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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:
9 June 2020 - 23 September 2020
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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-3-{4-[2-hydroxy-3-(prop-2-enoyloxy)propoxy]butoxy}propyl prop-2-enoate
EC Number:
701-230-0
Cas Number:
52408-42-1
Molecular formula:
C16H26O8
IUPAC Name:
2-hydroxy-3-{4-[2-hydroxy-3-(prop-2-enoyloxy)propoxy]butoxy}propyl prop-2-enoate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl CD® (SD) IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: 192.3 - 253.8 g (males) and 138.2 - 181.7 g (females)
- Housing: The animals were group-housed in two from the same sex and group in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust (Le comptoir des sciures, Meyzieu, France). Animals were isolated where experimental procedure dictated. This was documented in the study file. Each cage contained rat hut and nylabone for environmental enrichment.
- Diet: ad libitum, SSNIFF rat/mouse pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: 14 days

CONTAMINANT ANALYSES
No contaminants were present in the diet, drinking water or sawdust at levels which could have been expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approx. 8-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 9 June 2020 To: 23 September 2020

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since it is a route of administration which is requested by the Regulatory Authorities for this type of test item.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the administration procedure.
The control dose formulations were stirred just before administration, the test item dose formulations for at least 30 minutes before administration, and then throughout the whole administration procedure.
The test item dose formulations were administered within 4 hours after the end of their preparation.

VEHICLE
- Concentration in vehicle: 2, 6, 20 mg/mL
- Amount of vehicle: 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On three occasions during the study (in Weeks 1, 6 and 12) a sample was taken from control and test item dose formulations and analyzed using the validated method. The samples were analyzed with an LC/MS-MS.
Acceptance criterion: Measured concentration = nominal concentration ± 15%
Duration of treatment / exposure:
at least 13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
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 in agreement with the Sponsor, based on the results of a previous Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (OECD 422) in Wistar Rats (Gavage), in which the test item was administered by daily oral gavage to male and female rats at dose levels of 50, 150 or 500 mg/kg bw/day. Because of clinical findings and the premature death of 3 male animals at the high dose level, the dose was reduced at 300 mg/kg bw/day from Day 6 of the study.
At 500/300 mg/kg bw/day, parental toxicity consisted of premature deaths for 6 males and 3 females (one female was found dead and one female was prematurely euthanized for delivery difficulties at the end of the gestation period, and one female died during the lactation period), clinical signs (i.e. hypersalivation, breathing difficulties and/or piloerection in males and females), reduced body weight and body weight gain for males and affected hematological parameters (i.e. increased neutrophil count and decreased lymphocyte count) in males. Histopathological changes were seen in the forestomach (i.e. erosion/ulcer and/or related squamous hyperplasia or inflammation/edema in 9/10 males and 10/10 females) and in the stomach (i.e. erosions or ulcers in 3/10 males).
At 150 mg/kg bw/day, there were breathing difficulties in males and females, hypersalivation and/or transient piloerection in isolated males and females and reduced body weight in males on mating Day 8. Histopathological changes were seen in the forestomach (i.e. erosion/ulcer and/or related squamous hyperplasia or inflammation/edema in 9/10 males and 7/10 females) and in the stomach (i.e. erosions or ulcers in 1/10 females).
At 50 mg/kg bw/day, hypersalivation was noted in males and females. Histopathological changes were seen in the forestomach (i.e. erosion/ulcer and/or related squamous hyperplasia or inflammation/edema in 6/10 males and 1/10 females).
Pathological findings observed at all dose levels were related to the irritating potential of the test item.
Based on the above-mentioned results and the histopathological changes observed at 150 mg/kg/day in most animals after 4-8 weeks of exposure, 100 mg/kg bw/day was selected as the high-dose level in the 13-week exposure study. The low- and intermediate-dose have been selected using a ratio representing approximately a 3-fold interval (i.e. 30 and 10 mg/kg bw/day).
- Fasting period before blood sampling for clinical biochemistry: at least 14 hours but not exceeding 18 hours
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Each animal was checked for mortality and morbidity at least once a day during the acclimation period and at least twice a day during the treatment period, including weekends and public holidays.
- Each animal was observed for the recording of clinical signs: once a day before the beginning of the treatment period, at least once a day during the treatment period, after completion of treatment of the last animal in the animal room.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once before the beginning of the treatment period, at least once a week during the treatment period.
- Scabs and alopecia localized on the cervical region (neck) were observed in one female of group 3 in Week 6. These lesions required medical treatment which consisted of the application of an antiseptic/cicatrizing agent for 5 days (containing essential oils, Cothivet®, Vetoquinol, France).

BODY WEIGHT: Yes
- Time schedule for examinations: at least once a week before the beginning of the treatment period, on the first day of treatment, and at least once a week until the end of the study.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- 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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: on all animals, before the beginning of the treatment period, and on control and high-dose animals on one occasion during Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13 before daily treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked in table were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13 before daily treatment
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked in table were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: week 13
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: T3, T4 and TSH

URINALYSIS: Yes
- Time schedule for collection of urine: week 13 during overnight period of at least 14 hours
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

MONITORING OF ESTROUS CYCLE:
The estrous cycle stage was determined daily from a fresh vaginal lavage (stained with methylene blue), on the day of euthanasia and daily during the 4 days before euthanasia.
Only individual estrous cycle data were presented in the study report. No statistical analyses were performed on those data.
Sacrifice and pathology:
GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

ORGAN WEIGHTS:
The body weight of each animal was recorded before euthanasia at the end of the treatment period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

HISTOPATHOLOGY:
- For all study animals, the tissues specified in the Tissue Procedures Table were preserved in 10% buffered formalin (except for the eyes and optic nerves and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's Fixative).
- A microscopic examination was performed on: all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4), all macroscopic lesions, forestomach, and spleen (females) for all low- and intermediate dose animals (groups 2 and 3).
- In addition, testicular staging was performed for control and high-dose males (groups 1 and 4). A detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with hematoxylin-PAS in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.

BONE SMEARS:
Two bone marrow smears were prepared from the femoral bone (at necropsy) of all animals euthanized on completion of the treatment period. In absence of abnormalities at the haematological investigations or histopathological examination requiring a bone marrow differential cell count determination, the smears were archived without further investigations.
Statistics:
Provantis software was used to perform the statistical analysis of body weight, food consumption, haematology, coagulation, blood biochemistry, thyroid hormones and urinalysis. See illustration for sequence followed.
PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01) according to the following sequence:
- Non parametric testing: Test for group differences Kruskal-Wallis if > 2 groups; Dunn test if > 2 groups Wilcoxon test if 2 groups
- Parametric testing: Test for group differences in means One-way analysis of variance if > 2 groups; Dunnett test if > 2 groups t-test if 2 groups



Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only test item-related clinical sign observed during the study was minimal to severe hypersalivation in 2/10 males and 2/10 females at 30 mg/kg bw/day and in 9/9 males and 3/10 females at 100 mg/kg bw/day. While observed at a low frequency (up to 6 occasions) in males and females at 30 mg/kg bw/day and in females at 100 mg/kg bw/day, hypersalivation was reported almost throughout the treatment period in most males at 100 mg/kg bw/day. Hypersalivation, a common observation when a test item is administered by gavage, had no impact on the general condition of the animals and was therefore considered not adverse. This was considered likely to be related to the irritating potential of the test item.
All other clinical signs observed during the study (i.e. reflux at dosing, chromodacryorrhea, chromorhynorrhea, soiled coat, alopecia, scabs, nodule, thin or erected fur, bent tail, half-closed eyes and/or short, broken or abnormal growth of teeth) were considered incidental as they were occasional, observed with no dose-relationship and/or can commonly be encountered in laboratory rats dosed by gavage.
Mortality:
mortality observed, treatment-related
Description (incidence):
There was one unscheduled death during the study.
One male dosed 100 mg/kg bw/day died on Day 85 (during the ophthalmological examination). Except for repeated hypersalivation and occurrences of loud breathing on Days 73, 74 and 76, no clinical signs were observed before death. Microscopically, test item-related moderate multifocal erosion/ulceration (down to submucosa and with bacteria) along with marked inflammation/oedema (mixed inflammatory cells infiltrates in the submucosa) were noted in the forestomach, and correlating with many raised white masses up to 0.2 cm in diameter at necropsy. These changes most likely contributed to the death of this animal, which was considered as test item related. Moderate squamous cells hyperplasia and hyperkeratosis in the forestomach was considered a reaction to ulceration.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on body weights or body weight gains during the study.
Any differences between control and test item-treated groups, including those that were statistically significant, were transient, observed sporadically with no dose-relationship and/or were consistent with normal biological variations, and were therefore not considered to be test item-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item-related effects on food consumption during the study.
Any differences between control and test item-treated groups, including those that were statistically significant, were transient, observed sporadically with no dose-relationship and/or were consistent with normal biological variations, and were therefore not considered to be test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related ophthalmological findings at the end of the treatment period in animals at 100 mg/kg bw/day. Animals at 10 and 30 mg/kg bw/day were therefore not examined at the end of the treatment period.
Hyaloid remnants were observed in the right eye vitreous body of one male at 100 mg/kg bw/day but as they were already observed pre-test, as well as in one control male, these findings were considered to be incidental and not related to the test item.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on haematology parameters at the end of the treatment period.
Any differences between control and test item-treated groups were not statistically significant and not dose-related, of minimal amplitude and/or consistent with normal biological variations, and were therefore not considered to be test item-related.
There were no changes in prothrombin time at the end of the treatment period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on blood biochemistry parameters at the end of the treatment period.
Any differences between control and test item-treated groups, including those that were statistically significant, were not dose-related, of minimal amplitude and/or consistent with normal biological variations, and were therefore not considered to be test item-related.
Specifically, the statistically significantly higher mean total bilirubin concentration in males at 100 mg/kg bw/day compared to controls (1.26 µmol/L vs. 0.80 µmol/L in controls) was consistent with normal biological variations. The non statistically higher total bilirubin concentration, alanine aminotransferase, aspartate aminotransferase activity and/or biliary acids concentration in females at 10 or 30 mg/kg bw/day were isolated, related to one or two females and not dose-related; they were all considered incidental.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Higher TSH (mainly in males at 10 mg/kg bw/day due to a high interindividual variability), T3 and T4 levels were observed in test item-treated males when compared to controls, reaching statistical significance in T4 levels at 100 mg/kg bw/day. These changes which were within the HCD, were observed with no dose relationship and/or had no histopathological correlates and were therefore not considered to be test item-related.
There were no relevant changes on thyroid hormones levels in females at the end of the treatment period.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related findings on quantitative and qualitative urinary parameters at the end of the treatment period.
Any differences between control and test item-treated groups were not statistically significant and not dose-related, of minimal amplitude and/or consistent with normal biological variations and were therefore not considered to be test item-related.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no organ weight changes related to the test item administration at the end of the treatment period.
The statistical organ weight changes for kidneys in females were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates and were not consistent for the sexes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic observations were noted in the forestomach at 100 mg/kg bw/day in both sexes.
At 100 mg/kg bw/day, the forestomach showed white masses and/or was gelatinous in both sexes. It correlated with microscopic inflammation/edema and/or squamous cell hyperplasia.
None of the remaining macroscopic findings were test item-related because they were consistent with spontaneously occurring findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to findings found in controls.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic observations were observed in the forestomach from 30 mg/kg bw/day at the end of the treatment period.
From 30 mg/kg bw/day in males and at 100 mg/kg bw/day in females, minimal to moderate erosion/ulcer, inflammation/oedema (mixed inflammatory cells infiltrates in the submucosa) and squamous cell hyperplasia were observed in the forestomach. Incidence and/or severity grades were higher in males when compared to females.
There were no test item-related changes noted in the testes or epididymides at microscopic evaluation. Unilateral testicular hypoplasia was noted in a single male at 100 mg/kg/day; this is a congenital lesion of the testes which can be observed in controls rats and was therefore considered as incidental and unrelated to the test item administration.
No test item-related microscopic changes were observed in the thyroid glands. The few microscopic findings observed in test item-treated animals (minimal infiltrate of mononuclear inflammatory cells and ectopic thymic tissue both observed in isolated males at 100 mg/kg bw/day) were of those commonly seen in the thyroid glands of control rats and were considered to be incidental.
In females at 100 mg/kg bw/day, minimal to slight increased lymphoid cellularity (germinal centers) was observed in the mandibular lymph nodes of 2/10 animals. In view of the low incidence and magnitude of this change, it was considered as incidental and not test item-related.
None of the remaining microscopic findings were considered as test item-related since they were consistent with spontaneously occurring findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to findings found in controls.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
MONITORING OF ESTROUS CYCLE:
There were no test item-related effects on estrous cycle during the 5 days of evaluation at the end of the treatment period, with comparable data between control and test item-treated females.

Effect levels

Dose descriptor:
NOAEL
Effect level:
30 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

Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Dose formulation analysis


The test item concentrations in the administered dose formulations analyzed in Weeks 1, 6 and 12 remained within an acceptable range of variations (-4.7% to +6.5%) when compared to the nominal values (± 15% of the nominal concentrations).


No test item was observed in the control dose formulations.


 


 


Incidence and Severity of Selected Microscopic Findings in the Forestomach at the End of the Treatment Period











































































































































































Organ Finding



Male



 



 



 



Female



 



 



 



Dose level (mg/kg bw/day)



0



10



30



100



0



10



30



100



Num. of exam. animals/Num. of treated animals



10/10



10/10



10/10



9/10



10/10



10/10



10/10



10/10



Forestomach



 



 



 



 



 



 



 



 



Erosion/ulcer



 



 



 



 



 



 



 



 



Minimal (Grade 1)



-



-



2



-



-



-



-



2



Slight (Grade 2)



-



-



-



1



-



-



-



-



Inflammation/Edema



 



 



 



 



 



 



 



 



Minimal (Grade 1)



-



-



1



-



-



-



-



-



Slight (Grade 2)



-



-



1



2



-



-



-



2



Moderate (Grade 3)



-



-



-



5



-



-



-



1



Hyperplasia; squamous cell



 



 



 



 



 



 



 



 



Minimal (Grade 1)



-



-



1



3



-



-



-



3



Slight (Grade 2)



-



-



1



3



-



-



-



2



Moderate (Grade 3)



-



-



-



3



-



-



-



-



-: not observed.

Applicant's summary and conclusion

Conclusions:
In a 90-day repeated dose toxicity study the oral administration of the test item in rats induced erosion/ulcer, inflammation/oedema and secondary squamous cell hyperplasia in the forestomach from 30 mg/kg bw/day in males and at 100 mg/kg bw/day in females. These changes were considered as adverse at 100 mg/kg bw/day in both sexes and contributed to the premature death of one male at 100 mg/kg bw/day. Based on these adverse findings at 100 mg/kg bw/day, the No-Observed-Adverse-Effect Level (NOAEL) in this study was considered to be 30 mg/kg bw/day.
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item,
1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl) diacrylate, following daily oral administration (gavage) to rats for 13 weeks.
Four groups of ten males and ten females Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item, 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl) diacrylate, at dose levels of 0, 10, 30 or 100 mg/kg/day for 13 weeks (i.e. 91 or 92 days according to the necropsy schedule). The test item was administrated as an emulsion in the vehicle (corn oil) at a constant dosage volume of 5 mL/kg/day.
The following parameters and endpoints were evaluated in this study: survival, clinical signs, body weights, body weight gains, food consumption, ophthalmology, clinical pathology (hematology, coagulation, blood biochemistry, and urinalysis as well as thyroid hormones), estrous cycle, gross necropsy findings, organ weights, and histopathologic examinations.
The actual test item concentrations in the analyzed dose formulations were within an acceptable range of variations (-4.7% to +6.5%) when compared with the nominal values (nominal ± 15%).
The unscheduled death of one male at 100 mg/kg/day on Day 85 was attributed to the test item and considered to be related to adverse moderate erosion/ulceration and marked inflammation/edema in the forestomach.
Test item-related clinical signs in surviving animals consisted of minimal to severe
hypersalivation, observed at a low incidence and frequency in males and females at 30 mg/kg/day and females at 100 mg/kg/day, and at a high incidence and frequency in males at 100 mg/kg/day. There were no test item related effects on body weight, food consumption, ophthalmology, estrous cycle and clinical pathology parameters. At the end of the treatment period in the forestomach, adverse microscopic erosion/ulcer, inflammation/edema and squamous cell hyperplasia correlating with macroscopic white masses and/or gelatinous aspect
were observed at 100 mg/kg/day in both sexes. Similar non adverse microscopic changes of lower severities and incidence were noted at 30 mg/kg/day in males. These changes were reported with higher incidence and/or severity in males than in females. 


In conclusion, the oral administration of 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl) diacrylate in rats induced erosion/ulcer, inflammation/edema and secondary squamous cell hyperplasia in the forestomach from 30 mg/kg/day in males and at 100 mg/kg/day in females.
These changes were considered as adverse at 100 mg/kg/day in both sexes and contributed to the premature death of one male at 100 mg/kg/day.
Based on these adverse findings at 100 mg/kg/day, the No-Observed-Adverse-Effect Level (NOAEL) in this study was considered to be 30 mg/kg/day.