Registration Dossier
Registration Dossier
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EC number: 217-565-6 | CAS number: 1888-91-1
Repeated dose toxicity: oral
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:
- 2 017
- 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
- Reference substance name:
- N-acetylhexanelactam
- EC Number:
- 217-565-6
- EC Name:
- N-acetylhexanelactam
- Cas Number:
- 1888-91-1
- Molecular formula:
- C8H13NO2
- IUPAC Name:
- 1-acetylazepan-2-one
- Test material form:
- liquid
- Details on test material:
- Batch: not yet known
Constituent 1
- 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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