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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2 May 2013 - 26 Nov 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline Study. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Didodecyl fumarate
- Physical state: solid
- Analytical purity: 93.8 %
- Lot/batch No.: 0008043725
- Storage condition of test material: Room temperature (15 - 25 °C)

Test animals

Species:
rat
Strain:
other: Han-Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 328 to 395 g; Females: 217 to 255 g
- Fasting period before study: no
- Housing: In groups of three to five in Makrolon type-4 cages with wire mesh tops during acclimatization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V. / Netherlands). During the prepairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch nos. 43/12 and 56/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland), ad libitum.
- Water: Community tap-water from Füllinsdorf in water bottles, ad libitum
- Acclimation period: minimum 5 days; under test conditions after health examination; only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily using the test item as supplied by the Sponsor. Didodecyl fumarate was weighed into a glass beaker on a tared precision balance and 80 - 90% of the warmed-up (40 ± 5 °C) vehicle was added (w/v) with a syringe in small amounts under continuously stirring. The dose formulation was heated on approx. 50 °C for approx. 20 minutes and then the remaining warmed-up vehicle was added. Each dose formulation was homogenized with an Ultraturrax and stirred again for approx. 20 minutes at 37 - 40 °C. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period by stirring at 37 - 40 °C temperature.
Dose Volume: 5 mL/kg bw
Dose Concentration: Group 1: 0 mg/mL; Group 2: 20 mg/mL; Group 3: 60 mg/mL; Group 4: 200 mg/mL

VEHICLE
- Source: Carl Roth GmbH
- Lot/batch no.: 103197718


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In the first week of dose formulation day a sample from the control group as well as three samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Due to high variation of the analytical homogeneity results, additional samples were taken in the first and second week from remaining formulations to evaluate optimized analytical conditions for sample workup and derivatization. These samples were analysed but results were not reported. Samples of each test item concentration were taken from the middle to confirm the stability (4 hours at room temperature 15 - 25 °C). Towards the end of the study, samples were taken from the middle to confirm concentration.
Since the dose formulation became not homogenous anymore at low temperature, samples of the exact amount of dose formulation were drawn and the entire sample was analyzed:
Groups 1 and 2: 500 mg dose formulation
Group 3: 250 mg dose formulation
Group 4: 100 mg dose formulation
The samples were analyzed by GC-FID. The test item was used as the analytical standard.
Duration of treatment / exposure:
Males: 48 days
Females: approx. 6 weeks
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
The study schedule can be found in "Any other information on materials and methods incl. tables".

Mating, Gestation and Lactation
During the pairing period, females were housed with sexually mature males from the same dose group (1:1) until evidence of copulation was observed. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.
If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.
All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Termination of the Study
Males were sacrificed after treatment of at least 48 days, when no longer needed for the assessment of reproductive effects. Pups were sacrificed on day 4 post partum. Dams were sacrificed on day 5 post partum.
If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
The following observations were recorded:
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Pup Data: The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

DETAILED CLINICAL OBSERVATIONS: Yes
Once prior to the first administration of the test item and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum), detailed clinical observations were performed outside the home cage in a standard arena. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Any changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Body Weights: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food Consumption: Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly. Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 and days 1 - 4 of the lactation. No food consumption was recorded during the pairing period.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HEMATOLOGY: Yes
Blood samples were obtained on the day before or on the day of the scheduled necropsy from 5 males randomly selected from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
The following hematology parameters were determined:
- Complete Blood Cell Count: Erythrocyte count, Hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, total Leukocyte count, Differential leukocyte count: Platelet count and Reticulocytes
- Coagulation: Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, total Bilirubin, total Cholesterol, Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Sodium, Potassium, Chloride, Calcium, Phosphorus, total Protein, Albumin, Globulin, Albumin/Globulin ratio.
Furthermore, an additional blood sample (0.5 mL) was collected into serum tubes for possible future measurement of thyroid releasing hormone (TSH), and the thyroid hormones triiodothyronine (T3) and thyroxine (T4). Serum samples were stored at -80 ± 10 °C. Any samples remaining at finalization of the study report were discarded. These measurements were not necessary, since there were no changes at microscopic examination of the thyroid glands or thyroid gland weights.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females randomly selected from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
• Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
• Hand-held observations: muscle tone, constitution, skin, pupil size, palpebral closure lacrimation, salivation, reaction to handling and general abnormalities.
• Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
• Reflexes: blinking, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
• Measurements / Counts: hind limb / fore limb grip strength, rectal temperature.
Any abnormal findings were recorded and, where appropriate, graded in severity. Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. Dead pups, except those excessively cannibalized, were examined macroscopically. All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

HISTOPATHOLOGY: Yes
Tissue Preservation
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution: Prostate, Seminal vesicles with coagulating gland, Testes (in modified Davidson Solution), Epididymides (in modified Davidson Solution).
The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution: Ovaries (with oviduct), Uterus (with vagina).
In addition, from all males and females the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: Gross lesions, Brain, Spinal chord (cervical, thoracic, lumbar), Small and large intestines1 (incl. Peyer’s patches), Stomach (forestomach and glandular stomach), Liver, Kidneys, Adrenals, Spleen, Lymph nodes (axillary and mesenteric), Aorta2, Eyes with optic nerve and harderian gland2, Lacrimal gland2, Larynx2, Nasal cavity2, Esophagus2, Heart, Thymus, Thyroids, and parathyroids if possible, Trachea and lungs (preserved by inflation with fixative and then immersion), Pituitary gland2, Urinary bladder, Peripheral nerve (sciatic), Bone marrow (femur), Femur with knee joint2, Mammary gland (male and female)2, Pancreas2, Salivary glands – mandibular, sublingual2, Skeletal muscle2, Sternum with bone marrow2, Pharynx2.
(1 = Duodenum, jejunum, ileum, colon, caecum, rectum; 2 = Only examined by histopathology in case of macroscopic findings indicative of potential toxicity)

Histotechnique
All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.

Histopathology
Macroscopical findings, testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high dose group were examined. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis. The remaining organs/tissues of 5 randomly selected males and females of the control and high dose group, respectively, were examined histopathologically. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. Since test item-related morphologic changes were detected in the liver at the high dose, these organs from the mid- and low-dose group were examined to establish a no-effect level. Histological examination of ovaries was carried out on any females that did not give birth. A histopathology peer review was performed.
Statistics:
The following statistical methods were used to analyze food consumption, body and organ weights, grip strength, rectal temperature, clinical laboratory and reproduction data, locomotor activity and macroscopical findings:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Description (incidence and severity):
Since the differences were minor, they were considered not to be toxicologically relevant.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The statistically significant differences from controls occurred in one gender only and the values were within the range of historical control data or showed no dose-dependency.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
ALAT levels in males were in the range of the historical control data. Nevertheless, the increase may be test item related but not adverse. Further statistically significant differences are considered not to be toxicologically relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Due to the observed morphological alterations in the liver of males (1000 mg/kg bw/d), the increase was considered to be test item related but not adverse.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were minor morphological alterations in the liver of males is considered as adaptive in nature, within physiological limits and not a manifestation of frank toxicity.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unplanned deaths. All animals survived until scheduled necropsy. No test item-related clinical signs were noted in males and in females treated at 100, 300 and 1000 mg/kg bw/day. The findings noted during the detailed weekly clinical observations of males and females did not indicate any test item-related effects.

BODY WEIGHT AND WEIGHT GAIN
Males: There were no effects on mean body weight gain and mean body weights at any dose level and in any study phase. Statistically significant differences in body weight gain occurred on several occasions in males, but were either not dose-dependent or represented slightly higher body weight gains. Therefore, these differences were considered to be fortuitous. The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +11%, +12%, +12% and +10% during the pre-pairing period, +6%, +7%, +6% and +7% during the pairing period and +3%, +3%, +4% and +3% during the after pairing period (percentages refer to the body weight gain within the period).
Females: There were no effects on mean body weight gain and mean body weights at any dose level and in any study phase. The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +6%, +6%, +7% and +7% during the pre-pairing period, +54%, +57%, +57% and +57% during the gestation period and +4%, +4%, +4% and +4% during the lactation period (percentages refer to the body weight gain within the period).

FOOD CONSUMPTION AND COMPOUND INTAKE
Males: There were no test item-related effects on mean food consumption in males at any dose level and in any study phase. At 1000 mg/kg bw/day, food consumption was slightly higher between day 8 and day 20 of the after pairing period (approximately 10%). Since the differences were minor, they were considered not to be toxicologically relevant.
Females: There were no effects on mean food consumption in females at any dose level and in any study phase.

HEMATOLOGY
The assessment of the hematology data did not reveal any test item-related effects in males and females at any dose level. The statistically significant differences from controls (higher numbers of neutrophils and reticulocytes in males at 1000 mg/kg bw/day; higher mean corpuscular hemoglobin concentration in females at 100 and 1000 mg/kg bw/day; lower MCV values in females at 100 mg/kg bw/day) occurred in one gender only and the values were within the range of historical control data or showed no dose-dependency.

CLINICAL CHEMISTRY
In males, the alanine aminotransferase levels were slightly, but dose-dependently increased at 300 and 1000 mg/kg bw/day (up to 2.2-fold). These levels were in the range of the historical control data. Nevertheless, due to the observed morphological alterations in the liver, the increase may be test item related but not adverse. Further statistically significant differences (higher glucose values at 100 and 1000 mg/kg bw/day; lower creatinine values at 1000 mg/kg bw/day; higher potassium values at 1000 mg/kg bw/day; higher protein levels at 100 and 1000 mg/kg bw/day, with higher globulin values and lower albumin/globulin ratio at 1000 mg/kg bw/day) were minor and within the historical control range except protein at 1000 mg/kg bw/day, which was borderline to historical control data. In the absence of corroborating changes (especially regarding histopathology) these differences are therefore considered not to be toxicologically relevant. In females, the assessment of the clinical biochemistry data did not reveal any test item-related effects at any dose level. Statistically significantly higher chloride values occurred at 100 mg/kg bw/day, but without dose-dependency.

NEUROBEHAVIOUR
None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect. A slightly but statistically significantly lower body temperature (-1.3%) was noted in males at 300 mg/kg bw/day. However, due to the lack of dose-dependency, this finding was deemed to be incidental.
Locomotor activity was not affected by the treatment with the test item at any dose level.

ORGAN WEIGHTS
In males at 1000 mg/kg bw/day, absolute and relative liver weights were statistically significantly increased. The value relative to body weight was in the range of the historical control data (2.39-3.56 g). Due to the observed morphological alterations in the liver, the increase was considered to be test item related but not adverse. Higher kidney values occurred in males at 100 and 1000 mg/kg bw/day. These differences were considered to be incidental since there was no dose-dependency. Further statistically significant differences comprised only derived relative weights or organ to brain ratios and were not accompanied by histopathological findings. No effects were observed at other dose levels or in females at any dose level.

GROSS PATHOLOGY
There were no test item-related findings noted at necropsy in males and females. All gross lesions recorded were considered to be within the range of normal background alterations and showed no dose dependency.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were minor morphological alterations in the liver of males. This consisted of a minimal degree of diffuse, midzonal/centrilobular hepatocellular hypertrophy recorded in 3/5 rats at 1000 mg/kg bw/day. This finding may be considered as adaptive in nature, within physiological limits and not a manifestation of frank toxicity.

Effect levels

Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects were observed up to the highest dose tested (1000 mg/kg bw/d).

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 2: Alanine Aminotransferase (ALAT) in males (on the day of the scheduled necropsy) and in females (on day 5 post partum)

Males

Females

 

ALAT [U/L]

ALAT [U/L]

Control

20.4

38.6

100 mg/kg bw/d

24.3-

32.5-

300 mg/kg bw/d

32.7*

46.3-

1000 mg/kg bw/d

44.1**

42.0-

*/**/- : Significant at 5% (*), 1% (**) or not significant (-)

Table 3: Liver weights and liver/body weight ratios in males

 

Control

100 mg/kg bw/d

300 mg/kg bw/d

1000 mg/kg bw/d

Liver weights

MEAN

11.33

11.57-

11.30-

13.56**

ST.DEV.

0.63

0.86

0.47

0.63

MINIMUM

10.63

10.85

10.82

12.67

MAXIMUM

12.08

12.94

11.86

14.22

N

5

5

5

5

Liver/body weight ratio (%)

MEAN

2.48

2.57-

2.54-

3.12**

ST.DEV.

0.12

0.11

0.08

0.14

MINIMUM

2.37

2.48

2.43

2.95

MAXIMUM

2.67

2.76

2.64

3.28

N

5

5

5

5

*/**/- : DUNNETT-Test based on pooled variance significant at 5% (*), 1% (**) or not significant (-)

Applicant's summary and conclusion