Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
Repeated Dose 28 Days Toxicity Testing swith additional testing on reproductive organs
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from a study report.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD No. 407
Principles of method if other than guideline:
The above study was performed to assess and evaluate the effects of the test chemical on reproductive organs of Sprague Dawley rats.
GLP compliance:
yes
Limit test:
no
Justification for study design:
No Data Available

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
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 %
Specific details on test material used for the study:
- 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
Details on species / strain selection:
No Data Available
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 exposure:
PREPARATION OF DOSING SOLUTIONS: Test chemical 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
Details on mating procedure:
Reproductive organ weight was observed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and concentration of the test chemical 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
Positive control:
No data available

Examinations

Parental animals: Observations and examinations:
Animals of all dose groups were observed for Clinical signs/ symptoms daily during the experimental period. Mortality and morbidity were observed and recorded twice a day Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for rats from main groups. Animals were weighed during randomization, at start of treatment and thereafter weekly, till the end of experimental period. Clinical pathology evaluation of all surviving rats from the main groups and recovery groups was performed just prior to necropsy. Hematological and clinical chemistry parameters were analyzed at the end of the treatment and recovery period. Urine samples were collected from all the survived rats of main and recovery group and urinary parameters were determined.
Oestrous cyclicity (parental animals):
No data available
Sperm parameters (parental animals):
No data available
Litter observations:
No data available
Postmortem examinations (parental animals):
Reproductive organ weights were recorded in all treated animals at 0, 125, 250 and 500mg/kg dose levels, gross pathology and histopathology were examined.
Postmortem examinations (offspring):
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.
Reproductive indices:
No data available
Offspring viability indices:
No data available

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period.
Detailed clinical examinations like Home cage observation, Handling observation and Open field observation all animals were observed normal during study period at 125, 250 and 500mg/kg dose groups
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No mortality and morbidity were observed among all the groups of animals throughout the study period
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In treated group with 500 mg/kg/day body weight, significant decreased in female rats were observed as compare to control.

No change in body weight were observed at 125 and 250 mg/kg/day in treated male and female rats as compare to control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No change was observed in food consumption of treated male and female rat at 125, 250 and 500mg/kg dose groups in treatment and recovery period as compare to control.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No abnormalities were observed in treated 125, 250 and 500 mg/kg male and female rat in treatment groups at and recovery period as compare to control.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Animalswhen 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 biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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, 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.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urine parameters did not show any significant difference among all the experimental animals of both the sexes and were comparable to animals of control group, except statistical significant increase observed in volume in (500 mg/kg body weight) in females as compared to control group the change were not attributed to the effect of test item administration.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Among all organs like testes, prostate with seminal vesicle, epididymis, ovaries with oviduct and uterus (g) and relative weights (%) were not statistically significant in both sexes compared to control group. Absolute and relative organ weight S.V. with coagulating gland and prostrate as whole was decreased in (250 mg/kg body weight) male as compared to control animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross necropsy of all male and female animals did not show any extermal and internal abnormal changes in any animal at 125, 250 and 500mg/kg.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
When treated 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. Significant decrease in absolute and relative organ weight of S.V. with coagulating gland and prostrate as whole was a incidental finding as histopathologically tissues were normal.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
other: Effect on reproductive organ weight, gross pathology and histopathology.

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: Not Spcified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

No Data Availab;e

Effect levels (F1)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Relative Organ Weight (%)

Sex: Male

Organs

 

Group 1

Group 2

Group 3

Group 4

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Brain

0.762

0.052

0.817

0.102

0.820

0.044

0.824

0.040

Adrenals

0.019

0.006

0.025

0.004

0.022

0.005

0.022

0.004

S.V. With Coagulating gland and prostrate

0.467

0.058

0.470

0.083

0.360↓

0.045

0.480

0.055

Testes

1.196

0.100

1.196

0.062

1.237

0.090

1.217

0.062

Epdidymides

0.356

0.046

0.420

0.037

0.401

0.058

0.413

0.026

Heart

0.416

0.041

0.425

0.056

0.415

0.016

0.395

0.025

Liver

4.155

0.252

4.933

0.358

5.417↑

0.275

5.850↑

0.690

Kidneys

0.915

0.105

0.949

0.046

1.071↑

0.053

1.051↑

0.087

Spleen

0.534

0.153

0.476

0.128

0.471

0.066

0.393

0.094

Thymus

0.120

0.032

0.132

0.032

0.125

0.034

0.104

0.023

Organs

Group 1-R

Group 4-R

Mean

SD

Mean

SD

Brain

0.710

0.063

0.687

0.023

Adrenals

0.021

0.004

0.023

0.003

S.V. With Coagulating gland and prostrate

0.572

0.184

0.535

0.115

Testes

1.181

0.133

0.974

0.345

Epdidymides

0.420

0.021

0.406

0.030

Heart

0.424

0.059

1.043

1.396

Liver

3.999

0.523

3.305

1.377

Kidneys

0.893

0.174

0.779

0.143

Spleen

0.367

0.036

0.297

0.082

Thymus

0.107

0.031

0.124

0.030

Sex: Female

Organs

Group 1

Group 2

Group 3

Group 4

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Brain

0.970

0.077

0.978

0.037

0.970

0.036

1.056

0.072

Adrenals

0.032

0.005

0.030

0.007

0.034

0.005

0.038

0.007

Ovary

0.0494

0.0049

0.0616

0.0189

0.0626

0.016

0.0536

0.0078

Uterus

0.392

0.081

0.373

0.062

0.313

0.081

0.325

0.061

Heart

0.463

0.071

0.419

0.015

0.410

0.038

0.428

0.050

Liver

4.443

0.280

4.107

0.341

4.342

0.342

4.954

0.298

Kidneys

0.855

0.061

0.815

0.044

0.824

0.046

0.936

0.111

Spleen

0.382

0.129

0.291

0.078

0.274

0.079

0.301

0.120

Thymus

0.162

0.026

0.153

0.046

0.131

0.016

0.143

0.026

Organs

Group 1-R

Group 4-R

Mean

SD

Mean

SD

Brain

0.913

0.053

0.956

0.043

Adrenals

0.040

0.005

0.048↑

0.004

Ovary

0.0837

0.0103

0.0901

0.0100

Uterus

0.395

0.151

0.385

0.068

Heart

0.431

0.033

0.415

0.012

Liver

3.786

0.379

3.929

0.313

Kidneys

0.859

0.060

0.809

0.069

Spleen

0.395

0.066

0.234↓

0.027

Thymus

0.144

0.036

0.170

0.025

N = Number of animals in the group, SD = Standard deviation,↓= statisticalsignificant decrease at 95% level of significance (p > 0.05),↑= statisticalsignificant increase at 95% level of significance (p < 0.05).

Applicant's summary and conclusion

Conclusions:
The NOAEL was considered to be 500 mg/kg/day body weight when male and female rat were exposed to test chemical for 28 days.
Executive summary:

In the present repeated dose toxicity study for 28 days, male and female Wistar rats in group of 5/sex were exposed to test chemical orally in the concentration of 0, 125, 250 and 500 mg/kg/day body weight. Recovery groups were dosed with 0 and 500mg/kg of test chemical. The animals were observed for clinical signs of toxicity; mortality and morbidity; feed consumption, haematological and clinical chemistry parameter analysis; urinalysis; and at the end of the study necropsy were performed in alll survived animals and change in organ weight, gross pathology and histopathology examination were performed. The results of the observed paramters revealed, No mortality and morbidity were observed among all the groups of animals throughout the study period; No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period. Detailed clinical examinations like Home cage observation, handling observation and open field observation all animals were observed normal during study period at 125, 250 and 500mg/kg dose groups; No change in body weight were seen in animals of 125 and 250mg/kg doses howver 500mg/kg group females shows significant decreased in body weight compared to control animals. No change in food consumption and opthalmological examination in treated male and female rat at 125, 250 and 500mg/kg dose groups in treatment and recovery period were observed. The haematological examination revealed statistically significant increase neutrophil count in 500 mg/kg in female rats; 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. The clinical biochemistry paramters changes were observed. 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, 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. Urine parameters did not show any significant difference, except statistical significant increase observed in volume in (500 mg/kg body weight) in females as compared to control group the change were not attributed to the effect of test item administration. The organ weight study revealed, not statistically significant in both sexes compared to control group among all organs like  testes, prostate with seminal vesicle, epididymis, ovaries with oviduct and uterus (g) and relative weights (%). However, absolute and relative organ weight S.V. with coagulating gland and prostrate as whole was decreased in (250 mg/kg body weight) male as compared to control animals. No external and internal gross pathological changes were seen at 125, 250 and 500 mg/kg body weight. In histopatholgy study, When treated 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. S.V. with coagulating gland and prostrate as whole was a incidental finding as histopathologically tissues were normal. Therefore, NOAEL was considered to be 500 mg/kg/day body weight when rats are exposed to test chemical orally for 28 days.