and litters were killed on Day 13/15 of lactation (the day before the corresponding female was killed). F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted control group received the vehicle, 1% CMCNa (medium viscosity) + 0.5% Tween 80 in water.

During the study, mortality, clinical signs, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology and coagulation, blood biochemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated.

Clinical signs, behavior assessment, litter size and survival, sex ratio, body weight and macropathology were also assessed for all offspring.

Results

The study results can be summarized as follows:

-         Formulations analyzed demonstrated their correct preparation during the study.

-         No mortality was observed during the study, and administration of Dihexyl fumarate at 100, 300 or 1000 mg/kg/day was considered not to have any relevant effects on clinical condition, body weight, food consumption, grip strength, sensory reactivity and motor activity, pre-coital interval, mating performance and fertility, or gestation length. Salivation was recorded occasionally at 300 and 1000 mg/kg/day; the incidence was dose-related in both sexes and is most likely due to taste aversion (from gavage dosing).

-         Estrous cycles before and during treatment were considered not to be altered by the test item. At termination, all reproductive phase females showed diestrus, with the exception of three females in the Control group.

-         There were no relevant effects observed in hematology or coagulation analyses. Clinical biochemistry analysis reveals a slight effect in cholesterol, HDL, triglycerides, creatinine and urea. Although they were not considered adverse due to their magnitude, dose relation, occasional nature and the fact that they were within the normal ranges observed in rats of this strain and age, it can be demonstrated that there is an adaptive response to the test item administration given that a slight effect in liver and kidney weights was also observed, mainly at 1000 mg/kg/day.

-         There were no differences in T4 analysis attributable to treatment.

-         There were no macroscopic findings that could be considered test-item-related.

-         Microscopic pathology reveals adaptive responses to oral administration (epithelial hyperplasia and hyperkeratosis in the forestomach) andto a test item-related hepatic enzyme induction (hypertrophy of centrilobular hepatocytes).

-         There were no adverse effects observed in the offspring. Thus, offspring survival, litter size, clinical signs, body weights, ano-genital distances or macropathology were not affected byDihexyl fumarate administration.

In conclusion, the effects of oral (gavage) administration ofDihexyl fumarate to Wistar rats receiving 100, 300 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

Reproductive / developmental toxicity:

-       The No Adverse Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on estrous cycle, pre-coital interval, mating performance, fertility and gestation length or in the offspring, on litter size, survival, sex ratio, clinical signs, body weights, ano‑genital distances or macropathology.