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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 Nov 2020 to 23 Apr 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Esterification products of fatty acids, C16 and C16-18 (even numbered, unsaturated) alkyl and adipic acid with pentaerythritol
EC Number:
923-900-3
Molecular formula:
Not applicable (UVCB substance)
IUPAC Name:
Esterification products of fatty acids, C16 and C16-18 (even numbered, unsaturated) alkyl and adipic acid with pentaerythritol
Specific details on test material used for the study:
- Physical Description: Yellow to brownish liquid

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-7 weeks
- Condition: Outbred, SPF-Quality
-Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L´ Arbresle Cedex, France
- Weight at study initiation: Males between 155 and 215 g and females between 115 and 146 g
- Housing: Polycarbonate cages (Makrolon type IV, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Up to 5 animals of the same sex and same dosing group together. During locomotor activity monitoring, animals are were individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Ad libitum, SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Municipal tap water, Freely available to each animal via water bottles.
- Acclimation period: at least 5 days
- For environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities. For animal welfare reasons, animals were provided with wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands).

DETAILS OF FOOD AND WATER QUALITY: Periodic analysis of the water was performed, and analysis for nutritional components and environmental contaminants in the feed were provided by the supplier. It is considered that there were no known contaminants in the water or feed that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 (Target), 19 to 21 (Actual mean)
- Humidity (%): 40 to 70 (Target), 39 to 59 (Actual mean)
- Air changes (per hr): Ten or more
- Photoperiod (hrs dark / hrs light): 12 /12 (except during designated procedures)

IN-LIFE DATES: From: 8 Dec 2020 To: 10 Mar 2021

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of exposure was selected because ECHA has evaluated this the most
appropriate route of administration for the study.
Vehicle:
paraffin oil
Remarks:
Specific gravity: 0.85 g/cm3
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were divided into aliquots where required to allow to be dispensed on each dosing occasion. Formulations (w/w) were prepared at least weekly, homogenized to visually acceptable levels, and stored at 2-8°C.
Test item dosing formulations were kept at room temperature until dosing. An adjustment was made for specific gravity of the test item and vehicle. No correction was made for the purity and composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was chosen based on trial preparations. These trial preparations were caried out prior to the start of the study to assess the suitability of the formulation procedure and used a representative dosing concentration and volume. These trials were not performed as part of this study and were not used for dosing.
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected in week 1, 6 and 13 of dosing. The accuracy of preparation (concentration) was analysed for all groups, homogeneity was checked for groups 2 and 4 from the top, middle and bottom of the preparations.
Analyses were performed using a validated method. Concentration results were considered acceptable if mean sample concentration results were within or equal to ±10% solutions of theoretical concentration. Homogeneity results were considered acceptable if the relative standard deviation (RSD) of concentrations of ±10% for each group.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20254769) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
(Group 2)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
(Group 3)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
(Group 4)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on results of a 28-day repeated dose toxicity study with oral exposure of the test substance in rats, performed by the Sponsor, and in an attempt to produce graded responses to the test item. In this 28-d rat study (OECD 407) rats received 0, 100, 300 and 1000 mg/kg bw/day test item by daily oral gavage with paraffin oil as vehicle. No effects on clinical signs, body weight, food consumption, functional observations or clinical pathology were seen. Based on histopathological finding in the mesenterial lymph nodes (foamy macrophage aggregates), the NOAEL was set at 300 mg/kg bw/day for males and 100 mg/kg bw/day for females. For the current study, dose levels were selected with the rational that the high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Fasting period before blood sampling for clinical biochemistry: yes

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily a check for cage side observations was performed; from Day 1 at 0 to 1 hours postdose. ; At least twice daily beginning upon arrival through termination a check was done for mortality, except on days of receipt and necropsy where frequency was at least once dialy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly; from Week 1 and throughout the study, and on the day of necropsy.


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study.

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly; from at least Day 1 and throughout the study.

FOOD EFFICIENCY: no
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION: Yes
- Time schedule for examinations: Monitored on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretreatment and week 13
- Dose groups that were examined: Pretreatment Period - All animals once (including spare animals); Dosing Period - All Group 1 and 4 animals during Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of necropsy between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (isoflurane )
- Animals fasted: Yes
- How many animals: all (10/sex/dose)
- Parameters examined: White blood cell (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC) (absolute), Red blood cell (RBC), Reticulocytes (absolute), Red blood cell distribution width (RDW), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets, Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all (10/sex/dose)
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST),
Alkaline phosphatase (ALP), Total protein, Albumin, Total bilirubin, Urea, Creatinine, Glucose, Cholesterol, Triglycerides, HDL and LDL Cholesterol,Sodium, Potassium, Chloride, Calcium, Inorganic phosphate (Inorg. Phos)

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time schedule for collection of blood: day of necropsy between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all (10/sex/dose)
- Parameters examined: Triiodothyronine (T3), Thyroxine (T4), Thyroid stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the Dosing Period, during Week 12-13. These tests were performed after clinical observations (including arena observation, if applicable).
- Dose groups that were examined: The first 5 animals per sex per group
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

ARENA OBSERVATIONS: yes
- Arena observations were performed on all animals, once before the first administration of the test item and weekly during the Treatment Period. Animals were observed for clinical signs outside the home cage in a standard arena.

ESTROUS STAGE DETERMINATION: yes
- Time schedule for examinations: End of Treatment - on the day of necropsy, a vaginal smear was taken to determine the stage of estrous from all animals. This was done for all females.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Study animals were subjected to a complete necropsy examination, which included evaluation
of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity
and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their
associated organs and tissues.


ORGAN WEIGHTS: Yes
- Organs weighed at necropsy: brain, epididymis, adrenal gland, pituitary gland, prostate gland,
seminal vesicle gland (including coagulation gland), thyroid gland, heart, kidney, liver, ovary,
spleen, testis, thymus, uterus/cervix.

HISTOPATHOLOGY: Yes
- The following tissues were examined for the control group and the 1000 mg/kg bw/day dose group: aorta artery, bone marrow, sternum, brain, cervix, epididymides, eye, adrenal gland, mammary gland, parathyroid gland, pituitary gland, prostate gland, seminal vesicle gland (including coagulation gland), thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, cecum, colon, rectum, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscles, optic and sciatic nerve, ovary, pancreas, skin, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testis, thymus, trachea, urinary bladder, uterus, vagina.
- The following tissues were examined for all groups: gross lesions/masses
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. The following pairwise
comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Data Collected in Provantis:
- Parametric/Non-parametric: Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
- Non-Parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.
- ANCOVA: data corresponding to a response variable of interest and to a related covariate were
submitted to an analysis of covariance (ANCOVA), and if found to be significant, then pairwise comparisons were conducted using Dunnett’s test.
- Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Data Collected/Processed in Toxdata
- Parametric/Non-parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing.
- Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
- Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The abovepairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
See attachment 1 - clinical signs
Abnormal breathing sounds were noted in one female at 100 mg/kg bw/day on Day 36. As this only occurred in one animal at a single occasion and lacked a dose-related response, it was considered not test item-related.
Any other clinical signs noted during the Dosing Period (e.g. fur loss, skin lesions, scabs and discolored eye lid) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See attachment 2- body weight
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related ophthalmology findings in Week 13. The nature and incidence of ophthalmology findings noted during the Pre-treatment Period and in Week 13 was similar among the groups and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See attachment 3- haematology
No test item-related hematology parameter changes were observed in males up to 300 mg/kg
bw/day and in females at 100 mg/kg bw/day. At 1000 mg/kg bw/day an increase in white blood cell, neutrophil, lymphocyte, monocyte, basophil and large unstained cell count was observed in males and females. The neutrophil and monocyte counts in females at 300 mg/kg bw/day were also increased.
No test item-related effects on coagulation parameters were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See attachment 4- Clinical biochemistry
No test item-related clinical chemistry parameter changes were observed in males and females at 100 mg/kg bw/day. An increase in alanine aminotransferase and aspartate aminotransferase (ALT and AST) activities were noted in males at 300 and 1000 mg/kg bw/day (1.74x and 2.49x of control for ALT, and 2.01x and 3.30x of control for AST, respectively, not statistically significant at 300 mg/kg bw/day). ALT and AST activities were also increased in females at 1000 mg/kg bw/day (each 1.58x of control). Furthermore, an increased alkaline phosphatase (ALP) was noted in males at 1000 mg/kg bw/day (1.12x of control, not statistically significant) and an
increase in calcium concentration was noted in females at 300 and 1000 mg/kg bw/day (1.06x
and 1.07x of control, respectively, not statistically significant) (see Table 1 in "Any other information on results incl. tables").
Other values despite of achieving a level of statistical significance, when compared to controls, occurred in the absence of a dose-related distribution and/or were, given the magnitude of change, considered to be of no toxicological significance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
See attachment 5 - Behaviour
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See attachment 6 - Organ weight findings including organ body weight ratios
In females at 1000 mg/kg bw/day, a slightly higher liver weight was observed and was statistically significant relative to body weight and correlated with the microscopic observation of histiocytic infiltrates.
In males and females at 1000 mg/kg bw/day, mildly higher spleen weight of similar
magnitude was observed and was statistically significant relative to body weight and/or
absolute value. In males this correlated with a slightly higher incidence/severity of
extramedullary hematopoiesis; there was no microscopic correlate in females.
There were no other test item-related organ weight changes (see Table 2 in "Any other information on results incl. tables").
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See attachment 7 - Gross pathology
There were few macroscopic observations in males at 1000 mg/kg bw/day that were interpreted to be test item-related and included pale foci in the liver (caudate process and medial lobe) of one male and an enlarged spleen in one male.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See attachment 8 - Histopathology
In the jejunum of males and females at 1000 mg/kg bw/day, minimal histiocytic infiltrates were present multifocally in the lamina propria of the villi.
In the liver of males and females at 300 and 1000 mg/kg bw/day, multifocal histiocytic infiltrates were present scattered throughout the sections. Additionally, at 1000 mg/kg bw/day, minimal or mild necrosis was present.
In the mesenteric lymph node of males and females at 300 and 1000 mg/kg bw/day multifocal
histiocytic infiltrates were present ranging from minimal to moderate. A low incidence and severity of histiocytic infiltrates was present in females at 100 mg/kg bw/day however this was generally comparable to that observed in the control animals and not ascribed to the test item.
In the spleen of males at 300 and 1000 mg/kg bw/day, there was a slightly higher incidence and severity of extramedullary hematopoiesis present when compared to the control males which was considered test item-related. In females the incidence of extramedullary hematopoiesis was more variable and without a dose relationship thus a relationship to the test item was not suspected.
In the testis, a low incidence of mononuclear cell infiltrates consisted of small individual foci in the capsular or subcapsular region were noted in a few males at 300 mg/kg bw/day (1/10) and 1000 mg/kg bw/day (3/10). This was interpreted to be within the range of background findings and not test item-related due to the low incidence and focal nature and often unilateral nature and was primarily composed of lymphocytes (not histiocytic cells as the test item-related infiltrates seen in other organs).
In the brain of one control female axonal dystrophy was noted in the medulla oblongata (specifically, the cuneate and gracile nuclei). This observation was bilaterally symmetrical and characterized by the presence of numerous spheroids in these nuclei. Axonal dystrophy was also present bilaterally in the dorsal funiculus of the spinal cord as a few spheroids present at each examined segment (cervical, thoracic and lumbar). This is a spontaneous lesion that is reported to occur in ageing rats (Kaufman et al., Eisenbrandt et al).
The remaining histologic changes were considered to be incidental findings or were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
There were no toxicologically significant changes in the estrous stage.
Details on results:
As the hematology changes (white blood cell, neutrophil, lymphocyte, monocyte, basophil and large unstained cell count) occurred in absence of a histopathological correlation, these findings were considered to be not adverse.
Microscopically, histiocytic infiltrates were observed in the jejunum of males and females at 1000 mg/kg bw/day, and in the mesenteric lymph node and liver of males and females starting at 300 mg/kg bw/day. In the liver of both sexes at 1000 mg/kg bw/day these findings were accompanied by increased liver enzymes (aspartate aminotransferase and alkaline phosphatase) and by foci of necrosis. The foci of necrosis were considered to be adverse. Despite the observation of histiocytic infiltrates in the mesenteric lymph node up to a moderate degree in males and females at 300 and 1000 mg/kg bw/day, their presence was not accompanied by necrosis or degeneration, nor by indication of dysfunction (e.g. no indication
of obstruction of lymphatic drainage or morphologic alteration in the number or arrangement
of lymphoid cells), thus alone was considered non-adverse.
In the spleen a slight increase in the incidence and severity of extramedullary hematopoiesis in males at 300 and 1000 mg/kg bw/day correlated at 1000 mg/kg bw/day to higher spleen weight and a single instance of spleen enlargement. Higher spleen weight was also noted in females at 1000 mg/kg bw/day but without a histological correlation. The spleen findings were considered to be non-adverse due to the minimal degree and only the slight variation from background observations, or lack of histologic correlate.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Table 1 Test Item-Related Hematology Changes






















































































Groups223344
Dose level (mg/kg bw/day)10020030030010001000
SexMFMFMF
White blood cells----1,27x1,51x
Neutrophils---1,68x1,21x1,83x
Lymfocytes----1,28x1,47x
Monocytes---1,75x1,72x1,78x
Basophils----2,33x3,75x
Large unstained cells----1,54x2,71x

M = Males F = Females


A dash (-) indicates absence of change. Numerical values indicate fold change of the treated group mean value relative to the control group mean value. 


 


Table 2 Mean Percent Liver And Spleen Weight Differences from Control Groups













































































 MMMFFF
Dose level (mg/kg bw/dag)10030010001003001000
Liver      
Absolute3580110
Relative to body weight2370310*
Spleen      
Absolute21825*41230**
Relative to body weight01423*41430**

*: P<0.05, **: P<0.01; shaded values were considered test item-related.

Applicant's summary and conclusion

Conclusions:
In conclusion, administration of the test substance by once daily oral gavage for
90 days was well tolerated in Wistar Han rats at dose levels up to and including 1000 mg/kg
bw/day. An adverse morphologic alteration was present in the liver of males and females at
1000 mg/kg bw/day and consisted of foci of necrosis. The other alterations observed in
clinical pathology, organ weight changes and histopathology were considered to be not
adverse. Based on these results, the No Observed Adverse Effect Level (NOAEL) was
considered to be 300 mg/kg bw/day.