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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Experimental result using OECD recommended guidelines
Justification for type of information:
Data is from study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
Repeated Dose 28 days Study of the test chemical in rats.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Solid
Details on test material:
- Name of test material (as cited in study report): N-tert-butylacrylamide
- Molecular formula (if other than submission substance): C7H13NO
- Molecular weight (if other than submission substance): 127.19 g/mole
- Substance type: Organic
- Physical state: Solid
- Impurities (identity and concentrations): 0.29 %

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-House Bred at sa-FORD, Animal Facility
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: Male : 137-191 g, Female: 137- 165 g
- Fasting period before study: No data available
- Housing: Animals were housed in Polycarbonate cages. Cage rotation was carried out weekly and cleaned at regular intervals. Animals were bedded on sterilized corn cob produced from pure corn, dried and free from dust. Floor of the experimental room and work tops were swept and mopped with disinfectant solution every day or as on requirement.
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet, ad libitum.
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles, ad libitum.
- Acclimation period: Male: 6 days, Female: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.00 to 23.10 °C
- Humidity (%): 49.90 to 69.40%.
- Air changes (per hr): Adequately filtered air 12 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark artificial light.

IN-LIFE DATES: From: September 22, 2014
To: October 20, 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Substance was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was used as a vehicle based on the solubility testing.
- Concentration in vehicle: 0,125,250 and 500 mg/kg/day
- Amount of vehicle (if gavage): 1 ml/100g body weight
- Lot/batch no. (if required): MKBQ9948V and MKBG9426V
- Purity: No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and concentration of the substance in dose formulation was analysed by a validated analytical method, at the start of treatment (on day 1) and on day 21.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: mg/kg/day
Dose / conc.:
125 other: mg/kg/day
Dose / conc.:
250 other: mg/kg/day
Dose / conc.:
500 other: mg/kg/day
No. of animals per sex per dose:
Total : 60
0 mg/kg/day: 5 male, 5 female
125 mg/kg/day: 5 male, 5 female
250 mg/kg/day: 5 male, 5 female
500 mg/kg/day: 5 male, 5 female

Recovery:
0 mg/kg/day: 5 male, 5 female
500 mg/kg/day: 5 male, 5 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the information provided by Sponsor.
- Rationale for animal assignment (if not random): No data available
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (morning and evening)
- Cage side observations checked in table [No.?] were included: Morbidity and mortality were observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first treatment and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: At start of treatment and thereafter weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY: No data available
- 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 data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available
- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to treatment (During Acclimatization Period) and at the end of the dosing (main groups) and recovery periods (recovery group) were examined.
- Dose groups that were examined: All 60 animals were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On termination day, just prior to necropsy.
- Anaesthetic used for blood collection: No data available
- Animals fasted: Yes, overnight (approximately 16-18 hr).
- How many animals: All 60 animals were examined.
- Parameters checked in table [No.?] were examined: Total Erythrocyte Count (RBC), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Hemoglobin (HGB), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), Total Leukocyte count (WBC), Prothombin Time (PT) and Activated Partial Thromboplastin time (aPTT) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On termination day, just prior to necropsy.
- Animals fasted: Yes, overnight (approximately 16-18 hr).
- How many animals: All 60 animals were examined.
- Parameters checked in table [No.?] were examined: Glucose (Glu), Cholesterol (Chol), Triglycerides (TRIG), Alanine amino transferase (ALT), Aspartate amino transferase (AST), Gamma-Glutamyl Transpeptidase (GGT), Calcium, Creatine Kinase (CK), Albumin, Total Protein (TP), Creatinine (Crea), Total Bilirubin (T.Bil), Phosphorus, Alkaline phosphatase (ALP), Urea, Lactate Dehydrogenase (LD), Sodium (Na), Potassium (K), Chloride (Cl), Blood urea nitrogen (BUN), Globulin (Glob), A/G and Bile acids were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined: Colour, appearance, urine volume, Blood / Blood Cell, Bilirubin, Urobilinogen, Ketone, Protein, Nitrite, Glucose, pH, Specific Gravity, Leucocytes, Microscopical Parameters were examined.

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Yes, Sensory reactivity to stimuli, grip strength, hind limb foot splay and motor activity were tested.

OTHER:
Organ weight: Absolute and relative organ weights were examined.

Organ examined: Liver, kidneys, adrenals, testes, epdidymides, Prostate and Seminal vesicle with coagulating glands, uterus with cervix, ovary with oviduct, thymus, spleen, brain and heart were examined.
Bone Marrow Smear were also examined
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were fasted overnight before necropsy. Animals were sacrificed by using over dose of CO2 and examined externally and macroscopically.

HISTOPATHOLOGY: Yes
All tissues were preserved in 10 % neutral buffered formalin (except eyes and testes; which were fixed using Modified Davidson fluid for 24 hr and then transferred to 10 % neutral buffered formalin (NBF) for preservation) for subsequent histopathological examintion.

Organ examined:
Adrenals, Bone (femur) with joint, Brain (cerebrum,cerebellum,mid brain), Cecum, Colon, Duodenum, Epididymides, Eyes with optic nerve, Gross lesion, Ileum, Jejunum, Kidneys, Liver, Lung, Mammary glands, Mesenteric and Mandibular lymph node, Oesophagus and Ovary with oviduct were examined.
Other examinations:
No data available
Statistics:
Statistical analysis of raw data was performed by using statistical software Sigma Plot 11.0. The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, haematology, clinical chemistry, absolute and relative organ weights) were checked for their homogeneity using Bartlett’s test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
Mortality:
No mortality and morbidity was observed in treated male and female rat as compare to control.

Clinical signs:
No clinical sign were observed in treatment and recovery dosed male and female rat as compare to control.

Body weight and weight gain: When treated with 500 mg/kg/day body weight, in female rat statistically significant decreased in body weight gain were observed as compare to control.

No change was observed in 125 and 250 mg/kg/day body weight treated male and female rats as compare to control.

Food consumption and compound intake: No change was observed in food consumption of treated male and female rat in treatment and recovery period as compare to control.

Food efficiency: No data available

Water consumption and compound intake: No data available

Opthalmoscopic examination: No abnormalities were observed in treated male and female rat in treatment and recovery period as compare to control.

Haematology: When treated with 500 mg/kg/day body weight, in female rat statistically significant increase neutrophil were observed.

In recovery, statistically significant decrease was observed in MCV and WBC and increase observed in PLT in females and statistically significant decrease in APTT in male rat as compare to control.

Above changes were not related to the test substence and may due to the preanalytical and analytical variables.

Clinical chemistry: When treated with 125, 250 and 500 mg/kg/day body weight, in male rat statistically significant increase were observed in Sodium (Na) and decrease in Lactate Dehydrogenase (LD) and Creatine Kinase (CK) when treated with 125 and 500 mg/kg/day body weight.

When treated with 250 and 500 mg/kg/day body weight, Statistically significant increase was observed in Alkaline phosphatase (ALP) in male rat.

When treated with 500 mg/kg/day body weight, Alanine amino transferase (ALT) increased in male rat as compare to control.

In recovery, Statistically significant increase were observed in Blood urea nitrogen (BUN), Urea, Albumin (ALB), Alkaline phosphatase (ALP), Potassium (K), Chloride (Cl) and Bile acids and statistically significant decrease in Cholesterol (Chol) in male rat and Sodium (Na) in female rat as compare to control.

Urinanalysis: No change was observed in urine analysis of treated animals in treatment and recovery period as compare to control.

Neurobehaviour: No data available

Organ weights: In male rat, relative weight of liver were significantly increase in 250 and 500 mg/kg/day body weight.

Relative organ weight of kidney showed statistically significant increase when treated with in 250 and 500 mg/kg/day body weight.

Absolute and relative organ weight of S.V. with coagulating gland and prostrate as whole was decreased in 250 mg/kg/day body weight in male rat as compare to control.

In female absolute weight of liver showed significantly decrease in 125 mg/kg/day body weight.

In recovery, in female absolute and relative organ weight of Adrenals was increased and absolute organ weight of kidney was decreased when treated with 500 mg/kg/day body weight and absolute and relative organ weight of spleen was decreased in 250 mg/kg/day body weight.

In male, significant difference were observed in absolute & relative weight of liver in male rat when treated with 250 and 500 mg/kg/day body weight.

Gross pathology: No external and internal abnormal changes were observed in treated male and female rats in treatment and recovery period as compare to control.

Histopathology: When treaed with 500 mg/kg/day body weight, hyperplasia of BALT in Lung and MNC infiltration in trachea were observed in male and female this effect was reversed in 14 days recovery period.

These observed changes are inflammatory changes and are the most common observed changes in rodents.

Effect levels

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effect on survival, body weight, food consumption, battery test, hematology, clinical chemistry, organ weight, gross pathology and histopathology.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mortality and Morbidity

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Day of Observations

Observation

G1

Control

0

5

1-29

NMM

G2

Low

125

5

1-29

NMM

G3

Mid

250

5

1-29

NMM

G4

High

500

5

1-29

NMM

G1-R

Control -Recovery

0

5

1-43

NMM

G4-R

High- Recovery

500

5

1-43

NMM

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Day of Observations

Observation

G1

Control

0

5

1-29

NMM

G2

Low

125

5

1-29

NMM

G3

Mid

250

5

1-29

NMM

G4

High

500

5

1-29

NMM

G1-R

Control -Recovery

0

5

1-43

NMM

G4-R

High- Recovery

500

5

1-43

NMM

Key:NMM = No mortality and morbidity observed.

Mean Absolute Organ Weight (g)

Sex:Male

Organs

 

Group 1

Group 2

Group 3

Group 4

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Body weight (g)

251.80

21.09

239.20

24.78

240.60

18.37

234.60

11.48

Brain

1.910

0.062

1.935

0.053

1.966

0.051

1.900

0.057

Adrenals

0.046

0.013

0.059

0.007

0.053

0.009

0.051

0.010

S.V. With Coagulating gland and prostrate

1.1831

0.22882

1.11382

0.167243

0.8658

0.1214

1.1246

0.12127

Testes

3.003

0.246

2.857

0.296

2.966

0.167

2.810

0.150

Epdidymides

0.902

0.164

1.004

0.135

0.958

0.106

0.954

0.045

Heart

1.044

0.088

1.007

0.078

0.998

0.069

0.911

0.043

Liver

10.450

0.919

11.804

1.553

13.062

1.580

13.492

1.428

Kidneys

2.294

0.219

2.271

0.261

2.575

0.202

2.427

0.192

Spleen

1.356

0.434

1.142

0.332

1.136

0.196

0.905

0.198

Thymus

0.301

0.071

0.311

0.044

0.299

0.071

0.241

0.056

Organs

 

Group 1-R

Group 4-R

Mean

SD

Mean

SD

Body weight (g)

273.40

20.21

280.80

15.39

Brain

1.935

0.120

1.927

0.067

Adrenals

0.056

0.009

0.064

0.008

S.V. With Coagulating gland and prostrate

1.5541

0.4754

1.4925

0.260007

Testes

3.210

0.166

3.168

0.254

Epdidymides

1.145

0.063

1.112

0.153

Heart

1.148

0.080

1.175

0.125

Liver

10.861

0.878

10.788

0.658

Kidneys

2.417

0.304

2.368

0.199

Spleen

1.002

0.106

1.048

0.285

Thymus

0.296

0.106

0.349

0.090

Sex:Female

Organs

Group 1

Group 2

Group 3

Group 4

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Body weight (g)

185.40

15.13

173.00

4.00

178.40

7.37

169.60

5.41

Brain

1.791

0.076

1.692

0.091

1.728

0.040

1.789

0.087

Adrenals

0.059

0.011

0.051

0.011

0.061

0.011

0.064

0.012

Ovary

0.092

0.012

0.106

0.032

0.111

0.024

0.091

0.015

Uterus

0.720

0.120

0.645

0.107

0.561

0.152

0.553

0.115

Heart

0.853

0.118

0.725

0.027

0.731

0.063

0.725

0.079

Liver

8.231

0.754

7.106

0.640

7.743

0.646

8.403

0.586

Kidneys

1.582

0.132

1.412

0.098

1.470

0.097

1.585

0.177

Spleen

0.719

0.279

0.502

0.122

0.485

0.120

0.516

0.225

Thymus

0.300

0.049

0.266

0.081

0.235

0.036

0.242

0.042

Organs

Group 1-R

Group 4-R

Mean

SD

Mean

SD

Body weight (g)

196.60

9.96

188.40

8.50

Brain

1.792

0.087

1.800

0.059

Adrenals

0.077

0.007

0.090

0.006

Ovary

0.164

0.015

0.170

0.018

Uterus

0.777

0.305

0.723

0.120

Heart

0.846

0.054

0.782

0.045

Liver

7.429

0.677

7.394

0.560

Kidneys

1.684

0.058

1.522

0.110

Spleen

0.776

0.126

0.441

0.054

Thymus

0.281

0.062

0.320

0.037

Applicant's summary and conclusion

Conclusions:
The No observed adverse effect level (NOAEL) was considered to be 500 mg/kg/day body weight when male and female rat were exposed to the test chemical.
Executive summary:

In a repeated dose toxicity study, male and female Wistar rats were exposed to the test chemical orally in the concentration of 0, 125, 250 and 500 mg/kg/day body weight. No substance related toxic changes were obsrved in survival, body weight, food consumption,hematology, clinical chemistry,battery test and urinalysis of treated rats. Significant change in liver, kidney,Adrenals and spleen of 250 and 500 mg/kg bw/day treated rats as compared to control. In addition, no gross pathological and histopathological changes were observed in male and female rats. Some changes were observed but they are not test substance related.Therefore, No observed adverse effect level (NOAEL) was considered to be 500 mg/kg/day body weight when rats are exposed to the test chemical orally for 28 days.