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REACH

N-tert-butylacrylamide

EC number: 203-505-6 | CAS number: 107-58-4

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

Repeated dose toxicity

OECD 407:

NOAEL in male rats was considered at 500 mg/kg. The observed effects in female rats treated at 500 mg/kg in the main study included significant decreases in body weight gain and indications of minor kidney toxicity. These effects were considered to be reversible based on lack of similar effects in the recovery groups of female rats treated at 500 mg/kg. The observed effects in female rats treated at 500 mg/kg in the main study were considered adverse and only reversible upon cessation of treatment. NOAEL in female rats was therefore considered at 250 mg/kg.

OECD 408:

NOAEL in male rats was considered to be 120 mg/kg bw/day, based on clinical signs of toxicity (lethargy) and significant decreases in body weight and body weight gain at 450 mg/kg bw/day. NOAEL in female rats was considered to be 450 mg/kg bw/day, as no adverse effects were observed at this dose level in the female rats.

Repeated dose toxicity- Inhalation route

According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the particle size distribution of N-tert-butylacrylamide and that the exposure via the inhalation route is unlikely, this end point was considered for waiver.

Repeated dose toxicity- Dermal route

The acute toxicity value for N-tert-butylacrylamide (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Hence this end point was considered for waiver.

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2022 - Jan 2023

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CPCSEA approved animals (under license from Charles River Labs) from Hylasco Biotechnology (India) Pvt. Ltd.
Hyderabad, 500 078
- Females (if applicable) nulliparous and non-pregnant: nulliparous & non pregnant
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 179.59 – 229.62 g (Males) and 132.12 – 164.82 g (Females)
- Housing: Animals of the same sex and group was housed in a maximum of three per cage in standard polycarbonate cages (size: 412mm x 290mm x 190mm). The cages were fitted with stainless steel mesh top grill with facilities for holding pelleted food and drinking water bottle.
- Diet (e.g. ad libitum): The pellet feed (Purina Lab Diet 5L79 RAT AND MOUSE 18% (Lot No.25MAR20223) manufactured by PMI Nutrition International) was available ad libitum throughout acclimatization and experimental period. Feed sample were analysed and determined to comply with the requirement of FSSC 22000.
- Water (e.g. ad libitum): Reverse osmosis water was available ad libitum throughout the study period. The test results of collected water samples met acceptance criteria as per IS 10500:2012 for drinking water.
- Acclimation period: The male animals were acclimatized for seven days and female animals were acclimatized for eight days to laboratory conditions and was observed for clinical signs once daily. Veterinary examination of all the animals was performed during acclimatization. Ophthalmological examination of all animals was performed during acclimatization.
- Bedding material: Autoclaved corncob (Lot No.13 and 14) was used as bedding material and was changed along with the cage at least once a week. The material was manufactured by Rowan Agro Nature Pvt Ltd. The corncob samples passed the requirement of the Food Safety Standard Regulation (2011) with respected to tested parameters.

ENVIRONMENTAL CONDITIONS: Animals was housed in an environmentally monitored air-conditioned room maintained at a temperature of 19.6 to 23.9°C, relative humidity of 46 to 71 % and with 12 hours light and 12 hours dark cycle. Temperature and humidity was monitored round the clock and recorded once daily.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC in Milli-Q water.

Details on oral exposure:

The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight. The volume of formulation administered was adjusted based on the recent body weight of the individual rat.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation analysis was performed by the Analytical chemistry department at Vipragen Biosciences Private Limited. The stability of the test item was analyzed in a separate study by HPLC (Study No.: VBPL-ANL-168-G-U-2022) before the start of treatment. The samples were collected in duplicates (5 mL each) from the top, middle and bottom layers from all dose concentrations, covered with aluminium foil and stored at room temperature (bench top) for 6 hours. Further, the prepared formulation samples were covered with aluminium foil and stored at 2 to 8 °C for 24 hours, 72 hours and 8 days. An aliquot was taken at post-storage for each dose concentration and analyzed for stability evaluation. Results of all dose concentrations indicated that the test item formulations in 0.5% CMC in Milli-Q water were stable for 6 hours under room temperature, and for 8 days at 2 to 8 °C. Sampling and analysis of formulations were performed during week 1, week 7 and week 13 of the treatment to determine the homogeneity and concentration using HPLC. The samples were collected in duplicates (5 mL each) from the top, middle and bottom layers from all dose concentrations, and in duplicates (5 mL each) from the middle layer from vehicle control. Results of all dose concentrations were found to be acceptable as the overall mean concentrations were within ±15% (from 95.13% to 99.29%) of the nominal concentrations, and the relative standard deviation (RSD) of top, middle and bottom layers were less than 10% (from 0.21% to 1.3%).

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 (Vehicle control)

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

Healthy rats were weighed and grouped before the initiation of treatment and allocated to their respective treatment groups using a body weight based stratified randomization method using MS Excel spreadsheets. Mean body weights of each group before the start of the treatment did not exceed ±20% of the mean body weight in each sex and group. Body weights of the animals was analyzed statistically to rule out the statistically significant differences between groups of same sex.

Positive control:

None

Observations and examinations performed and frequency:

Mortality and morbidity:
The animals were observed for morbidity and mortality twice a day. On the weekends and public holidays, the animals were observed at least once a day.

Clinical signs:
All animals were observed for clinical signs of toxicity during the treatment period. General clinical signs of toxicity were carried out once per day throughout the treatment period. Detailed clinical signs of toxicity were carried out before dosing (during acclimatization) and once a week thereafter. These observations were made outside the home cage, in a standard arena and at similar times on each occasion. Care was taken to ensure that variations during observations are minimal. Observation included, but not be limited to, changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, and 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 behaviour (e.g., self-mutilation, walking backwards).

Body weight:
Body weights were recorded on day 1 of treatment, once a week thereafter, on the day before fasting, and on the day after fasting (before terminal sacrifice).

Food intake:
Leftover feed was recorded once a week (coinciding with body weight recordings) and feed input was weighed and recorded based on consumption requirements. Feed consumed was calculated and presented as feed consumption per rat per day (g/rat/day).

Functional parameters:
The animals were subjected to functional observatory battery (FOB) examinations during the last week of treatment. The FOB included the following:
a) Home Cage Observations
Each rat was observed in the home cage for activity level and scored.
b) Handheld Observations
Observations were made while holding the subject animal. Animals were observed for lacrimation and salivation, hair coat characteristics (color/staining, alopecia, and piloerection), degree of eyelid closure, ocular abnormalities and muscle tone or mass. Each observation was recorded using a scoring/ranking system.
c) Open Field Observation
Each rat was placed in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each rat. During this observation period, each rat was evaluated as it moved freely/unperturbed, and the following parameters were recorded using a scoring/ranking system:
• Activity (locomotion).
• Rearings for a standard time.
• Observation of reactivity and arousal.
• Observation of gait and postural characteristics.
• Observation of involuntary/abnormal motor movements (tremors, twitches, clonic
convulsions, tonic convulsions, Stereotypic behavior and bizarre behavior).
d) Sensory Reactivity Measurements
While the rat was in the open field arena and after two minutes of observation, the following sensory reactivity measurements were performed and the observations were recorded using a scoring/ranking system:
• Approach response
• Touch response
• Click response
• Tail-pinch response
• Pupil response
• Aerial righting reflex
e) Landing Hind limbs Foot splay
The landing hind limbs foot splay was performed by dropping each rat on a horizontal surface (e.g., table surface) from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were gently pressed to an ink pad just prior to testing. The rat was dropped from a height of approximately 30 cm on to a recording paper sheet, which had the details viz., Study No., animal No. and group. A clean recording paper sheet was used for each rat. A total of 3 readings was recorded for each rat and the average of three individual foot splay values were presented in the report.
f) Grip Strength Performance
Forelimbs and hind limbs grip strength performance was measured using a grip strength meter (Orchid Scientific & Innovative India Pvt. Ltd; Model: GSM 02RS). Three trials for both fore and hind limb will be conducted for each rat. The average value (forelimb and hind limb, respectively) was calculated and presented in the report.
g) Motor Activity
Locomotor activity was tested for 5 minutes using an activity measuring system (Orchid Scientific & Innovative India Pvt. Ltd; Model: ACT01). The displayed values for each rat were documented in the raw data and presented in the report.

Ophthalmic examinations:
Ophthalmic examination were performed in all animals of G1, G2, G3, and G4 before initiation of treatment and in all animals of G1 and G4 during the last week of treatment, prior to blood collection for clinical pathology. Ophthalmic examinations were carried using a 3.5v Coaxial Ophthalmoscope (Welch Allyn Inc., USA). Before examination, mydriasis was induced using a 1% solution of Tropicamide.

Serum hormone levels:
Total serum levels of T4, T3, and TSH, respectively, were measured on samples obtained from each animal at study termination on day 91. For this, separate blood samples were collected in a plain tube and serum was collected after centrifugation. Aliquots of serum samples were stored at -20˚C or lesser until measurement. Serum thyroid hormone levels were measured using a commercially available assay kit.

Urinalysis:
At the last week of treatment, all rats were housed in metabolic cages overnight (reverse osmosis water available ad libitum without feed) and urine samples were collected early in the morning before blood collection. Color, appearance and volume of the urine were recorded after visual observation. The other parameters were analyzed using a Cobas u411 Urine Analyzer, Roche, USA.

Table 1. Urine Parameters
Sr. No. Parameters Unit
1 Appearance -
2 Volume mL
3 Specific gravity -
4 pH -
5 Leucocytes (WBC) Cells/µL
6 Protein mg/dL
7 Glucose -
8 Erythrocytes (RBC) Cells/µL

Sacrifice and pathology:

CLINICAL LABORATORY INVESTIGATIONS:
At the end of the experiment period, blood and urine samples were collected from all surviving animals. All animals were placed individually in metabolic cages and fasted overnight for approximately 16 hours with access to water ad libitum. Urine samples were collected early in the morning. Blood samples were collected early in the day to reduce biological variation caused by circadian rhythms. Blood samples were collected from the retro-orbital plexus using micro glass capillary tube under isoflurane anaesthesia. Samples were collected for hematology, coagulation assessment and clinical chemistry. For hematology and coagulation assessment, blood samples were collected in tubes containing 10% dipotassium ethylene diamine tetra acetic acid (K2-EDTA) and sodium citrate, respectively. For clinical chemistry, blood samples were collected in a plain tube and serum was collected after centrifugation.

HEMATOLOGY
The following haematological parameters were analyzed at the end of the experimental period by using a SCIL Vet ABC hematology analyzer, Horiba Medical ABX S.A., USA.

Table 2. Hematology Parameters
Sr. No. Parameters Unit
1 Total Leukocyte Count (WBC) 103 cells/mm3
2 Erythrocyte Count (RBC) 106 cells/mm3
3 Hemoglobin (HGB) g/dL
4 Hematocrit (HCT) %
5 Platelet Count 103 cells/mm3
6 Mean corpuscular volume (MCV) µm3
7 Mean Corpuscular Hemoglobin (MCH) pg
8 Mean Corpuscular Hemoglobin Concentration (MCHC) g/dL
9 Differential Leucocytes Count (DLC) %
10 Reticulocytes Counts %

Reticulocytes and the differential leucocyte counts were carried out manually. Blood smear was prepared by using standard techniques and stained with Giemsa stain (For DLC) and Reticulocyte diluting fluid (for reticulocyte). The cell count will be expressed as %. A total of 100 cells will be counted per blood smear and the following cells count will be recorded: Lymphocytes (LYM), Monocytes (MOM), Neutrophils (NEU), Eosinophils (EOS), and Basophils (BAS).

COAGULATION PARAMETERS
Blood samples collected at the end of the experimental period from all animals were subjected to coagulation analysis. Blood samples were centrifuged for 10 minutes at 2500 rpm for the isolation of plasma by using Stago Max coagulation analyser (Diagnostica stago, 92600 Asnieres, France).

Table 3. Coagulation Parameters
Sr. No. Parameters Unit
1 Prothrombin Time (PT) Seconds
2 Activated Partial Thromboplastin Time (APTT) Seconds

CLINICAL CHEMISTRY
The following clinical chemistry parameters were measured at the end of the experiment period for all surviving animals by using a Randox Daytona Plus, London, UK. Electrolytes were analyzed by using a 9180 Electrolyte Analyzer, Roche, USA.

Table 4. Clinical Chemistry Parameters
Sr. No. Parameters Unit
1 Total Protein (T.Pro) g/L
2 Albumin (ALB) g/L
3 Alanine Aminotransferase (ALT) U/L
4 Aspartate Aminotransferase (AST) U/L
5 Alkaline Phosphatase (ALP) U/L
6 Total Bile acids (TBA) µmol/L
7 Globulin – Calculative (GLOB) g/L
8 Glucose (GLU) mg/dL
9 Gamma Glutamyl Transpeptidase (GGT) U/L
10 High Density Lipoprotein (HDL) mg/dL
11 Low Density Lipoprotein (LDL) mg/dL
12 Total Cholesterol (TCHO) mg/dL
13 Creatinine (CRE) mg/dL
14 Serum Urea (URE) mg/dL
15 Total bilirubin (TBIL) mg/dL
16 Triglycerides (TRIG) mg/dL
17 Blood Urea Nitrogen (BUN) – Calculative mg/dL
18 Calcium (Ca) mg/dL
19 Phosphorous (I.Phos) mg/dL
20 Chloride (Cl) mmol/L
21 Sodium (Na+) mmol/L
22 Potassium (K+) mmol/L

PATHOLOGY
All animals were subjected to detailed pathological examination at the end of the experiment period. Vaginal smears were examined from all females on the day of necropsy to determine the stage of estrous cycle and to correlate that data with the histopathological findings.

NECROPSY AND GROSS PATHOLOGY
At the end of the observation period, all the surviving animals were euthanized by exsanguination under CO2 asphyxiation. All animals in the study were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

ORGAN WEIGHTS
On the completion of the gross pathological examination, the tissues and organs noted in the following table were collected and weighed from all the rats. 10 % Neutral Buffered Formalin (NBF) was used for fixation.

Table 6. List of Organs for Weighing
Sr. No. Tissue
1 Liver
2 Kidneys
3 Adrenals
4 Spleen
5 Heart
6 Thymus
7 Brain
8 Testes/ Ovaries
9 Epididymides/Uterus
10 Prostate + seminal vesicles with coagulating glands
11 Thyroid gland (after fixation)
12 Pituitary gland (after fixation)

Note 1: Paired organs were weighed together.
Note 2: Tissues were trimmed off for any adherent tissue and their wet weight was taken as soon as possible after dissection to avoid drying.

TISSUE COLLECTION AND PRESERVATION
The following tissues were collected from all the surviving animals and preserved in 10% neutral buffered formalin.

Table 7. List of Organs for Histopathology
Sr. No. Tissue
1 Adrenal glands
2 Aorta
3 Axillary/neck lymph nodes
4 Bone marrow smear (femur)
5 Brain (cerebrum, cerebellum, medulla/pons)
6 Cecum
7 Colon
8 Duodenum
9 Epididymides
10 Oesophagus
11 Eyes (with optic nerve) a
12 Femur bone with bone marrow along the joint
13 Gross lesions
14 Heart
15 Ileum with Peyer’s Patch
16 Jejunum
17 Kidneys
18 Liver
19 Lungs b
20 Mammary gland (male and female)
21 Mesenteric lymph nodes
22 Nerve, sciatic
23 Ovaries
24 Pancreas
25 Pituitary gland
26 Prostate + seminal vesicles with coagulating glands
27 Rectum
28 Salivary gland
29 Skeletal muscle
30 Skin
31 Spinal cord (cervical, thoracic and lumbar)
32 Spleen
33 Stomach
34 Testes a
35 Thymus
36 Thyroid with Parathyroids
37 Trachea
38 Urinary bladder b
39 Uterus with cervix
40 Vagina
Key: a = were collected in modified Davidson’s fluid; b= infused with formalin at necropsy.

HISTOPATHOLOGY
The histopathological examination was carried out on the preserved organs/tissues of vehicle control (G1) and high dose group (G4) rats. In addition, all gross lesions were examined microscopically. As no test item related histopathological changes were observed in high dose groups (G4), histopathological examinations were not performed for lower dose groups (i.e. G2 and G3). The tissues were processed for routine paraffin embedding and tissue sections were stained with Mayer’s Haematoxylin and Eosin stain. In addition, testes were sectioned at 4 micron and stained with PAS (Periodic acid–Schiff) reagent and Haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.

Statistics:

The following statistical methods were used to analyze the body weight, feed consumption, organ weights as well as clinical pathology data.
• Data was summarized in tabular form. Statistical analysis was performed using Graphpad Prism (Version 8.1.2).
• All the data was checked for normality and homogeneity before the comparisons.
• Data for each group of animals were subjected to parametric tests such as one way ANOVA (analysis of variance) along with multiple comparisons. Non normal/non homogenous data was subjected to non-parametric tests. Values were given as mean ± standard deviation (SD).
• All analyses and comparisons were evaluated at the 5% (P ≤ 0.05) level.
• All the statistically significant values were indicated with * asterisk symbol in the respective summary tables.

Clinical signs:

effects observed, treatment-related