Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-acetylhexanelactam
EC Number:
217-565-6
EC Name:
N-acetylhexanelactam
Cas Number:
1888-91-1
Molecular formula:
C8H13NO2
IUPAC Name:
N-acetylhexanelactam
Test material form:
liquid
Details on test material:
Batch: not yet known
Specific details on test material used for the study:

- Batch No.of test material: ESD0024613
- Expiration date of the batch: After 06 May 2018
- Purity as per Certificate of analysis: 98.10GC area%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +29°C)
- Stability under test conditions: The stability and homogeneity of the test item in the vehicle was established at 1 and 200 mg/mL under Advinus Study No. G13342. Based on the results, the test item was stable and re-suspendible in the vehicle up to 4 days when stored at room temperature.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 6 weeks
- Weight at study initiation:
Males Females
G1: 175.230 ± 10.432 142.490 ± 7.145
G2: 179.608 ± 6.056 140.840 ± 4.741
G3: 177.548 ± 9.087 139.160 ± 7.063
G4: 173.876 ± 8.971 140.476 ± 3.549
G1R:175.692 ± 8.334 140.324 ± 5.973
G4R:178.354 ± 9.549 140.982 ± 7.724

- Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in each group and sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week.
- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet – Pellet (Certified) manufactured by Envigo, P.O.Box 44220, Madison WI 53744-4220, was provided ad libitum to the rats.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: 27 May 2017 to 31 May 2017


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 24°C
- Humidity (%): 65 to 68%
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 01 June 2017 To: 28 June 2017

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Route of test item administration was through oral gavage. The oral route was chosen because it will provide an exaggerated model of the normal exposure in humans.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Required quantities of the test item was weighed in to a pre-calibrated beaker and the vehicle (Corn oil) was added up to the pre-mark and mixed using glass rod. The final concentration achieved was 20, 60 and 200 mg/mL for the G2, G3 and G4 groups, respectively. The suspensions were mixed well by stirring using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the solubility test, the test item formed a clear solution in Corn oil, while it did not dissolve/suspend in Milli-Q water and 0.1% carboxymethyl cellulose sodium salt (medium viscosity). Hence, Corn oil was used as vehicle for dose formulation preparation
- Amount of vehicle (if gavage): 5 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during week 4 of the treatment period and analysed in-house. For each set, duplicate sample was drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Advinus Study No. G13342. One set of samples were analysed for concentration (a.i) analysis.
Dose formulations were considered acceptable as the overall mean results were within ± 15.0% of the theoretical concentration and the overall relative standard deviation (RSD) was less than 10.0%.
Duration of treatment / exposure:
28 day
Frequency of treatment:
Daily once
Doses / concentrationsopen allclose all
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:
5 males + 5 female per dose group
Control animals:
yes
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The dose levels of 100 (G2), 300 (G3) and 1000 (G4/G4R) mg/kg/day were selected for this study based on the results of 14-Day Repeated Dose Oral Toxicity Study in Wistar rats (Study No. N3336) and in consultation with the Sponsor.

In addition to the test doses, vehicle control and vehicle control recovery groups were included. Animals in the vehicle control and vehicle control recovery groups were handled in a manner similar to the treatment groups except for test item administration.

- Rationale for animal assignment (if not random): Animals were randomly distributed to different groups by body weight stratification method using ProvantisTM software. Rats with extreme body weights were discarded. Grouping was done one day prior to start of treatment during acclimatization.

- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.2] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

- Food consumption: Weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: ophthalmoscope prior to start of the treatment, at the end of the treatment period for main groups and at the end of recovery period for recovery groups
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment for main groups and end of recovery for recovery groups
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in main report [Section 8.7.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment for main groups and end of recovery for recovery groups
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in main report [Section 8.7.4] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:End of treatment for main groups and end of recovery for recovery groups
- Animals fasted: Yes
- Parameters checked in main table [Section 8.8] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 4th week of treatment period for main groups and towards the end of recovery period for recovery period
- Dose groups that were examined: All groups
- Battery of functions tested: sensory activity / open field observation / grip strength / motor activity : Yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see in Main report section 8.9)

HISTOPATHOLOGY: Yes (see in Main report section 8.10)
Statistics:
Data captured using Provantis™ for the parameters body weight and organ weights; laboratory Investigations - Haematology (Coagulation tests PT and APTT which were entered retrospectively in ProvantisTM) and Clinical Chemistry were analyzed using built-in statistical tests.
Derived data like net body weight change, food consumption and organ weight ratios were also be analyzed using above mentioned methods.
The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights) were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA is performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test is found to be significant
The data pertaining to males and female rats was evaluated separately.
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated by the following symbols throughout the report:
+/-: Significantly higher/lower than the respective control group

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed both during the treatment and recovery period at all the treated groups in both sexes.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities observed both during the treatment and recovery period at all the treated groups in both sexes.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weights and net body weight gains were unaffected by the treatment at all the doses tested in both sexes either during the treatment or recovery periods.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The mean food consumption was not altered by the treatment in any of the tested doses either during the treatment or recovery period in both sexes.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmological examination was carried out with an ophthalmoscope prior to start of treatment, at the end of the treatment and recovery period did not reveal any abnormalities in the eyes of the experimental rats.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related alterations observed in any of the haematological parameters evaluated in the treated rats of main and recovery groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
An increase in triglycerides levels (57%) at 1000 mg/kg Bwt/day dose in females was attributed to test item administration and reversible at the end of recovery period. In females, increased bile acid at ≥ 300 mg/kg Bwt/day were considered as test item-related non adverse effects as individual values falls within normal range of historical control data and was reversible at the end of recovery period.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in any of the urine parameters in males and females at all the doses tested.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related abnormalities were observed in any of the doses tested in both sexes.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The treatment-related higher liver weight (absolute and/or relative) was observed in both males and females at 1000 mg/kg Bwt/day dose and was reversible at the end of the 14 days recovery period. Microscopically, hepatocellular hypertrophy (centrilobular) was observed in both sexes and considered as test item-related change.
The treatment-related higher thyroid weight (absolute and/or relative) was observed at ≥ 300 mg/kg Bwt/day in males and at 1000 mg/kg Bwt/day in females and microscopically associated with follicular epithelial cell hypertrophy. At the end of recovery period, these changes were reversible.
Decreased thymus weight (absolute and relative to brain weight) was observed in males at 1000 mg/kg Bwt/day dose and considered as toxicologically insignificant in the absence of related microscopic changes and also no alterations in terminal body weights and lymphocyte cell count were observed.
Increased kidney weights were noticed in males at 1000 mg/kg Bwt/day without any microscopic correlates. Increased ovaries weight was observed in recovery group females at 1000 mg/kg Bwt/day, which was not present at the end of treatment period. Hence these alterations were considered as incidental findings
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related multifocal thickening noted in the non-glandular stomach at 1000 mg/kg Bwt/day dose in females was microscopically found to be epithelial hyperplasia and hyperkeratosis.
The dilatation of uteri noted in the recovery females was attributed to normal estrous cyclicity. Further, single incidence of uterine horn malformation (focal atresia between body and horn; unilateral) was found to be normal histologically and the incidence was considered as a spontaneous finding, not related to treatment.
Skin hair loss observed in one female at 1000 mg/kg Bwt dose was found to be normal on microscopic evaluation and was considered as spontaneous in nature, not related to test item administration.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In stomach, reversible non-glandular mucosa epithelial hyperplasia and hyperkeratosis observed in females at 1000 mg/kg Bwt/day was considered due to the local irritation induced by the test item.
In liver, the hepatocellular hypertrophy noted in both the sexes at ≥ 300 mg/kg Bwt/day dose, was a likely reflection of an adaptive response to test item and considered as non-adverse effect. The pattern of hepatocellular hypertrophy was mainly centrilobular in nature with minimal to moderate in severity at ≥ 300 mg/kg Bwt/day in both the sexes and reversible at the end of recovery period.
In thyroid, follicular epithelial hypertrophy was noted in both sexes at ≥ 300 mg/kg Bwt/day and in pituitary gland, basophil cell hypertrophy was observed in ≥ 300 mg/kg males and at 1000 mg/kg in females. The changes observed in thyroid gland and pituitary glands were considered as secondary to hepatocellular hypertrophy in liver. However, these changes were reversible at the end of recovery period.
Hepatocellular hypertrophy was often seen concomitantly in rats with thyroid gland follicular cell hypertrophy and pituitary gland basophil cell hypertrophy. Due to hepatic enzyme induction which was reflected morphologically as hepatocellular hypertrophy in liver, there could be increased excretion of thyroid hormones leading to negative feedback response to pituitary and thyroid gland and stimulate them to release the TSH (basophil cell hypertrophy) and T3 (hypertrophy of follicular cells) respectively. Hypertrophy of thyroid gland indicates a systemic response to increased pituitary secretion of thyroid-stimulating hormone. These changes could be due to treatment with N-Acetylhexanelactam and considered as a species specific finding for the rat and not relevant to human as pointed by Tanja S. et al., 2011 and Kuei-Meng Wu, 2006.
All the other randomly distributed microscopic findings observed in some of the rats were considered as incidental/spontaneous background findings commonly noted in this age group

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
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
urinalysis

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Note: Study G13343 - additional Summary tables including Haematology, clin. Chemistry and Pathology are attached.

Summary of Body Weights (g) – Males

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                              Group Sex           1        8       15       22       28 

                                              ----------------------------------------------------------------

                                              G1     m  Mean     175.230   209.964  236.422  259.608  277.572

                                                          S.D.    10.432  15.195   18.167   18.058   20.561

                                                          N          5        5        5        5        5   

                                                           ------  ------- ------- ------- ------- -------

                                              G2     m  Mean     179.608  208.266  232.128  251.138 267.224

                                                           S.D.  6.056  11.992  14.307  18.625  19.566

                                                          N         5       5       5       5       5   

                                                          ------  ------- ------- ------- ------- -------

                                              G3     m  Mean    177.548 208.996 231.916 254.660 268.822

                                                          S.D.   9.087  15.015  18.086  20.531  24.657

                                                           N         5       5       5       5       5   

                                                          ------  ------- ------- ------- ------- -------

                                               G4     m  Mean    173.876 206.276 226.402 248.354 261.024

                                                          S.D.    8.971   8.396  14.201  13.270  14.069

                                                          N         5       5       5       5       5   

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                      Day numbers relative to Start Date                                                      

                                     Group Sex              1       8      15      22      28      35      42 

                                      ----------------------------------------------------------------------------------

                                     G1R    m  Mean    175.692 200.398 222.836 245.796 262.722 284.366 300.024

                                                  S.D.  8.334  11.667  12.485  15.334  16.746  20.117  21.191

                                                 N         5       5       5       5       5       5       5   

                                                 ------  ------- ------- ------- ------- ------- ------- -------

                                     G4R    m  Mean    178.354 205.220 222.516 238.552 248.876 268.262 284.836

                                                 S.D.   9.549   7.569   10.706  14.388  18.138  26.783  24.337

                                                 N         5       5       5       5       5       5       5   

---------------------------------------------------------------------------------------------------------------------------------------------------------

Group G1 - 0 mg/kg/day    Group G2 - 100 mg/kg/day   Group G3 -300 mg/kg/day   Group G4 - 1000 mg/kg/day                                                                             

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day                                                                                                                   

 

 


 

 

 

Summary of Body Weights (g) – Females

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                              Group Sex              1       8      15      22      28 

                                              ----------------------------------------------------------------

                                              G1     f  Mean    142.490 157.916 171.268 185.518 188.422

                                                          S.D.   7.145   9.426  16.715  16.295  16.499

                                                           N         5       5       5       5        5   

                                                          ------  ------- ------- ------- ------- -------

                                               G2     f  Mean    140.840 163.852 177.844 192.264 198.148

                                                          S.D.    4.741   6.110  10.006  10.292  10.314

                                                          N         5       5       5        5        5   

                                                          ------  ------- ------- ------- ------- -------

                                              G3     f  Mean    139.160 157.486 170.124 184.542 189.994

                                                          S.D.   7.063  10.475  12.481  17.107  15.557

                                                          N         5       5       5        5        5   

                                                           ------  ------- ------- ------- ------- -------

                                              G4     f  Mean    140.476 163.516 171.720 180.828 187.602

                                                           S.D.  3.549   6.417   8.961  11.621  10.246

                                                          N         5        5        5        5       5   

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                      Group Sex              1       8      15      22      28      35      42 

                                     ----------------------------------------------------------------------------------

                                     G1R    f  Mean    140.324 159.224 169.668 182.768 190.446 197.248 205.058

                                                 S.D.    5.973   2.326   7.545   6.642   6.255  10.997  11.173

                                                 N          5        5        5        5        5        5        5   

                                                 ------  ------- ------- ------- ------- ------- ------- -------

                                     G4R    f  Mean    140.982 158.826 174.616 182.874 190.366 193.304 198.556

                                                 S.D.    7.724   8.690  11.510  11.538  16.692  16.613  15.626

                                                 N          5       5        5        5        5        5        5   

---------------------------------------------------------------------------------------------------------------------------------------------------------

Group G1 - 0 mg/kg/day    Group G2 - 100 mg/kg/day   Group G3 -300 mg/kg/day   Group G4 - 1000 mg/kg/day                                                                             

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day       

 

 


 

Summary of Net Body Weight Gains (g) – Males

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                                  Base                                                         Abs      %  

                                                  Weight                                                      Gain    Gain 

                                                  Day     From:     1         8        15        22         1        1 

                            Group   Sex         1        To:       8        15        22        28        28       28  

                            ----------------------------------------------------------------------------------------------------

                            G1        m         175.230  Mean     34.734    26.458    23.186    17.964   102.342   58.58

                                                  10.432  S.D.      7.740     8.529     3.905     3.588    17.634   10.72

                                                     5      N          5          5          5          5          5         5  

                            -----   ---        -------  ----    --------  -------   -------   -------   -------  ------

                             G2       m         179.608  Mean     28.658    23.862    19.010    16.086    87.616   48.74

                                                   6.056  S.D.      9.124     3.067     4.994      4.040    16.513    8.58

                                                     5      N          5          5          5          5          5        5  

                            -----   ---        -------  ----    --------  -------   -------   -------   -------  ------

                             G3       m         177.548  Mean     31.448    22.920    22.744    14.162    91.274   51.19

                                                   9.087  S.D.      6.373     4.435     2.610     4.762    16.302    7.02

                                                     5      N          5          5          5         5          5         5  

                            -----   ---        -------  ----    --------  -------   -------   -------   -------  ------

                             G4       m         173.876  Mean     32.400    20.126    21.952    12.670    87.148   50.24

                                                   8.971  S.D.      3.356    10.006     4.093     2.935    11.388    7.15

                                                     5      N          5         5          5         5          5         5  

---------------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                          Base                                                                        Abs       %  

                                         Weight                                                                      Gain     Gain 

                                          Day      From:      1        8       15       22       28       35        1        1 

                    Group   Sex         1        To:        8       15       22       28       35       42       42       42  

                    --------------------------------------------------------------------------------------------------------------------

                    G1R      m         175.692  Mean     24.706   22.438   22.960  16.926   21.644   15.658  124.332   70.75

                                           8.334  S.D.     3.810     1.984    4.884    1.775    6.734    1.405   16.087    8.34

                                            5       N         5         5         5         5         5         5         5         5  

                    -----   ---        -------  ----    -------  -------  -------  -------  -------  -------  -------  ------

                    G4R      m         178.354  Mean     26.866   17.296   16.036   10.324   19.386   16.574  106.482   60.20

                                           9.549  S.D.      5.224    9.057    5.094    4.577   13.134    3.511   28.195   18.02

                                            5       N          5         5         5          5        5         5         5         5  

---------------------------------------------------------------------------------------------------------------------------------------------------------------

Abs Gain = absolute bodyweight gain between base period and end of the analysis period                                                                        

% Gain = percentage bodyweight gain between base period and end of the analysis period                                                                         

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day                                                                                                                   

Group G1 - 0 mg/kg/day    Group G2 - 100 mg/kg/day   Group G3 -300 mg/kg/day   Group G4 - 1000 mg/kg/day   

 

 

 


 

Summary of Net Body Weight Gains (g) – Females

---------------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                                   Base                                                      Abs        %  

                                                 Weight                                                     Gain     Gain 

                                                   Day     From:    1         8         15         22       1         1 

                            Group   Sex         1        To:       8         15        22         28       28        28  

                            ----------------------------------------------------------------------------------------------------

                            G1       f         142.490  Mean     15.426    13.352    14.250     2.904    45.932   32.14

                                                  7.145  S.D.      4.679     7.694     2.787      3.986    11.914    7.65

                                                   5      N          5          5          5          5          5         5  

                             -----   ---        -------  ----    --------  -------   -------   -------   -------  ------

                            G2       f         140.840  Mean     23.012+   13.992    14.420     5.884    57.308   40.76

                                                  4.741  S.D.      3.908      4.772     3.652     3.722     9.879    7.52

                                                   5      N          5          5          5          5          5         5  

                             -----   ---        -------  ----    --------  -------   -------   -------   -------  ------

                            G3       f         139.160  Mean     18.326    12.638    14.418     5.452    50.834   36.42

                                                  7.063  S.D.      4.707     3.680     5.281      4.825    10.263    6.37

                                                   5      N          5         5          5          5          5        5  

                             -----   ---        -------  ----    --------  -------   -------   -------   -------  ------

                            G4       f         140.476  Mean     23.040+    8.204     9.108     6.774    47.126   33.49

                                                  3.549  S.D.      3.535     3.974     3.632     4.101     7.394    4.62

                                                    5      N          5          5          5          5          5         5  

---------------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                          Base                                                                         Abs        %  

                                         Weight                                                                         Gain     Gain 

                                          Day     From:      1        8       15       22       28       35        1        1 

                    Group   Sex          1        To:       8       15       22       28       35       42      42       42  

                    --------------------------------------------------------------------------------------------------------------------

                     G1R      f         140.324  Mean     18.900   10.444   13.100    7.678    6.802    7.810   64.734   46.54

                                           5.973  S.D.      6.650    5.830    3.228    1.403    5.385    3.913   15.953   13.25

                                           5      N         5        5        5        5        5        5        5        5  

                    -----   ---        -------  ----    -------  -------  -------  -------  -------  -------  -------  ------

                    G4R      f         140.982  Mean     17.844   15.790    8.258    7.492    2.938    5.252   57.574   40.92

                                           7.724  S.D.      4.839    4.562    3.518    7.426    0.561    3.658   13.163    9.59

                                            5      N          5         5         5         5         5         5         5         5  

---------------------------------------------------------------------------------------------------------------------------------------------------------------

+: significantly higher than the vehicle control group

Abs Gain = absolute bodyweight gain between base period and end of the analysis period                                                                         

% Gain = percentage bodyweightgainbetween base period and end of the analysis period                                                                        

Group G1 - 0 mg/kg/day    Group G2 - 100 mg/kg/day   Group G3 -300 mg/kg/day   Group G4 - 1000 mg/kg/day                                                                             

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day                                                                                                             

 


 

Summary of Cagewise Average Food Consumption (g/rat/day) – Males

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                        Day numbers relative to Start Date

                                                              From:      1        8        15       22 

                                                  Group Sex    To:      8       15       22       28 

                                                  ---------------------------------------------------------

                                                   G1    m   Mean   19.368   19.030   18.929   19.004

                                                              S.D.    0.402    2.498    2.657    1.346

                                                               N        3        3        3        3   

                                                                      ------   ------   ------   ------

                                                  G2    m   Mean   17.850   16.251   15.551   16.307

                                                              S.D.   1.134    1.843    1.275    1.183

                                                              N        3         3         3         3   

                                                                       ------   ------   ------   ------

                                                  G3    m   Mean   18.437   16.508   15.813   16.179

                                                              S.D.    1.429    0.888    0.683    1.161

                                                              N        3        3        3        3   

                                                                      ------   ------   ------    ------

                                                  G4    m   Mean   17.857   16.747   15.927   15.366-

                                                              S.D.    1.187    0.611    0.473    1.116

                                                               N        3        3        3        3   

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date

                                                    From:      1        8       15       22       28       35 

                                        Group Sex     To:      8       15       22       28       35       42 

                                        -----------------------------------------------------------------------------

                                        G1R   m   Mean   18.160   16.591   16.158   16.328   19.484   20.180

                                                    S.D.    0.541    0.330    1.481    1.145    1.266    0.710

                                                    N        3        3        3         3        3        3   

                                                            ------   ------   ------   ------   ------   ------

                                        G4R   m   Mean   16.808   15.546-  14.319   15.828   17.941   18.631

                                                    S.D.    1.377    0.487    2.403    0.841    2.506    1.625

                                                    N        3        3        3        3        3        3   

---------------------------------------------------------------------------------------------------------------------------------------------------------

-: significantly lower than the vehicle control group

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day                                                                                                                   

Group G1 - 0 mg/kg/day    Group G2 - 100 mg/kg/day   Group G3 -300 mg/kg/day   Group G4 - 1000 mg/kg/day


 

Summary of Cagewise Average Food Consumption(g/rat/day) – Females

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                        Day numbers relative to Start Date

                                                              From:      1        8       15       22 

                                                  Group Sex    To:      8       15       22       28 

                                                  ---------------------------------------------------------

                                                  G1    f   Mean   13.513   13.275   13.629   15.001

                                                              S.D.    0.863    1.017    0.444    3.214

                                                              N        3        3        3        3   

                                                                       ------   ------   ------   ------

                                                  G2    f   Mean   13.955   13.352   13.668   13.558

                                                               S.D.    0.943    0.100    1.339    0.981

                                                              N        3        3        3        3   

                                                                      ------   ------   ------   ------

                                                  G3    f   Mean   12.892   12.871   12.300   12.403

                                                              S.D.    0.849    0.429    1.024    0.461

                                                               N        3        3        3        3   

                                                                      ------   ------   ------   ------

                                                  G4    f   Mean   13.754   12.950   12.510   12.629

                                                              S.D.    0.991    1.160    1.516    0.984

                                                              N        3        3        3         3   

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                      Day numbers relative to Start Date

                                                    From:      1        8       15       22       28       35 

                                        Group Sex    To:      8       15       22       28       35       42 

                                         -----------------------------------------------------------------------------

                                        G1R   f   Mean   13.094   12.374   12.142   12.383   13.782   14.057

                                                     S.D.    0.363    0.298    0.614    0.373    0.683    0.359

                                                    N        3        3        3        3        3        3   

                                                             ------   ------   ------   ------   ------   ------

                                        G4R   f   Mean   13.120   13.038+  11.799   12.653   13.485   13.985

                                                    S.D.     0.639    0.130    1.282    1.264    1.986    0.938

                                                    N        3        3        3        3        3        3   

---------------------------------------------------------------------------------------------------------------------------------------------------------+: significantly higher than the vehicle control group

Group G1 - 0 mg/kg/day    Group G2 - 100 mg/kg/day   Group G3 -300 mg/kg/day   Group G4 - 1000 mg/kg/day                                                                              

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day                  

Applicant's summary and conclusion

Conclusions:
To summarise, the results of the study indicate that the oral administration of test item N-Acetylhexanelactam for 28 days in Wistar rats at dose level of 100 300 and 1000 mg/kg Bwt/day doses did not cause any toxicological effect on general health, body weights, food consumption, neurological findings, haematology, coagulation and urine parameters.
Treatment related increase in the triglycerides levels (57%) was observed at 1000 mg/kg Bwt/day dose and bile acid at ≥ 300 mg/kg Bwt/day dose in females and were reversible at the end of recovery period.
At 1000 mg/kg Bwt/day dose, increased liver weight was microscopically associated with hepatocellular hypertrophy in both sexes and considered as metabolic adaptive response of the test item. Secondary to hepatocellular hypertrophy, thyroid and pituitary gland revealed follicular cell hypertrophy (associated with increased weight) and basophil cell hypertrophy, respectively in both sexes. In females, due to local irritation with test item, single or two incidences of epithelial hyperplasia/hyperkeratosis noticed in non-glandular stomach (grossly multifocal thickening). All these changes were considered as non- adverse and reversible at the end of recovery period.
At 300 mg/kg Bwt/day dose, hepatocellular hypertrophy in liver, increased thyroid weight associated with follicular cell hypertrophy in both sexes and basophil cell hypertrophy in pituitary were observed in males.
There were no test item- related changes noticed at 100 mg/kg Bwt/day dose group.
In conclusion, daily oral administration of N-Acetylhexanelactam to Wistar rats by oral gavage for 28 consecutive days at the dose levels of 100, 300 and 1000 mg/kg Bwt/day did not cause treatment-related adverse changes in any of the parameters evaluated. Hence, the evaluated No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg/day under the test conditions and doses employed.
Executive summary:

The purpose of this repeated dose toxicity study was to evaluate the systemic toxicity profile of the test item (N-Acetylhexanelactam) in Wistar rats when administered orally by gavage for 28 consecutive daysand to assess the reversibility of any effects following 14 days recovery period. This study was intended to provide information on major toxic effects, target organs and an estimation of No Observed Adverse Effect Level (NOAEL).

The test item was weighed and suspended in vehiclei.e.,Corn oil and administered to rats at the graduated dose levels of 100, 300, and 1000 mg/kg Bwt/day to low (G2), mid (G3), and high (G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/ vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 5 mL/kg body weight. Each group in the experiment was comprised of five male and five female rats.

Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption was measured during the course of the in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment for main groups and towards the end of recovery period for recovery groups.The clinical laboratory investigations such as haematology, coagulation, clinical chemistry and urine analysis were performed at termination.

All the rats in the experiment were subjected to detailed necropsy and the organ weights and their ratio were derived as percent fasting body weights. Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose group animals (G4). In addition, all gross lesions from all the animals were examined microscopically. Further, liver, thyroid and pituitary in both sexes and stomach in females showing test item-related histopathological changes in high dose (G4) group were examined in the respective lower dose groups and recovery groups.

Under the experimental conditions employed, the following results were obtained:

·        Clinical Signs, Mortalities and Ophthalmological Examination: No clinical signs or mortalities or ocular abnormalities were observed at any of the doses tested.

·        Neurological Findings: No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.

·        Body Weights: The mean body weights and net body weight gains were unaffected by the treatment in both the sexes at all the doses tested.

·        Food Consumption:There were no treatment-related changes observed in the food consumption at all the doses tested in both sexes throughout the experimental period.

·        Haematology, Coagulation, Clinical chemistry and Urine Parameters: No test item-related changes in haematology, coagulation and urine analysis parameters were observed in both sexes at the end of treatment and recovery period. In females, test item relatedincrease in triglycerides and bile acid (non-adverse effect) were observed at 1000 mg/kg Bwt/day dose and were reversibleat the end of 14 days recovery period.

·        Fasting Body Weights, Organ Weights and its Ratios: Treatment-related higher liver weight (absolute and/or relative) was observed at 1000 mg/kgBwt/day dose in both sexes and was reversible at the end of the 14 days recovery period.The treatment also resulted in higher thyroid weight(absolute and/or relative)at ≥ 300 mg/kgBwt/day in males and at 1000 mg/kgBwt/day in females and these changes were reversible at the end of 14 days recovery period.

·        Gross and Histopathology: At 1000 mg/kgBwt/day dose, the hepatocellular hypertrophy was observed in both the sexes and considered as metabolic adaptive response of the test item. Secondary to hepatocellular hypertrophy, thyroid and pituitary glands revealed follicular cell hypertrophy (associated with increased weight) and basophil cell hypertrophy, respectively in both sexes. In females, due to local irritation of the test item, single or two incidences of epithelial hyperplasia/hyperkeratosis were observed in non-glandular stomach (grossly multifocal thickening). All these changes were considered as test item related non-adverse effects and were completely reversible at the end of 14 days recovery period.

 

At 300 mg/kg Bwt/day dose, hepatocellular hypertrophy in liver, increased thyroid weight associated with follicular cell hypertrophy in both sexes and basophil cell hypertrophy in pituitary were observed in males.

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