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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: 24 Mar 2021 To: 25 Aug 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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2018
Deviations:
no
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:
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

1
Chemical structure
Reference substance name:
2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate
EC Number:
239-701-3
EC Name:
2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate
Cas Number:
15625-89-5
Molecular formula:
C15H20O6
IUPAC Name:
2,2-bis[(acryloyloxy)methyl]butyl acrylate (non-preferred name)
Test material form:
solid - liquid: suspension
Details on test material:
- Name of test material (as cited in study report): TMPTA
- Physical state: Clear, colorless liquid
- Analytical purity: >80%
- Lot/batch No.: 200818307
- Expiration date of the lot/batch: 17 August 2021
- Storage condition of test material: At room temperature protected from light
Specific details on test material used for the study:
- Source: Miwon Specialty Chemical Co., Ltd., Yongin-si, Korea
- Storage condition of test material: at a temperature <45°C

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han), Outbred, SPF-Quality
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: 151 - 194 g (males) and 116 - 150 g (females)
- Fasting period before study: overnight with a maximum of 24 hours before sample collection for haematological and clinical chemistry, and overnight before the scheduled necropsies.
- Housing: Polycarbonate cages (Makrolon type IV, height 18 cm or Makrolon type 2000P, height 21.5 cm Type 2000P) 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 housed 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.
- Animal Enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany, ad libitum, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Tap water, ad libitum.
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY:
- Food: Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21°C (actual)
- Humidity (%): 45 to 77% (actual)
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 05 Apr 2021 To: 06 Jul 2021

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
- The first day diets were available was designated as Day 1.
- The actual test item intake was estimated based on the body weight and food consumption values.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was mixed directly with the required amount of powder feed.

DIET PREPARATION
- Frequency of preparation of diet: Diets containing the test item at a level of 300 ppm: up to 4 days prior to use. Diets containing the test item in the range of 500-15000 ppm: up to 3 weeks in advance.
- Mixing appropriate amounts with: Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Stability of diet preparations and storage temperature of food: Stability in the freezer (≤-15°C) over 3 weeks was confirmed for a 500-15000 ppm range under Test Facility Study No. 20265399 (Analytical Method Development and Validation Study). Diets were prepared up to 3 weeks in advance of first use. Diets were kept in the freezer (≤-15°C) until use, if not used on the day of preparation. Stability at room temperature for 4 days was confirmed at 300 ppm. Diets at this dose level were prepared up to 4 days prior to use and diets were kept at room temperature until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 4 days (stability is confirmed under Test Facility Study No. 20265399 (analytical Method Development and Validation Study)) for supplementing food during the respective food consumption measurement interval. The diet of Group 2 (300 ppm) was prepared up to 3 weeks in advance of first use, based on the confirmed stability at 300 ppm.
- Corrections for purity of the test item: No corrections
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLE COLLECTION
- Intervals: Week 1, 6 and 12, directly after preparation
- Samples for concentration analysis: All groups, 2 x approximately 5 g, samples collected from approximately middle
- Samples for homogeneity analysis: Groups 2 and 4, 2 x approximately 5 g, sample collected from approximately top, middle and bottom
- Samples taken from: Dosing container
- Backup samples: Duplicate middle samples for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis) wee taken on the same day samples are collected for analyses for possible future analysis.

ANALYTICAL METHOD
- According to a validated analytical procedure (Test Facility Study No. 20265399).

ACCEPTANCE CRITERIA
- For concentration: Mean sample concentration results within or equal to ± 20% of theoretical concentration.
- For homogeneity: Relative standard deviation (RSD) of concentrations of ≤ 10% for each group.

STABILITY ASSESSMENT
The Sponsor provided data to demonstrate that the test substance was stable in the diet.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
Continuously (via food)
Doses / concentrationsopen allclose all
Dose / conc.:
300 ppm
Remarks:
Equals mean intake level of 21 and 22 mg test item/kg body weight for males and females, respectively
Dose / conc.:
900 ppm
Remarks:
Equals mean intake level of 64 and 70 mg test item/kg body weight for males and females, respectively
Dose / conc.:
2 500 ppm
Remarks:
Equals mean intake level of 173 and 190 mg test item/kg body weight for males and females, respectively
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, plain diet
Details on study design:
ROUTE RATIONALE:
The oral route of exposure via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item. The oral route of exposure via dietary inclusion was selected, as effects on the stomach, due to irritating properties of the test item are suspected.

DOSE RATIONALE:
The dose levels were selected based on the results of a (14-day repeated dose toxicity study with oral exposure of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate in rats (Test Facility Study No. 20265400), and in an attempt to produce graded responses to the test item. In this 14-day study, at 10000 and 15000 ppm, animals were prematurely euthanized based on excessive toxicity. In addition, at 5000 ppm, body weight and food consumption were affected in both sexes. As the three previous dose levels exceeded the maximal tolerated dose (MTD), additional groups were treated at 1500 and 3500 ppm.
In males treated at 3500 ppm, a lower bodyweight gain was observed and was associated with a decreased food consumption. No relevant effect was observed in the females treated with 3500 ppm. At 1500 ppm, no effect on body weight or food consumption was observed in males or in females. In addition, at neither 1500 nor 3500 ppm, clinical signs were observed during the 14-day treatment period.
Histopathological examination showed test item-related findings in the stomach of the animals at both dose levels. At 1500 and 3500 ppm, the stomach alterations included squamous cell hyperplasia and hyperkeratosis in males and females, and ulcerations in the non-glandular stomach of females. Based on the severity of the stomach findings (hyperplasia/hyperkeratosis up to moderate degree and ulceration at mild degree) after 14 days of treatment with 3500 ppm, it was expected that at this dose, the stomach findings would become more severe after 90 days of treatment which could result in severe toxicity and possible mortalities. The stomach findings in animals treated at 1500 ppm were only minimal and the risk of severe toxicity resulting in mortalities is expected to be low after 90 days of treatment. Based on these findings, and in order to select a dose level which could lead to some toxic effect without excessive lethality, 2500 ppm is selected as the high dose concentration. The mid-dose and low-dose levels are set at 900 and 300 ppm.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily.

ARENA OBSERVATIONS: Yes
- Time schedule: Once before the first administration of the test item and weekly during the treatment.
- Approach: Animals were observed for clinical signs outside the home cage in a standard arena.

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

INDIVIDUAL BODY WEIGHT: Yes
- Time schedule: Days 1, 3, 6, 10, 13, 16 and 19 and starting from Week 4 twice weekly throughout the study.

FOOD CONSUMPTION: Yes
- Time schedule: Over Day 1-3, 3-6, 6-10, 10- 13, 13-16, 16-19, 19-22 and starting from Week 4 twice weekly throughout the study.
- Procedure: Quantitatively measured per cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule: Regular basis throughout the study.
- Procedure: Water consumption was monitored by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once during the pretreatment period (all animals, included unused replacement animals); Once during week 13 (group 1 and 4 animals)
- Procedure: The eyes were examined using an ophthalmoscope after application of a mydriatic agent (tropicamide 0.5%).

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: Once during the dosing period, during Week 12-13. Tests were performed after clinical observations (including arena observation, if applicable).
- Dose groups that were examined: The first 5 animals per sex per group.
- Parameters examined:
• hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength (recorded as the mean of three measurements).
• locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

ESTROUS STAGE DETERMINATION: Yes
- Time schedule: End of Treatment - on the day of necropsy (all surviving animals)
- Procedure: By examining the vaginal cytology of the samples obtained by vaginal smears procedures.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of the scheduled necropsy (between 7.00 and 10.30)
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: All surviving animals.
- Parameters examined: White Blood Cell Count (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC) (absolute), Red Blood Cell Count, Reticulocytes (absolute), Red Blood Cell Distribution Width (RDW), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets

COAGULATION: Yes
- Time schedule for collection of blood: On the day of the scheduled necropsy (between 7.00 and 10.30)
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: All surviving animals.
- Parameters examined: Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of the scheduled necropsy (between 7.00 and 10.30)
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: All surviving animals.
- 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)

THYROID HORMONES: Yes
- Time schedule for collection of blood: On the day of the scheduled necropsy (between 7.00 and 10.30)
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: All surviving animals.
- Parameters examined: Triiodothyronine (T3), Thyroxine (T4), Thyroid-Stimulating Hormone (TSH)

URINALYSIS: No
Sacrifice and pathology:
SACRIFICE
- Premature and scheduled euthanasia: Deeply anesthetized using isoflurane and subsequently exsanguinated
- Fasting: yes (overnight with a maximum of 24 hours)

POST-MORTEM EXAMINATIONS: Yes
- Surviving animals: 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 according to guideline
- Paired organs weighed together. Organ weight as a percent of body weight (using the terminal body weight) and organ weight as a percent of brain weight were calculated.

HISTOPATHOLOGY: Yes
- Organs according to guideline
- Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
Statistics:
The following statistical methods were used to analyze the data:
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% and or 5% levels. The pairwise comparisons are of the exposed groups (Group 2, 3, and 4) against the control group (Group 1).
Analysis were performed (only with 3 or more observations) according to the Parametric/ Non-parametric method for: Body Weight, Body Weight Gains, Hematology Variables, Coagulation Variables, Clinical Chemistry Variables, FOB Quantitative Variables, Organ Weights and Organ Weight relative to Body Weight. The Incidence method was used for FOB Qualitative Variables.
See for more information, section "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical signs noted during the administration period (scabs and skin lesions) 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 the test item.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of the test item administered animals remained in the same range as controls over the study period.
A slightly lower body weight gain was seen in males and females at 2500 ppm in the first three days of administration, which recovered thereafter. As this only occurred in the first three days of the study, this finding was considered not toxicologically relevant. The other statistically significant changes in body weight gain were minimal increases in body weight gain and/or lacked a dose-related response and were therefore considered to be not test item related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No effects on food consumption were seen in females up to 2500 ppm.
A slightly lower food consumption was observed in males at 2500 ppm between Days 1-3 and in all test item-treated males between Days 60-67 (at 900 ppm also between Days 57-60), which recovered over time (not statistically significant). As these were only temporary food consumption changes and food consumption was comparable with the control group over the entire administration period, these findings were considered to be not toxicologically relevant.
Over Days 3-6, food consumption in males at 300, 900 and 2500 ppm were higher when compared to the control group. This change was caused by low control group values and was therefore not test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At day 89, the following findings were noted:
- One Male, Group 1: Retina, Irregular Reflection, Right, 2 Slight, Focal
- One Male, Group 4: Iris, Pers Pup Membrane, Remnant, Left
- One Female, Group 1: Iris, Pers Pup Membrane, Remnant, Left
The nature and incidence of ophthalmology findings noted were similar among the groups and occurred within the range considered normal for rats of this age and strain. The findings were therefore considered to be unrelated to the administration of the test item.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
HEMATOLOGY
One female at 2500 ppm had marked platelet clumps. As this was only seen in one animal and lacked any other corroborating findings, this was considered to be not test item related.
Other values in males and females regardless of achieving a level of statistical significance, when compared to controls, were considered to have arisen as a result of slightly low control values, occurred in the absence of a dose-related distribution and/or were, given the magnitude of change, considered to be of no toxicological significance.

COAGULATION
A shorter prothrombin time was seen in males at 900 ppm (0.94x of control), which lacked a dose-related response and was therefore considered to be not test item-related. Overall, coagulation parameters of treated rats were considered not to have been affected by the test item.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related clinical chemistry changes were noted in males and females up to 900 ppm.
In males, a slight increase in alanine aminotransferase (ALT) activity (1.17x of control, not statistically significant) and calcium concentration (1.04x) were observed at 2500 ppm. At the severity observed and in absence of a histopathological correlation, these findings were considered to be not adverse.
Remaining differences in clinical chemistry parameters were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Due to a technical error during the measurement of thyroid-stimulation hormone (TSH), only the results of three out of ten females of the control group and four out of ten females at 2500 ppm were available. Therefore, historical control data was also used to determine the toxicological relevance of the result. The mean TSH levels in females at 2500 ppm were higher than the control group but remained well within the historical control range. Therefore, this result was considered to be not toxicologically relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly higher mean liver weight was noted in males at 2500 ppm that was statistically significant relative to body weight only. Given the small magnitude of the change (6% relative to body weight) and the lack of histologic correlate, this small difference was interpreted to represent biologic variability or a reflection of the slightly lower body weight (-5%, not statistically significant) rather than a test item related effect.
Other statistically significant changes (higher ovary absolute and relative to body weight and higher adrenal gland relative to body weight) in females at 900 ppm were considered to be not test item related as they occurred in the absence of a dose-related trend and microscopic correlate.
Overall, there were no test item-related alterations in organ weights.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period test item-related irregular surface was observed in the non-glandular stomach. This was recorded in all males and females at 2500 ppm and correlated with histopathological findings (see respective sections).
The remainder of the recorded macroscopic findings were considered incidental and/or were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic, non-neoplastic findings were noted in the non-glandular stomach of males and females dosed at 2500 ppm and are summarized in Table 2 of "Any other information on results incl. tables". Some females at 2500 ppm presented findings including mucosal erosion (4/10, minimal, focal), submucosal edema (6/10, minimal, regionally extensive to diffuse) and submucosal mixed cell infiltrates (7/10, up to mild degree, regionally extensive to diffuse). The alterations were considered local test item effects and likely resulted from the slightly irritating properties of the test item.
The low incidence of mucosal erosion (minimal, focal) noted in the non-glandular mucosa of 1/10 males each at 900 and 2500 ppm and 1/10 females at 900 ppm, and submucosal edema (minimal, diffuse) in 1/10 males at 2500 ppm were within the normal range of background pathology encountered in rats of this age and strain and were therefore regarded to be unrelated to the test item.

Miscellaneous findings:
In one female dosed at 2500 ppm, axonal dystrophy was noted in the medulla oblongata (specifically, in the cuneate and gracile nuclei). This observation was focal and characterized by the presence of numerous spheroids in these nuclei. Axonal dystrophy was also present in the dorsal funiculus of the spinal cord as a few spheroids present at each examined segment (cervical, thoracic and lumbar). Axonal dystrophy at this location is a well described background finding in aged rats (Fujisawa and Shiraki 1978; Kaufmann W, 2012), but also has been reported in rats from sub-chronic (90-day) studies (Eisenbrandt et al., 1990).Given the single incidence in this study, this observation was thus interpreted to be a spontaneous change and not related to the test item.
There were no other test item-related histologic changes. Remaining histologic changes 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.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Squamous cell hyperplasia and hyperkeratosis (mild, diffuse) was observed in all males and females in the 2500 ppm group (see Table 2 of "Any other information on results incl. tables"). This was test-item related. However, the alterations were considered local test item effects and likely resulted from the slightly irritating properties of the test item.
Other effects:
no effects observed
Description (incidence and severity):
ESTROUS CYCLE:
Estrous cycle was unaffected by test substance administration.

MOTOR ACTIVITY:
Motor activity was similar between control and high dose animals. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
2 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: Effect level corresponds to an actual test article intake of 173 and 190 mg/kg body weight/day for males and females, respectively

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

CONCENTRATION AND HOMOGENEITY ANALYSIS 


No test item was detected in the control group diet.


The mean accuracies for the concentrations in the formulations of the 300, 900 and 2500 ppm group (Week 1, Week 6 and Week 12 formulations) were between 92% to 100% and therefore in agreement with the target concentrations for suspensions (80% and 120%).


The coefficient of variation for the formulations of the 300 and 2500 ppm group (Week 1, Week 6 and Week 12 formulations) and 900 ppm (week 1 formulation) were between 0.51 and 1.7% and therefore the formulations were considered homogeneous (i.e. coefficient of variation ≤ 10%).


 


STABILITY RESULTS





Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 500 to 15000 ppm was confirmed for at least 3 weeks in the freezer (≤-15°C) and for at least 4 days at room temperature (Test Facility Study No. 20265399 (method development and validation study)). In addition, stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) at 300 ppm is confirmed for at least 3 weeks in the freezer (≤-15°C) and for at least 4 days at room temperature (Test Facility Study No. 20265399 (method development and validation study)).


 


TEST ARTICLE INTAKE


Table 1: Test Article Intake







































Group



Nominal Dietary Inclusion Level [ppm]



Mean over Means Intake [mg test item/kg body weight]


(mean range indicated within brackets)



Males



Females



2



300



21



(16-33)



22



(17-31)



3



900



64



(47-102)



70



(54-97)



4



2500



173



(127-270)



190



(143-275)



 


MICROSCOPIC PATHOLOGY


Table 2: Summary Test Item-Related Microscopic Findings – Stomach
















































































































































































 



Males



Females



Dose level (ppm)



0



300



900



2500



0



300



900



2500



 



 



 



 



 



 



 



 



 



STOMACH (non-glandular) a



10



10



10



10



10



10



10



10



   Hyperplasia squamous cell



 



 



 



 



 



 



 



 



      Mild



-



-



-



10



-



-



-



10



   Hyperkeratosis



 



 



 



 



 



 



 



 



      Mild



-



-



-



10



-



-



-



10



   Infiltration mixed cell, submucosal



 



 



 



 



 



 



 



 



      Minimal



-



-



-



-



-



-



-



4



      Mild



-



-



-



-



-



-



-



3



    Edema, submucosal



 



 



 



 



 



 



 



 



      Minimal



 



 



 



1



 



 



 



5



      Mild



-



-



-



-



-



-



-



1



    Erosion, mucosal



 



 



 



 



 



 



 



 



      Minimal



-



-



1



1



-



-



1



4



a  =  Number of tissues examined from each group. Bold values indicate a test item-related effect.




Applicant's summary and conclusion

Conclusions:
Based on the results obtained in this study, the No Observed Adverse Effect Level for 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate was 2500 ppm (corresponding to an actual test article intake of 173 and 190 mg/kg body weight/day for males and females, respectively).
Executive summary:



A 90-day dietary rat toxicity study was conducted according to OECD/EC guidelines and in accordance with GLP principles. Wistar Han rats were administered with 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate for 90 days by dietary administration at dose levels of 300, 900 and 2500 ppm. The animals of the control group received the standard rodent diet alone.





No test item-related effects were observed in males and females up to 900 ppm.











At clinical chemistry assessment, a test item-related increase in alanine aminotransferase activity and calcium concentration were observed in males at 2500 ppm. At the severity observed and in absence of a histopathological correlation, these findings were considered to be not adverse.


At histopathological assessment, test item-related findings in the non-glandular stomach were noted in males and females at 2500 ppm consisting of squamous cell hyperplasia and hyperkeratosis. These findings correlated macroscopically with irregular surface described in the same stomach region. In addition, some females at 2500 ppm presented a few other microscopic findings in the non-glandular stomach including mucosal erosion, submucosal edema and submucosal mixed cell infiltrates. The combination of these findings at the recorded incidences, severities and distribution are considered non-adverse. The recorded alterations in the non-glandular stomach were local test item effects and likely resulted from the slightly irritating properties of the test item.


No test item-related toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, clinical appearance, body weight, food consumption, functional observations, ophthalmoscopy, hematology, coagulation and organ weights).








Based on these results, the No Observed Adverse Effect Level was considered to be at least 2500 ppm (corresponding to an actual test article intake of 173 and 190 mg/kg body weight/day for males and females, respectively). Selecting higher dose levels for this 90-day dietary study was considered to be not justified, based on the toxicity observed in the 14-day dietary rat study (Test Facility Study No. 20265400).