Reaction products of acrylic acid with 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Vapour pressure was predicted using QSAR models of EPI Suite and US EPA T.E.S.T (US EPA, 2018).

Vapour pressure:

0 Pa

at the temperature of:

25 °C

Predicted VP from MPBPWIN v.1.44 of EPiSuite v.4.11: VP range: 3.89E-26 to 2.84E-06 Pa at 25°C; weighted average: 1.47E-06 Pa at 25°C. Considering the likelihood of prediction errors for very low VP values below the cut-off of 1.33E-4 Pa in EPISuite, the individual and weighted average VP values were presented generically as less than the cut-off i.e., <1.33 E-4 Pa.

di-TMPTTA predictions at higher temperatures: 8.13E-05 Pa at 40°C; 3.18E-04 Pa at 60°C;1.03E-03 Pa at 70°C

Predicted VP from US EPA T.E.S.T v.4.2.1: VP range: 2.95E-09 to 1.33E-01 Pa at 25°C; weighted average: 0.07 Pa at 25°C.

Based on the individual or weighted average VP predictions from EPISuite and T.E.S.T, the test substance is overall considered to have low volatility. The individual prediction for the most volatile constituent, di-TMPTTA, which is also the major constituent of the test substance present at 20-70%, has been considered, to represent the worst case scenario. However, given that the test substance has operating conditions at higher temperatures (such as 60 and 70°C), and that the T.E.S.T QSAR model only allows prediction at 25°C, the individual VP predictions for di-TMPTTA at 25°C and higher temperatures from EPISuite, has been considered further for risk assessment.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Physical state, appearance and colour was determined based on safety data sheet information of the test substance.

Physical state at 20°C and 1013 hPa:

liquid

Colourless to pale yellow liquid with an ester acrylate odour.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 09, 2020 to December 09, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

The deviations have no effect on the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany)
- Sex: Male/females, untreated animals were used at initiation of the study

Animal Screening
Only healthy animals were used for the test, as verified by the clinical Veterinarian. Females were nulliparous and non-pregnant.

Sex:

male/female

Details on test animals or test system and environmental conditions:

40 males and 40 females, 10 animals/group/sex, 6 weeks old (at study initiation), Males: 194 – 227 g, females: 159 – 198 g at start of treatment, 2-3 animals of the same sex and group/cage, 20.6 to 24.2℃, 24 to 77% humidity, 12 h light/dark, Autoclavable complete diet ad libitum, bottled tap water

Environmental Acclimation
Acclimation period in the study was 6/7 days.

Selection, Assignment, Replacement, and Disposition of Animals
At the end of the acclimatisation period, the animals were assigned to their respective dose groups by randomisation based on body weights. It was checked that all animals are within 20% of the overall mean at the start of the study. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; PROVANTIS v.9 software was used in order to verify homogeneity/variation among/within groups. Males and females were randomised separately.

Environmental Conditions
Environmental parameters (temperature and relative humidity) were continuously monitored and the minimum and maximum values were recorded twice a day during the study and acclimation period. The actual ranges are given below.

Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: from 20.6 to 24.2℃ (target: 22 ± 3 ℃) Relative humidity: from 24 to 77% (target: 30 - 70%) Ventilation: 15-20 air exchanges/hour

Food
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany) during study, ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate for the batch used (batch number: 713 70882, expiry date: 30 April 2021 and batch number: 560 65984, expiry date: 31 October 2020).

Water
Animals received tap water from the municipal supply, as for human consumption from a 400- 500 mL bottle, ad libitum. The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Water quality control analysis was performed at least once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, Address: H-8200 Veszprém, József Attila utca 36., Hungary). The quality control results are retained in the Archives of the Test Facility, one certified copy is kept and archived in the raw data binder.

Route of administration:

oral: gavage

Details on route of administration:

Selection of dose: The dose levels were selected based on the available information of the chemical nature, characteristics of the test item, and available data and information from previous experimental work, including the results of an OECD 422 and its Dose Range Finding (DRF) study, with the aim of inducing toxic effects but no death or suffering at the highest dose (1000 mg/kg bw/day).
Preparation of formulations: The formulations were prepared fresh prior to administration to animals. No correction for purity of the test substance was applied during formulation.

Vehicle:

corn oil

Details on oral exposure:

Administration of Test and Control Items
Dose formulations were administered daily starting from Day 1 for 90 consecutive days by oral gavage, using a tipped gavage needle attached to a syringe. A constant dose volume of 5 mL/kg bw was administered to all animals/in all groups. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight. Control animals were treated concurrently with the vehicle only. Test Item formulations were mixed with magnetic stirrer during treatment.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Top, middle and bottom duplicate samples were taken from each test concentration: 20, 60 and 200 mg/mL
The concentration of the formulations was within the acceptance criteria of the solutions (100 ± 15% of the nominal concentration).
All formulations proved to be homogeneous. Acceptance criteria for homogeneity is that the relative standard deviation (RSD%) of the replicates must be less than 15%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily for 90 consecutive days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals/group/sex

Control animals:

yes, concurrent vehicle

Details on study design:

40 male and 40 female Wistar rats were treated once daily for 90 consecutive days by oral gavage administration. The first day of dosing of each animal was regarded as Day 1. Control animals received the vehicle only. Blood samples were collected before necropsy for Clinical Pathology and Thyroid hormone analysis. All animals underwent necropsy upon completion of the 90-day treatment period (Day 91).

Observations and examinations performed and frequency:

The examinations included clinical signs, mortality, body weights, food consumption, ophthalmoscopy, neurological assessment (including landing foot splay, grip strength and motor activity assessment), vaginal smears, clinical pathology (including haematology, coagulation, clinical chemistry, urinalysis, thyroid hormone analysis), gross pathology, sperm analysis, organ weights and histopathology. Full histopathology was performed in Group 1 (Control) and Group 4 (High dose).
-Mortality/Morbidity: Animals were inspected for signs of morbidity and mortality twice daily
-Clinical Observations: clinical sign noted during dosing or at any other occasions were recorded at the time seen
-Cage Side Observations: General (routine) clinical observations were made once daily after treatment
-Detailed Clinical Observations: Observations were made on all animals outside the home cage in a standard arena prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter
-Body Weights: Body weights were recorded at randomisation (pre-treatment period), on the first day of treatment (Day 1, prior to start of treatment), then weekly, including on Day 90. The growth curve was prepared graphically, body weight gain was calculated weekly and for the total duration of the study.
-Food Consumption: Observation of food consumption was performed for all groups weekly. Animal food consumption per cage was measured on a cage-basis and the average daily feed intake per rat was calculated on a weekly basis and for the study duration. Based on food consumption data, food conversion efficiency (g/g) was calculated as [weekly body weight gain (g)/weekly food consumption (g)].
-Ophthalmic Examinations: Ophthalmoscopic examination was conducted in all animals before treatment on Week -1 and last week of treatment (during Week 13) in the Control (Group 1) and High dose (Group 4) animals. Mydriasis were produced after instillation of “Cicloplegicedol” (10 mg/mL cyclopentolate hydrochloride) eye drops into the conjunctival sac. The evaluation was performed using an Omega 500 ophthalmoscope. No treatment related alterations were found in the Control and High dose group animals, the remaining animals were not examined at termination.
-Neurological Assessment (Functional Observational Battery and SMART): Towards the end of the treatment period, during Week 12-13, each animal was subjected to the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted, and the general physical condition and behaviour of animals was tested. A modified Irwin test was performed. A detailed assessment for neurotoxicity effects was made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968). Parameters such as, but not limited to body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated. To measure the landing foot splay, the fore and hind paws of the rat was painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. This was performed 3 times for each animal on each test day. The distance between the two resulting ink spots of the hind paws was measured. The fore paws of the rat were painted for any possible additional measurements. Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and pulled back until they release the bar; the device measures the maximum grip strength. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data. Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. The data from all groups was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.
-Examination of Vaginal Smears: Prior to the necropsy (Day 91), the oestrus cycle of all females was determined by taking vaginal smears, which was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.
- Organ weight: Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights was calculated and reported. The eyes with the optic nerve and the testes with epididymides were preserved in modified Davidson’s fixative; all other organs in 10% buffered formalin solution.

Sacrifice and pathology:

Method of Euthanasia: At study termination euthanasia was performed under pentobarbital anaesthesia by exsanguination.
Unscheduled Euthanasia: No unscheduled death was during the study
Scheduled Euthanasia: Necropsy and macroscopic examination was performed on all animals, at the end of treatment period, on Day 91 (after the sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia.

CLINICAL PATHOLOGY:
-Sample Collection: At the end of the treatment period on Day 91, clinical pathology investigations (haematology, coagulation, clinical biochemistry, urinalysis and thyroid hormone analysis) were conducted in all animals
-Thyroid Hormone Analysis (T3 and T4): T4 and T3 hormone measurement was done
-Thyroid Hormone Analysis (TSH): TSH level was analysed
-Histology and Microscopic Evaluation: Tissues and organs for histopathology evaluation were embedded in paraffin wax, sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Full histopathology was performed in Groups 1 (Control) and 4 (High dose). In addition, any organs or tissues with macroscopic abnormalities was subjected to histological examination from all groups.

Statistics:

Statistical evaluation of data was performed using the statistical program package of SAS 9.2 (when using Provantis). The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an ANOVA / ANCOVA (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test. For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control. For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related clinical signs were noted during study. The following clinical observations were made during the observation period of the study: alopecia in 2/10 Mid and 1/10 High dose males and in 1/10 Control and 1/10 Mid dose females, crust in 1/10 Mid dose male, scar in 1/10 Control, 2/10 Low dose, 2/10 Mid dose and 1/10 High dose males and in 1/10 Control and 1/10 Mid dose females, wound in 1/10 Control, 2/10 Low dose, 1/10 Mid dose and 1/10 High dose males and in 1/10 Control female, noisy respiration in 1/10 High dose female, red discharge in 1/10 High dose male and tonic convulsion in 2/10 Low dose, 1/10 Mid dose and 1/10 High dose males and in 1/10 Mid dose and 1/10 High dose females. None of these clinical signs were considered to be clearly test item related. Although the incidence of tonic convulsion was higher than the average study, the detailed neurotoxicology endpoints (Irwin test, foot splay, grip strength, quantitative locomotor activity) showed no evidence of effects, there was no dose response in the pattern of the data. Also there were no other clinical signs suggesting an effect and no clinical pathology, necropsy or histopathology indications that it could be possibly attributed to test item. Although convulsions were not seen in the concurrent controls, this finding is a known background clinical sign in Wistar rats. These findings were considered to be incidental and related to a few randomly distributed individual animals, not regarded as being related to treatment.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in the study at any dose level.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related effects were detected on bodyweight or bodyweight gain during the study in any of the treated groups when compared to the controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related differences in the mean food consumption in any of the treated groups when compared to the controls. Some sporadic statistical or numerical differences were considered to be random differences, unrelated to the treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related changes were observed in the haematology parameters. The observed sporadic statistically significant differences were considered to be incidental, were not related to dose and/or all recorded values were within the historical control ranges.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

An apparent, statistically significant increase in creatinine in Low and High dose males was noted, without a clear dose dependence. Furthermore, the control values in this study were higher than our historical control values and all treated groups were normal, therefore this statistical difference was not considered to be test item related.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant differences among groups in terminal body weights and the weights of organs measured when compared to controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related macroscopic findings were detected at necropsy. The lung showed dark red focus in 1/10 Mid dose and 1/10 High dose males. The mandibular lymph node showed dark red discoloration in 1/10 Low dose and 2/10 Mid dose males and in 1/10 Control, 1/10 Low dose and 3/10 High dose females. Enlargement of the mandibular lymph node was seen in 1/10 Control and 1/10 High dose males. Small seminal vesicles were noted in 1/10 Mid dose males. In the stomach, focal, dark red discoloration of the glandular mucosa in 1/10 Mid dose males and pale, multifocal discoloration of the glandular mucosa in 1/10 Mid dose females was recorded during necropsy. Thickness of the stomach was seen in 1/10 High dose females. Raised area in the non-glandular region of the stomach wall in 2/10 High dose males was also noted. The low incidence of the observations and the lack of histological change indicates the gastric findings were background pathology. Dark red, focal discoloration of thymus in 1/10 Mid dose males and small thymus in 2/10 High dose males was observed. Dilatation of the uterine body and horn in 3/10 Control, 2/10 Low dose, 1/10 Mid dose and 2/10 High dose females was recorded during the study. The above-mentioned macroscopic changes were not considered to be test item induced adverse effects.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related microscopic findings were detected during histopathology evaluation. All findings, without meaningful differences in severity and incidence through dosed and control groups were considered to be procedure related, incidental or a common background.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Neurological assessment (Functional observational battery and quantitative assessments)
There was no adverse effect of treatment noted during the assessment of grip strength, foot splay, Irwin Test or locomotor activity in any dose group when compared to the control. In the Mid and High dose males, an apparent dose-related decrease in grip strength of the fore limbs only was measured, however these values were within the normal historical control range, hind limb strength is the main parameter of importance for neural effects, and this decrease did not show statistical significance. All animal results were considered to be normal; these non-significant differences are not considered to be an adverse effect of the treatment. The locomotor activity was considered to have showed a normal response in the High group, it was initially high then reduced to a plateau at approximately 30 minutes. All locomotor activity data were considered as normal; therefore, the analysis of the Mid and Low dose group was considered to be not required.

Thyroid hormone
T3, T4 and TSH values were considered to be normal in all treated groups.

Examination of vaginal smears
There were no test item-related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

Sperm sample analysis
No statistically significant, treatment-related changes were observed in the sperm number, morphology or motility in any of the treated males. Higher sperm motility was observed in all treated dose groups and control values tended to be lower than our historical control data base. All treated groups had normal sperm motility.

Details on results:

Dose formulation analysis
All test item formulations were shown to be stable under the conditions of this study. The measured concentrations were all in the range of 99 to 107%. These results were within the acceptable ranges (85% - 115%) and were considered suitable for the study purposes.
All formulations proved to be homogeneous, meeting the acceptance criteria (relative standard deviation (RSD%) of the replicates <15%).

Discussion
No systemic toxicity was identified in any of the measured parameters in the animals or the clinical pathology data; sporadic differences between groups were all attributed to normal individual animal variation, with not relationship with the test item. The organ weights and macroscopic findings showed no differences that could be attributed to treatment; histopathology showed no effects in any treatment group.

Conclusion
In conclusion, under the conditions of this study, the no observed effect level for systemic toxicity (NOEL) for Di-Trimethylolpropane Tetraacrylate is considered to be 1000 mg/kg bw/day.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

serum/plasma biochemistry

sperm measures

urinalysis

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

For detailed study result tables, kindly refer to the attachment section.

Conclusions:

Under the study conditions, the test substance NOEL for repeated dose toxicity was determined to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance in Wistar rats, according to the OECD Test Guideline 408, in compliance with GLP. The test substance, formulated in corn oil, was administered daily at dose levels of 0, 100, 300 and 1000 mg/kg bw/day for at least 90 days by oral gavage to Wistar rats (10 animals/sex/group). The examinations included clinical signs, mortality, body weights, food consumption, ophthalmoscopy, neurological assessment (including landing foot splay, grip strength and motor activity assessment), vaginal smears, clinical pathology (including haematology, coagulation, clinical chemistry, urinalysis, thyroid hormone analysis), gross pathology, sperm analysis, organ weights and histopathology. Full histopathology was performed in Group 1 (Control) and Group 4 (High dose). There was no mortality in the study at any dose level. No treatment-related changes were noted in clinical signs, bodyweight, body weight gain, and food consumption of the treated groups. No treatment-related changes were notedin functional observation battery, ophthalmoscopy, clinical pathology and urinary parameters, oestrus cycle, T3, T4 and TSH values. No macroscopic, organ weight or microscopic findings were reported. No test substances related effected were reported on sperm number, sperm morphology and motility in any of the treated males or in any sexual organs. Under the study conditions, the test substance NOEL for the repeated dose toxicity was determined to be 1000 mg/kg bw/day (Krajcs, 2021).

Results and discussion

Target system / organ toxicity

Critical effects observed:

not specified