(2,4,6-trioxo-1,3,5-triazine-1,3,5(2H,4H,6H)-triyl)tri-2,1-ethanediyl triacrylate

Endpoint summary

Description of key information

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rat performed according to OECD TG 422 and  in accordance with GLP principles, the NOAEL parental for Tris(2-hydroxyethyl) Isocyanurate Triacrylate was considered to be 50 mg/kg bw/day based on the lesions in the forestomach which were adverse from 100 mg/kg bw/day.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 November 2019 - 13 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

RjHan:SD (CD®)

Details on species / strain selection:

The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected since background data from previous studies are available at Charles River Laboratories Evreux.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: approx. 10 weeks; females: approx. 11 weeks
- Weight at study initiation: males 361 g - 432 g; females 223 g - 317 g
- Fasting period before study: no
- Housing: F0 animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust (Le comptoir des sciures, Meyzieu, France). Individual housing of F0 animals was chosen as group housing for pregnant animals can adversely affect gestation and lactation, and to avoid aggressive behavior around mating.
Toward the end of gestation and during lactation, autoclaved wood shavings (Le comptoir des sciures, Meyzieu, France) were provided to females and their litter as nesting material. Each cage contained nylabone and rat hut for environmental enrichment.

- Diet: ad libitum, SSNIFF rat/mouse pelleted maintenance diet, batch Nos. 26453330 and 10657438 (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, tap water (filtered with a 0.22 µm filter)
- Acclimation period: males 7 days before treatment; females 5 days before the beginning of estrous cycle monitoring during the pre-treatment period

DETAILS OF FOOD AND WATER QUALITY: The batches of diet, sawdust and wood shavings were analyzed by the suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 december 2019 To: 10 February 2020

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since it is a route of administration which is recommended by the Regulatory Authorities for this type of study.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were maintained under delivery condition (protected from light) throughout the administration procedure.
The control and test item dose formulations were kept in warm water (approximately +37 ± 2°C) without magnetic stirring no longer than 20 ± 5 minutes (delivery conditions).
The control and test item dose formulations were continuously stirred in a water bath at +37°C for at least 15 and 30 minutes, respectively, before administration and throughout the dosing procedure. There was no stopping in water bath heating from preparation to the end of administration.
The test item dose formulations were administered within 3 hours and 30 minutes after the end of their preparation.

VEHICLE
- Justification for use and choice of vehicle: The control item for administration to the control group (control dose formulation) was selected in agreement with the Sponsor, based on previous experimental work and/or preliminary study(ies).
- Concentration in vehicle: 0, 7.1, 14.3 or 28.6 mg/mL
- Amount of vehicle: 7 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Three times during the study: Day 1, Weeks 5 and 8.
A sample was taken from control and test item dose formulations and analyzed using the validated method.

Acceptance criteria: Measured concentration = nominal concentration ± 15%.

Duration of treatment / exposure:

males: 2 weeks before mating, during the mating period (until evidence of mating or 2 weeks had elapsed), until euthanasia (at least 4 weeks in total)
females: 2 weeks before mating, during the mating period (until evidence of mating or 2 weeks had elapsed), during gestation, during lactation until Day 13 p.p. inclusive, until euthanasia for females with no evidence of mating or no delivery.

Frequency of treatment:

once daily

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

group 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

group 3

Dose / conc.:

200 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 in agreement with the Sponsor, on the basis of the results of a previous toxicity study (Study No. 47277 TSR) performed in the same species, see also additional information on results).
- Fasting period before blood sampling for clinical biochemistry: Prior to blood sampling and during urine collection, the animals were deprived of food for an overnight period of at least 14 hours.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also evaluated.

BODY WEIGHT: Yes
- Time schedule for examinations: once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until euthanasia. The body weight of each female was recorded once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until mated (or until euthanasia for females with no evidence of mating), on Days 0, 7, 14 and 20 p.c. (post coitum) (and on the day of euthanasia for females which did not deliver), and on Days 1, 4, 8 and 13 p.p.
Animals euthanized prematurely were weighed before euthanasia.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period.
The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1 4, 4-8 and 8-13 p.p.
During the mating period, food consumption was not measured for males or females.

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 AND COMPOUND INTAKE : Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the first half of the morning (between 7.5 and 10 am)
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters: According to the guideline

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters: According to the guideline

URINALYSIS: Yes
- Time schedule for collection of urine: overnight period of at least 14 hours
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters: According to the guideline

THYROID HORMONES:
- Time schedule for collection of blood:
- at termination on Day 14 p.p. from all F0 females (approximately 0.5 mL of blood was collected from the orbital sinus under isoflurane anesthesia),
- at termination from all F0 males (approximately 0.5 mL of blood was collected from the orbital sinus under isoflurane anesthesia).
- Parameters: The levels of the thyroid hormone (T4) and thyroid stimulating hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for F0 males sampled at termination. Plasma samples obtained on Day 14 p.p. from F0 females were kept at -80°C pending possible analysis.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the treatment period. For females, this was performed on Day 13 p.p. after euthanasia of the pups.
- Dose groups that were examined: all, first five males and lactating females from each group
- Battery of functions tested: a detailed clinical examination, the assessment of reactivity to manipulation and to different stimuli (touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature) and motor activity.

MONITORING ESTROUS CYCLE:
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning (between 7.5 and 10 a.m.):
- during the 2 weeks of the pre-treatment period, from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated and on the day of sacrifice before euthanasia, to allow correlation with reproductive organ histopathology.

Sacrifice and pathology:

On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
- males: after the end of the mating period (at least 4 weeks of treatment in total),
- females: on Day 14 p.p.
The following F0 females were euthanized by the same way without overnight fasting:
- females which did not deliver: on Day 25 p.c. (after a body weight recording to check for a possible un-noticed delivery),
- females with no evidence of mating: 24-25 days after the end of the mating period if no delivery occurred (after a body weight recording),
- female with total litter loss: as appropriate.

GROSS PATHOLOGY: Yes (see table)
A complete macroscopic post-mortem examination was performed on all F0 animals including those euthanized prematurely. 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. Special attention was paid to the reproductive organs.
The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 14 p.p. and for one female (group 3) euthanized 25 days after the end of the mating period with no evidence of mating.
For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

HISTOPATHOLOGY: Yes (see table)
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Based upon the results of the microscopic examination of the high-dose group and after the agreement of the Sponsor, the forestomach of the low- and intermediate-dose groups (groups 2 and 3) was examined.

Statistics:

Data are recorded and calculated using computerized validated software (REPROTOX and PATHDATA) and/or EXCEL software.
Data are expressed as group mean values ± standard deviation (body weight, body weight change, food consumption, number of corpora lutea, number of implantation sites, number of pups and pup body weight, gestation length) or as proportions (pre-implantation loss, post-implantation loss, pup observations, mating index, fertility index, gestation index, live birth index, viability index). Whenever appropriate, the experimental unit of comparison was the litter.
Data of non-pregnant females are not included in group mean calculations (body weight, body weight change, food consumption).
Data of pregnant females with no evidence of mating (i.e. not assigned a Day 0 p.c. date) are not included in the group mean calculations such as body weight and food consumption for the p.c. period, but all data from the dams and pups are included in the group mean calculations for the p.p. period.

Body Weight, Food Consumption and Reproductive Data
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

Hematology, Blood Biochemistry, Urinalysis, Hormones, Motor Activity, Anogenital Distance, Nipples/Areolae, Live Birth Index, Sex-Ratio and Post-Implantation Loss
CITOX software was used to perform the statistical analysis of these data according to the sequence decribed in the background material.

Organ Weight
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 sequence described in teh backgroud material.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Loud breathing was observed in 2/10 males given 200 mg/kg bw/day and in a few females given 50, 100 or 200 mg/kg bw/day during the pre-mating, gestation or lactation period. This sign was observed from the first week of the treatment period and was often associated in females with abdominal breathing, piloerection and/or round back, and in a single animal with half-closed eyes. These signs were either observed for a few days (1 to 12 days) or were present until the terminal sacrifice (one female at 100 mg/kg bw/day and one male and one female at 200 mg/kg bw/day).
They were considered to be most probably secondary to reflux/regurgitation of the test item formulations, worsen by the irritating properties of the test item (reflux at dosing was sometimes noted the day before the onset of the signs).
Ptyalism was observed in test item-treated males and females during the premating, pregnancy and lactation periods. This non-adverse finding, commonly noted when a test item is administered by the oral route, was attributed to the taste and/or texture of the test item dose formulations.
All the other clinical signs i.e. cutaneous observations on various parts of the body (areas of hair loss, cutaneous lesions, scabs), reflux at dosing, dacryorrhea, chromodacryorrhea and/or reddish vaginal discharge) were considered to be unrelated to the test item as they were present both in control animals and/or were reported sporadically in only a few animals and/or were attributed to the gavage procedure.

Mortality:

mortality observed, treatment-related

Description (incidence):

No unscheduled death occurred in males at any dose level during the study.
Pre-mating period
At 200 mg/kg bw/day, four females were prematurely euthanized due to their poor clinical condition on Days 7, 9, 12 and 17, respectively.
One to four days before death, piloerection, round back, cold to the touch and loud breathing were observed in all females, sometimes associated with pallor of the extremities, hypoactivity, dyspnea, abdominal breathing, swollen abdomen, soiled urogenital area and/or half-closed eyes. Ptyalism was noted in all females from Day 6 or 8. Body weight loss was recorded for all females (between -22 and – 8%, when compared to their last recorded body weight), accompanied by reduced food consumption in one female (12 g/day on Days 1-15 vs. 17 g/day in controls).
In all four females, test item-related microscopic changes were observed in the gastrointestinal tract, in the forestomach and stomach mainly. They consisted of minimal to moderate squamous cell hyperplasia (all four females), minimal to moderate hyperkeratosis (three females), minimal or slight mixed inflammatory cell infiltrate (two females) and/or slight ulcer (one female) in the forestomach, and minimal or slight erosion and/or hemorrhage in the stomach (all four females) and/or jejunum (one females). These lesions in the gastrointestinal tract likely contributed to the clinical signs in these females, particularly in three females which showed the most pronounced gastric lesions. However, as lesions of similar or higher severity were observed in the forestomach of terminally euthanized high-dose animals, the gastrointestinal lesions, due to irritant properties of the test item, were not considered to completely explain the poor clinical status of these females. In one female, in addition to gastric lesions, marked inflammation and slight erosion/ulcer were noted in the trachea, with inflammation in the adjacent tissues. In view of the clinical signs (loud breathing) observed in high dose females, a reflux due to irritant properties of the test item (Damsch et al., 2011) is suspected, and although nasal cavities were not microscopically examined, death of these four females was considered to be most probably related to the test item administration.
Gas distension and/or thin wall was noted at necropsy in one or several gastrointestinal segments in all prematurely euthanized females; in the absence of microscopic correlates, these findings were considered to be of minor toxicological significance.
In one female, slight multifocal degeneration/necrosis of the sternal bone marrow was observed. In the absence of similar changes in the bone marrow in other animals at 200 mg/kg bw/day, relationship with the test item was considered to be unlikely.
In addition, the following microscopic findings were interpreted as stress-related and/or secondary to debilitation: microscopic decreased lymphoid cellularity and/or lymphocyte apoptosis/necrosis occurring in the thymus, spleen (both macroscopically reduced in size in two females), G.A.L.T., and/or mandibular and mesenteric lymph nodes in all four females, tubular dilation of the renal tubules in two females, adrenal cortical hypertrophy in one female, and pancreatic acinar secretory depletion (correlating with brown discoloration) in one female.

Mating period
Due to absence of mating, two females were sacrificed on Day 52 or 53 (only ptyalism was noted during the treatment period).

Gestation period
Due to no delivery, the two females were euthanized on Day 25 p.c. At necropsy, these females were found to be non-pregnant.

Lactation period
One control female was prematurely euthanized on Day 1 p.p. due to the death of its litter (2 pups at birth). At histopathological examination, one uterine horn was thickened, which correlated with moderate subacute inflammation, with hemorrhage and focal ulcer.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day, when compared to controls, slightly lower mean body weight gain was recorded in males on Days 1-29 (+97 vs. +116 g in controls). Although a relationship to the test item treatment cannot be excluded, this difference, not statistically significant, was mainly due to transient lower mean body weight gain at treatment initiation with no significant impact on the body weight, and was therefore considered to be non-adverse.
The lower mean body weight gain in females during the pre-mating period was mainly due to some individual body weight losses. These effects on body weight change were attributed to the test item treatment.
At 50 or 100 mg/kg bw/day, no effects were noted on mean body weight or mean body weight change.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No relevant effects on mean food consumption were observed in males at any dose level and in females given 50 or 100 mg/kg bw/day.
The statistically significant difference between controls and males given 50 or 200 mg/kg bw/day during the pre-mating period was not attributed to the test item treatment as this difference was of low magnitude and/or not dose-related.
The slightly lower mean food consumption noted in females given 200 mg/kg bw/day during the pre-mating period (not statistically significant) was mainly due to the contribution of one prematurely sacrificed female (12 g/day on Days 1-15) and two other females (11 g/day on Days 8-15); these episodes of reduced food consumption were concomitant of clinical signs (mainly loud breathing).

Description (incidence and severity):

n/a

Description (incidence and severity):

n/a

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematology parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period.
At 200 mg/kg bw/day, when compared to controls, statistically significant, higher mean neutrophil count was noted in males (3.30 vs. 2.02 in controls), with the same tendency in females (5.14 vs. 3.52 in controls). Although these differences may be related to the administration of the test item, they were isolated and of low magnitude, and were therefore considered to be non-adverse.
The other statistically significant difference from controls, i.e. longer mean activated partial thromboplastin time in males given 200 mg/kg bw/day (16.4 s), was not attributed to the test item treatment as the values remained within the range of our historical control data (11.9-18.6 s as min-max) and no such finding was reported in females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day, when compared to controls, mean alanine aminotransferase activity was higher in males (57 vs. 46 U/L in controls) and females (123 vs. 72 U/L in controls) (5/5 and 4/5 individual values, respectively, were above the highest control value). While a relationship to the test item treatment cannot be excluded, the differences were of low magnitude and given the absence of correlated microscopic findings, they were considered as non-adverse.
The other statistically significant differences between control and test item-treated animals, namely higher mean chloride level in males at 200 mg/kg bw/day (106.7 vs.105.4 mmol/L in controls) and higher mean total protein level in males at 50 mg/kg bw/day (57.5 vs. 54.6 g/L in controls), were not attributed to the test item treatment as they were isolated and/or not dose-related and/or of minimal magnitude.

No relevant effects on T4 or TSH levels were noted in the adult males sacrificed at the end of the treatment period at any dose level.
The only statistically significant change from controls, i.e. higher mean T4 level in males given 50 mg/kg bw/day (45.1 vs. 39.76 ng/mL), was not attributed to the test item treatment as it was reported without any
dose-relationship and the values are included in the historical control data.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urinary parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No relevant test item-related effects were observed on motor activity (horizontal movements and rearing) in males or females at any dose level. Lower mean numbers of rearing movements recorded at 50 mg/kg bw/day in females (97 vs. 206 in controls) was not attributed to the test item treatment as this difference was not dose-related.
There were no test item-related neurologic abnormalities observed in the Functional Observation Battery.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No organ weight changes occurred that were considered to be related to the test item administration.
Higher mean absolute and relative spleen weights (up to +39%) were noted in males and females at 200 mg/kg bw/day. No microscopic correlates were observed in males. In females, these variations correlated with a higher incidence of congestion. This variable spleen congestion was considered to be related to pentobarbital anesthesia and/or inconsistency in exsanguination (Sellers et al, 2007) and not to be related to the test item administration.
Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic findings were observed in the forestomach in males and females at = 100 mg/kg bw/day. They consisted of several or many white masses, up to 0.3 cm in diameter, correlating with squamous cell hyperplasia and hyperkeratosis at microscopic examination.
The other macroscopic findings had no histological correlates or correlated with common histological findings in control rats, and were considered to be incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were observed in the forestomach.
Squamous cell hyperplasia, hyperkeratosis (i.e. increased thickness of the keratin layer on surface), and infiltrate of mixed inflammatory cells were present with dose-related incidence and severity in the forestomach in males and females at = 100 mg/kg bw/day. At these dose levels, they were variably associated with vesicles/pustules in the squamous epithelium and ulcers. At 50 mg/kg bw/day, only minimal focal or multifocal squamous cell hyperplasia was noted in 2/5 males, and no findings were present in the forestomach in females. All these lesions in the forestomach were attributed to local irritation of the test item (Nolte et al., 2016), and were considered adverse at = 100 mg/kg bw/day in view of their severity and presence of ulcers.
Marked ulceration and mixed inflammatory cell infiltrate were observed in the trachea of one female treated at 200 mg/kg/day. They may have been reflux-related (Damsch et al., 2011) and due to irritant properties of the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day, when compared to controls, a tendency towards lower mean number of cycles (2.1 vs. 2.8) associated with higher mean cycle length (4.4 vs. 4.1) was noted. While a relationship to the test item treatment cannot be excluded, this change was slight and did not affect the mating or fertility data and was therefore considered to be non-adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Key result

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

Results range finding study:

The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous toxicity study (Study No. 47277 TSR) performed in the same species, in which the test item was administered daily by gavage to five males and five females at dose levels of 100, 300 or 1000 mg/kg bw/day for 14 days.

At 1000 mg/kg bw/day, the test item induced ptyalism, lower body weight gain in males (-49% vs.controls) associated with lower food consumption during the first week of treatment (-15% vs.controls). One female, found dead, had test item-related gross gastro-intestinal changes and erosion in the forestomach that were considered to be related to the mortality, together with squamous cell hyperplasia, hyperkeratosis, submucosal infiltrate of mixed cells and fibroplasia.

Macroscopically, test item-related white masses were noted in the forestomach from terminally euthanized males and females and correlated with microscopic squamous cell hyperplasia and hyperkeratosis. There were also test item-related thickening in the stomach from one male and black discoloration and/or enlargement in the portal lymph node. At microscopy, test item-related findings were noted in forestomach (ulceration considered asadversein males, and minimal to moderate erosion, slight to severe squamous cell hyperplasia, slight to severe hyperkeratosis, minimal to moderate infiltrate of mixed cells, minimal to moderate fibroplasia, and minimal or slight perivascular inflammation in serosa in males and females) and in stomach (adverseatrophy/degeneration, and non-adverse infiltrate of mixed cells, edema and hemorrhage in males).

At 300 mg/kg bw/day, the test item induced ptyalism, lower body weight gain in males (-27% vs.controls) and body weight loss in females on Days 8-14, associated with lower food consumption (-13% vs.controls during the first week of treatment in males and -14%vs.controls on Days 8-14 in females). One female, prematurely euthanized, had test item-related gross gastro-intestinal changes and erosion in the forestomach that were considered to be related to the morbidity, together with submucosal infiltrate of mixed cells and fibroplasia.

Macroscopically, test item-related white masses were noted in the forestomach from terminally euthanized males and females and correlated with microscopic squamous cell hyperplasia and hyperkeratosis. There were also test item-related changes in the stomach (white masses, thickening and black content) and in the portal lymph node (enlargement in one female). In addition, one female was emaciated and had also small spleen and thymus, black content in duodenum, jejunum, ileum and cecum, together with distended ileum and colon (also noted in another female).

At microscopy, non adverse test item-related findings were noted in forestomach (minimal or slight erosion, infiltrate of mixed cells, fibroplasia, perivascular inflammation in serosa, and/or, slight or moderate squamous cell hyperplasia, hyperkeratosis and/or slight edema) in males and females. In stomach, adverse effects (severeatrophy/degeneration and moderate erosion) accompanied by other changes (minimal infiltrate of mixed cells, minimal edema and/or moderate hemorrhage) were observed in 2/4 females.

At 100 mg/kg/day, the test item was clinically well tolerated. Test item-related macroscopic white masses were noted in the forestomach from males and females and correlated with microscopic squamous cell hyperplasia and hyperkeratosis. There was also thickening in the stomach from one male that was test item-related although without microscopic correlates.

At microscopy, non-adverse test item-related findings were noted in forestomach (slight erosion, minimal to moderate squamous cell hyperplasia, minimal or slight hyperkeratosis, minimal or slight infiltrate of mixed cell and/or slight edema) in males and females.

The doses of 300 and 1000 mg/kg bw/day are considered to exceed the maximal tolerated dose in males and females. Therefore, 200 mg/kg bw/day was selected as the high-dose level. The low-dose and mid-dose were selected using a ratio representing approximately a 2-fold interval (i.e. 50 and 100 mg/kg bw/day).

 

 

Chemical analysis of the dose formulations

The test item concentrations in the administered dose formulations analyzed in Weeks 1, 5 and 9 remained within an acceptable range of -9.8% to +3.7% when compared to the nominal values (± 15%).

No test item was detected in the control dose formulation.

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rat performed according to OECD TG 422 and in accordance with GLP principles, the NOAEL parental for Tris(2-hydroxyethyl) Isocyanurate Triacrylate was considered to be 50 mg/kg bw/day based on the lesions in the forestomach which were adverse from 100 mg/kg bw/day.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item, Tris(2-hydroxyethyl) Isocyanurate Triacrylate, following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, through gestation until Day 13 post-partum (p.p.).

This study provides information  on the possible health hazards (including neurological effects) likely to arise from repeated exposure over a relative limited period of time and on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Tris(2-hydroxyethyl) Isocyanurate Triacrylate,daily by the oral route (gavage) at dose levels of 50, 100 or 200 mg/kg/day, at a constant dosage volume of 7 mL/kg/day.A control group often male and ten female ratsreceived the vehicle only (corn oil) under the same experimental conditions.

Males were treated for an overall period of approximately 4 weeks: 2 weeks before mating, during the mating period (up to 2 weeks) until the day before euthanasia. Females were treated for an overall period of 7 to 9 weeks: 2 weeks before mating, through mating (up to 2 weeks) and gestation (3 weeks) until Day 13post-partum(p.p.) inclusive.

The actual test item concentrationsin the dose formulations were determined in Weeks 1, 5 and 9 using a validated HPLC-UV analytical method.

Animals were checked daily for clinical signs and mortality. Functional Observation Battery and motor activity were performed on five males and five females during the last days of treatment. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stages were determined daily from two weeks before mating until the females had mated and on Day 14 p.p.before euthanasia.

The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 13p.p. The total litter sizes and numbers of pups of each sex were recorded after birth.The size of each litter was adjusted on Day 4p.p.by culling extra pups to obtain as nearly as possible four males and four females per litter.Pups not selected on Day 4p.p.were sampled, euthanized and discarded without further examination. The pups were observed daily for clinical signs or abnormal behaviour and were weighed on Days 1, 4, 8 and 13p.p.The physical development of pups was assessed by measuring the anogenital distance on Day 1p.p.and by counting the number of nipples and areolae in male pups on Day 12 p.p. Hematology, blood biochemistry and urinary investigations were performed on the first five males and females in each group at scheduled euthanasia.Thyroid hormones (TSH and T4) were determinedin males at termination and in at least two pups/litter euthanized on Day 13 p.p. Males were euthanized after completion of the mating period. Dams were euthanized on Day 14 p.p. A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from the first five control and high-dose males and lactating females as well as from four high-dose females sacrificed before mating, on all macroscopic lesions and on the forestomachof the low- and intermediate-dose groups.

Pups were euthanized on Day 13 p.p. and submitted to a detailed external examination with a particular attention to the external genital organs.

F0 animals: No unscheduled deaths occurred in males at any dose level during the study.

Four high dose females were prematurely euthanized before mating, between Days 7 and 17, due to their poor clinical condition. In all of them, test item-related microscopic changes were observed in the gastrointestinal tract, in the forestomach and stomach mainly. They consisted of squamous cell hyperplasia, hyperkeratosis, mixed inflammatory cell infiltrate and/or erosion or ulcer in the forestomach and stomach, and rarely jejunum. These lesions in the gastrointestinal tract, considered to be related to irritant properties of the test item, likely contributed to the clinical signs in these females. In addition in one of these females, marked inflammation and slight erosion/ulcer were noted in the trachea, with inflammation in the adjacent tissues. This was considered to be reflux-related due to irritant properties of the test item, and explained the clinical signs leading to premature euthanasia of this individual.

Clinical signs of poor clinical condition were observed at all dose levels of the test item, mainly loud breathing in 2/10 males at 200 mg/kg/day and in a few females at 50, 100 or 200 mg/kg/day during the pre-mating, gestation or lactation period. This sign was observed from the first week of the treatment period and in females was often associated with abdominal breathing, piloerection, round back and/or half-closed eyes. They were considered to be most probably secondary to reflux/regurgitation of the test item formulations, worsen by the irritating properties of the test item (reflux at dosing was sometimes noted the day before the onset of the signs). Ptyalism was observed in test item-treated males and females during the whole treatment period. This non-adverse finding, commonly noted when a test item is administered by the oral route, was attributed to the taste and/or texture of the test item dose formulations.

The functional observation battery and motor activity were unaffected by the test item treatment.

Body weight was not impaired by the test item treatment. Some episodes of reduced food consumption were recorded in isolated females given 200 mg/kg/day during the pre-mating period.

The estrous cycle was not significantly impacted by the test item treatment. The mating, fertility and delivery data were unaffected by the test item treatment.

All laboratory investigations, higher alanine aminotransferase activity was noted in males and females at 200 mg/kg/day (+24 and +71%, respectively,vs.controls). This isolated finding was considered as non-adverse.

Hematology and urinary investigations were not impacted by the test item treatment. Thyroid hormone analysis did not reveal any disturbances in males F0 at sacrifice.

At pathology, no test item-related organ weight changes were noted at any dose levels. Test item-related adverse lesions indicative of local irritation were observed in the forestomach at=100 mg/kg/day at terminal euthanasia in parent animals. They consisted of squamous cell hyperplasia and hyperkeratosis correlating with macroscopic white masses, and infiltrate of mixed inflammatory cells variably associated with vesicles/pustules in the squamous epithelium and ulcers.

At 50 mg/kg/day, only minimal, non-adverse squamous cell hyperplasia of the forestomach was present in 2/5 males.No test item-related macroscopic findings were observed. There were no histopathological findings in females at 50 mg/kg/day.

Pups: Observations of the pups from birth to Day 13p.p.did not show any effects on mortality, viability, clinical signs, sex ratio, anogenital distance or nipples and areolae numbers.

Lower body weight gain (-7 and -9%, respectively, on Days 4-13p.p.) and lower body weight (-5% and -8%, respectively, on Day 13p.p.) were recorded in male and female pups at 200 mg/kg/day.

Thyroid hormone analysis did not reveal any disturbances in pups on Day 13p.p.

 

Based on the experimental conditions of this study:

·        the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 50 mg/kg/day in males and females based on the lesions in the forestomach which were adverse from 100 mg/kg/day,

·        the No Observed Effect Level (NOAEL) for reproductive performance (mating and fertility) was considered to be 200 mg/kg/day based on the absence of adverse effects on mating or fertility at this dose level,

·        the NOAEL for toxic effects on progeny was considered to be 200 mg/kg/day given the slight and non-adverse effect on the body weight gain/body weight in pups at this dose level.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

This 28-day study is considered to be reliable because it was performed according to the OECD guideline (klimisch score = 1).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

28-day repeated toxicity study by oral route (OECD 422, 2020) :

 The objective of this study was to evaluate the potential toxic effects of the test item, Tris(2-hydroxyethyl) Isocyanurate Triacrylate, following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, through gestation until Day 13 post-partum (p.p.).

This study provides information  on the possible health hazards (including neurological effects) likely to arise from repeated exposure over a relative limited period of time and on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Tris(2-hydroxyethyl) Isocyanurate Triacrylate,daily by the oral route (gavage) at dose levels of 50, 100 or 200 mg/kg/day, at a constant dosage volume of 7 mL/kg/day.A control group often male and ten female ratsreceived the vehicle only (corn oil) under the same experimental conditions.

Males were treated for an overall period of approximately 4 weeks: 2 weeks before mating, during the mating period (up to 2 weeks) until the day before euthanasia. Females were treated for an overall period of 7 to 9 weeks: 2 weeks before mating, through mating (up to 2 weeks) and gestation (3 weeks) until Day 13post-partum(p.p.) inclusive.

 No unscheduled deaths occurred in males at any dose level during the study.

Four high dose females were prematurely euthanized before mating, between Days 7 and 17, due to their poor clinical condition. In all of them, test item-related microscopic changes were observed in the gastrointestinal tract, in the forestomach and stomach mainly. They consisted of squamous cell hyperplasia, hyperkeratosis, mixed inflammatory cell infiltrate and/or erosion or ulcer in the forestomach and stomach, and rarely jejunum. These lesions in the gastrointestinal tract, considered to be related to irritant properties of the test item, likely contributed to the clinical signs in these females. In addition in one of these females, marked inflammation and slight erosion/ulcer were noted in the trachea, with inflammation in the adjacent tissues. This was considered to be reflux-related due to irritant properties of the test item, and explained the clinical signs leading to premature euthanasia of this individual.

Clinical signs of poor clinical condition were observed at all dose levels of the test item, mainly loud breathing in 2/10 males at 200 mg/kg/day and in a few females at 50, 100 or 200 mg/kg/day during the pre-mating, gestation or lactation period. This sign was observed from the first week of the treatment period and in females was often associated with abdominal breathing, piloerection, round back and/or half-closed eyes. They were considered to be most probably secondary to reflux/regurgitation of the test item formulations, worsen by the irritating properties of the test item (reflux at dosing was sometimes noted the day before the onset of the signs). Ptyalism was observed in test item-treated males and females during the whole treatment period. This non-adverse finding, commonly noted when a test item is administered by the oral route, was attributed to the taste and/or texture of the test item dose formulations.

The functional observation battery and motor activity were unaffected by the test item treatment.

Body weight was not impaired by the test item treatment. Some episodes of reduced food consumption were recorded in isolated females given 200 mg/kg/day during the pre-mating period.

The estrous cycle was not significantly impacted by the test item treatment. The mating, fertility and delivery data were unaffected by the test item treatment.

All laboratory investigations, higher alanine aminotransferase activity was noted in males and females at 200 mg/kg/day (+24 and +71%, respectively,vs.controls). This isolated finding was considered as non-adverse.

Hematology and urinary investigations were not impacted by the test item treatment. Thyroid hormone analysis did not reveal any disturbances in males F0 at sacrifice.

At pathology, no test item-related organ weight changes were noted at any dose levels. Test item-related adverse lesions indicative of local irritation were observed in the forestomach at=100 mg/kg/day at terminal euthanasia in parent animals. They consisted of squamous cell hyperplasia and hyperkeratosis correlating with macroscopic white masses, and infiltrate of mixed inflammatory cells variably associated with vesicles/pustules in the squamous epithelium and ulcers.

At 50 mg/kg/day, only minimal, non-adverse squamous cell hyperplasia of the forestomach was present in 2/5 males.No test item-related macroscopic findings were observed. There were no histopathological findings in females at 50 mg/kg/day.

Based on the experimental conditions of this study,the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 50 mg/kg/day in males and females based on the lesions in the forestomach which were adverse from 100 mg/kg/day,

 

14-day repeated toxicity study by oral route (2020) :

The objective of this preliminary study was to evaluate the potential toxicity of the test item, Tris(2-hydroxyethyl) Isocyanurate Triacrylate, following daily oral administration (gavage) to rats for 14 days in order to assist the selection of dose levels for a further combined repeated and reprotoxicology study to be performed in this species.

Four groups of five male and five female Sprague-Dawley rats received the test item Tris(2 -hydroxyethyl) Isocyanurate Triacrylate at dose levels of 0, 100, 300 or 1000 mg/kg/day in the vehicle (corn oil) by oral administration (gavage) once a day for 14 consecutive days. Assessment of toxicity was based on mortality, clinical observations, body weight, food consumption, gross pathology and histopathology (limited to the stomach and forestomach).

At 1000 mg/kg/day, 1/5 females was found dead on Day 14. Before death, decreased activity, loud breathing, severe hypersalivation and erected fur were seen the day, associated with body weight loss on Day 11 (-16.8% when compared to Day 8 value), and lower food consumption between Days 11 and 14. This female showed test item-related gross gastro-intestinal changes and erosion in the forestomach that were considered to be related to the mortality, together with squamous cell hyperplasia, hyperkeratosis, submucosal infiltrate of mixed cells and fibroplasia.

Surviving animals at the same dose level showed loud breathing and hypersalivation on several occasions throughout the study, accompanied by erected fur or decreased activity in two surviving females. A lower mean body weight gain was recorded in males between Days 1 and 14 (-49.2% when compared to controls), leading to lower mean body weights when compared to controls, from Day 4 until the end of the study (up to -7.8% on Day 14). Lower mean food consumption was noted in males during the first week of treatment (up to -21.9% between Days 1 and 4 vs. controls). No test item relevant changes in body weight were observed in surviving females. Necropsy revealed white masses in the forestomach from terminally euthanized males and females andcorrelated with microscopic squamous cell hyperplasia and hyperkeratosis. There were also thickening in the stomach from one male and black discoloration and/or enlargement in the portal lymph node.Microscopic findings were noted in the forestomach (ulceration considered as adverse in males, and non-adverse squamous cell hyperplasia, hyperkeratosis, infiltrate of mixed cells, edema, fibroplasia and perivascular inflammation in serosa of various magnitudes in males and females) and in the stomach (adverse atrophy/degeneration, and non-adverse infiltrate of mixed cells, edema and hemorrhage in males).

 

At 300 mg/kg/day,1/5 females was prematurely euthanized due to poor clinical conditions on Day 2 (such as hypersalivation, loud and abdominal breathing).This female showed test item-related gross gastro-intestinal changes and erosion in the forestomach that were considered to be related to the moribundity, together with submucosal infiltrate of mixed cells and fibroplasia.

Surviving animals at 300 mg/kg/day showed loud breathing and hypersalivation throughout the study, associated with erected fur and hunched posture in one surviving female.Lower mean body weight gain were recorded in males between Days 1 and 14 (-26.8% on Day 14 vs. controls), leading to slightly lower mean body weightsfrom Day 4 until the end of the study (up to -4.5% when compared to controls on Day 14). Lower mean food consumption was noted in males during the whole treatment period (up to -14.9% between Days 1 and 4 vs. controls), and in females during the second week of treatment (up to -18.8% between Days 11 and 14 vs. controls). Necropsy revealed white masses in the forestomach from terminally euthanized males and females andcorrelated with microscopic squamous cell hyperplasia and hyperkeratosis. There were also macroscopic changes in the stomach (white masses, thickening and black content) and in the portal lymph node (enlargement in one female). In addition, one female treated at 300 mg/kg/day was emaciated and had also a small spleen and thymus, black content in duodenum, jejunum, ileum and cecum, together with distended ileum and colon (also noted in another female). Microscopic findings were noted in the forestomach (non-adverse erosion, squamous cell hyperplasia, hyperkeratosis, infiltrate of mixed cells, edema, fibroplasia and perivascular inflammation in serosa) in males and females, and in the stomach (adverse atrophy/degeneration and erosion, and non-adverse infiltrate of mixed cells, edema and/or hemorrhage in 2/4 females).

At 100 mg/kg/day, test item-related macroscopic white masses were noted in the forestomach from males and females andcorrelated with non-adverse microscopic squamous cell hyperplasia and hyperkeratosis. There was also thickening in the stomach from one male that was test item-related although without microscopic correlates.Non-adverse microscopic test item-related findings were noted in forestomach (erosion, squamous cell hyperplasia, hyperkeratosis, infiltrate of mixed cells, edema) in males and females.

Consequently, under the experimental conditions of the study, based on adverse effects observed at 300 and 1000 mg/kg/day (mortality, body weight losses, microscopic lesions in the forestomach and the stomach) after 14 days of exposure, the doses of 300 and 1000 mg/kg/day were considered to exceed the Maximum Tolerated Dose (MTD) in males and females.

 

Justification for classification or non-classification

Based on the available data, no classification of Tris(2-hydroxyethyl) Isocyanurate Triacrylate is required for the repeated toxicity according to the Regulation EC n°1272/2008.