Registration Dossier

Administrative data

Description of key information

Two repeated dose toxicity studies are available, in which consistent results were obtained.

For the purpose of doses determination for a 28 -day toxicity study, rats were treated per gavage with the registration substance once daily for 14 days at doses of 0, 100, 300 and 1000 mg/kg bw/day. At dose of 1000 mg/kg bw/day, reduced body weight in males and changes of hematological and clinical chemistry were found for males and females and gross pathological effects in stomach. The NOAEL of 300 mg/kg bw/day was obtained.

The repeated dose toxicity of the registration substance was investigated according to the Guideline OECD 407.

Five male and five female rats per dose group were treated per gavage once daily for 28 days at doses of 0, 100, 300 and 600 mg/kg bw/day. These animals were sacrificed on day 29. In addition, five male and five female rats were treated in the same manner at doses of 0 and 600 mg/kg bw/day and allowed to recover for 14 days.

No effect was found for animals treated at 100 and 300 mg/kg bw.

The animals treated at dose of 600 mg/kg bw/day exhibited changes in clinical chemistry (AST, ALT and CK in males and females). In stomach, focal to multifocal thickening of non glandular mucosa was found that was associated with epithelial hyperplasia, erosion and oedema.

The NOAEL of 300 mg/kg bw/day was obtained.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
The test material corresponded to the approximately 45% of the registration substance. The given dose refers to the amount of the registration substance.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Age at study initiation: 7 weeks.
- Weight at study initiation: Males: 190.70 to 229.43g and Females: 143.89 to 174.62g
- Housing: Rats were housed in a group of two animals of same sex per cage in sterilized suspended polysulfone cages with solid floor (size: L 425 x B 266 x H 185 mm) with stainless steel top grill having facilities for providing pelletted food and drinking water in polycarbonate bottle with stainless steel sipper tubes. Polycarbonate rat huts was 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 Harlan Laboratories (Envigo), P.O. Box 44220, Madison, Wi 53744-4220 was provided ad libitum to the animals.
- 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: five days before start of the treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature maintained during the experiment was between 20 and 24°C.
- Humidity (%): The relative humidity was between 65 to 67 %.
- Air changes (per hr): Adequate fresh air supply of 12 - 15 air changes/hour was maintained in the experimental room.
- Photoperiod (hrs dark / hrs light): The photoperiod was 12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From: 17 September 2016 To: 03 November 2016
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q® Water was used to prepare the dose formulations as the same vehicle was used in the 14 day repeated dose toxicity study with the same test item (Study No.N2857).
Details on oral exposure:
VEHICLE PREPARATION
10.0 g of Sodium carboxy methyl cellulose was added to about 1800 mL of Milli-Q® water in a 2000 mL pre-marked beaker* and stirred on a magnetic stirrer. The final volume was made up to the mark with Milli-Q® water. After obtaining uniform suspension, the suspension was stored at room temperature in the experimental room.

Details of components used for vehicle preparation are as follows:
Name of vehicle: Carboxymethylcellulose Sodium salt (medium viscosity)
Manufactured by: Sigma
Date of receipt: 17.12.2014
Lot No.: SLBF3845V
Date of expiry: Dec 2019

This vehicle was used for the preparation of the dose formulations and administration to the rats in vehicle control group.

*Pre-marking of beaker: The magnetic stir bar was placed in a clean beaker, measured the purified water (Milli-Q®) in a graduated cylinder to the final volume of 2000 mL. The water was transferred into the beaker containing magnetic bar and the upper meniscus of the water was marked. Once marking was done, water was discarded and the beaker and stir bar was allowed to dry.

The weights and volumes of the above reagents were varied proportionately depending on the volume of the vehicle prepared during the course of the study.

The weights and volumes of the above reagents were varied proportionately depending on the volume of the vehicle prepared during the course of the study.
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 and analysed in-house. Samples were drawn from top, middle and bottom layers of each preparation for treatment groups in duplicate sets and from middle layers of preparation for control group (three samples corresponding to each treatment group).

The analysis was done as per the method validated under Advinus Study No.: G11354. One set of samples were analysed for concentration (a.i) analysis.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
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 and 5 Females per group
Control animals:
yes
Details on study design:
The dose levels of 100 (G2), 300 (G3) and 600 (G4/G4R) mg/kg/day were selected for this study in consultation with the Sponsor and were based on the results of 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No.N2857) in which the animals treated at 100 mg/kg/day exhibited a significant change in body weight development.
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.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Morning
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights (g) were recorded prior to test item administration on Day 1 and weekly thereafter (± 1 day) for all groups of rats during treatment and recovery period. Fasting body weight was recorded prior to sacrifice for all animals.

FOOD CONSUMPTION:
- The food consumption was measured at weekly intervals (± 1 day) during treatment and recovery period. The cage wise average food consumption (g/rat/day) was calculated and presented in the report.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period for main groups and at the end of recovery period for recovery groups.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 60 animals
- Following parameters checked:
Red Blood Corpuscles
Haemoglobin
Haematocrit
Mean Corpuscular Volume
Mean Corpuscular Haemoglobin
Mean Corpuscular Haemoglobin Concentration
Reticulocytes count
White Blood Corpuscles
Differential leukocyte count
Platelets


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period for main groups and at the end of recovery period for recovery groups.
- Animals fasted: Yes
- How many animals: 60 animals
- Following parameters checked:
Alanine Aminotransferase
Albumin
Albumin/Globulin ratio [calculated values]
Alkaline phosphatase
Aspartate Amino Transferase
Blood Urea Nitrogen
Bile acids
Calcium
Chloride
Creatinine
Creatine Kinase
Gamma Glutamyl Transpeptidase
Globulin [calculated values]
Glucose
Inorganic phosphorous
Potassium
Sodium
Total Bilirubin
Total Cholesterol
Total protein
Triglycerides

URINALYSIS
- Time schedule for collection of urine: at the end of treatment period for main groups and at the end of recovery period for recovery groups.
- Animals fasted: Yes
- How many animals: 60 animals
- Following parameters checked:
Specific gravity
Nitrite
pH
Proteins
Glucose
Ketone bodies
Urobilinogen
Bilirubin
Appearance (colour and clarity)
Volume (approximate)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
Other examinations:
FUNCTIONAL OBSERVATION BATTERY TESTS
The following neurological examination was performed during 4th week of treatment period for main groups and towards the end of recovery period for recovery period.

HOME CAGE OBSERVATIONS
Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.

OBSERVATIONS DURING REMOVAL OF ANIMAL FROM HOME CAGE AND HANDLING
The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
- ease of removal from home cage
-handling reactivity
-palpebral closure
-eye examination
-piloerection
-lacrimation
-salivation
-skin/fur examination
-perineum wetness
-respiration
-muscle tone and
-extensor thrust response
The observations were recorded using scores/ranks.

OPEN FIELD OBSERVATION
Rat was placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each rat. During this observation period, rat was evaluated as it moves about freely/unperturbed and the following observations were made and observations were recorded using score/ranks:
-gait
-posture
-tremors
-mobility score
-arousal level
-clonic or tonic movements
-stereotypic behaviour
-bizarre behaviour
-urination
-defecation
-rearing
-abnormal vocalizations

FUNCTIONAL TESTS
Functional testing includes motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.

- Motor Activity
The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, horizontal counts, ambulatory counts were monitored. The Opto-Varimex 4 motor activity measurement system provided the data at 1 minute interval and the data was analyzed in blocks of 10 minutes interval and the same was reported.

- Sensory Reactivity Measurements
After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks.
-approach response
-touch response
-click response
-tail-pinch response
-pupil response
-aerial righting reflex

Landing Hindlimbs Footsplay:
The landing hind limbs foot splay was performed by dropping the rat on to a horizontal surface of the table top from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat was gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which contains the details such as Study no., Animal no, Group and Sex. A clean recording paper sheet was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values are presented in the report along with the individual footsplay values.

Grip Performance:
Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Average of three trials for both forelimb and hindlimbs are calculated and presented in the report along with the individual grip strength values.

Physiological Observations :
Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer.
At the end of the functional test, body weight of each rat was measured.
Statistics:
Data captured using Provantis™ for the parameters body weight, organ weights; laboratory investigations - haematology (Coagulation tests PT and APTT which was entered retrospectively in ProvantisTM) and clinical chemistry were analyzed using built-in statistical tests.
The integrated decision tree of ProvantisTM:
(i)Test variance homogeneity by Levene’s method was tested. When variances are heterogeneous, suitable transformation was performed automatically by the software.
(ii) Further one-way analysis of variance (ANOVA) was performed. When ANOVA was significant, Dunnett’s control versus treatment group mean comparisons was performed

Derived data like net body weight change, food consumption and organ weight ratios were 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) was 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 were designated by the following symbols throughout the report:

+/-: Significantly higher/lower than the respective control group
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
↑ ALT activity in 300mg/kg/day dose females and ALT, AST and creatine kinase activity in 600mg/kg/day dose in males and females were observed. ↑ ALT activity(1.71 folds) observed in 300mg/kg/day dose females was not associated with any microscopic changes
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Grossly, focal to multifocal thickening of non glandular mucosa of stomach in single male at mid dose, in four males and all females at 600 mg/kg/day dose and reversible at the end of 14 day recovery period.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
epithelial hyperplasia, erosion and oedema microscopically in a single male at 300 mg/kg/day dose and in four males and all females at 600 mg/kg/day dose and reversible at the end of 14 day recovery period
Histopathological findings: neoplastic:
not examined
Details on results:
To summarise, daily oral (gavage) administration of test item “C18unsat-AAPB” to Wistar rats at the dose levels of 100, 300 and 600 mg/kg/day did not cause any clinical signs or mortalities. There were no test item-related changes in body weights, net body weight gains, food consumption and neurological findings. There were no test item-related changes observed in haematological, coagulation and urine parameters. Clinical chemistry analysis revealed increased ALT activity in 300 mg/kg/day dose females and ALT, AST and creatine kinase activity in males and females at 600 mg/kg/day dose group. Increased ALT activity (1.71 folds) in 300 mg/kg/day dose females was not associated with any microscopic changes in liver and hence, considered as non adverse change. Grossly, focal to multifocal thickening of non glandular mucosa of stomach was observed associated with microscopic changes of epithelial hyperplasia, erosion and oedema in a single male at 300 mg/kg/day and 600 mg/kg/day dose males (4/5) and females (5/5). The changes were considered as test item-related reversible changes. Single incidence of focal thickening of non glandular mucosa of stomach in 300 mg/kg/day dose male was considered as non-adverse as it was associated with only minimal to moderate epithelial hyperplasia, erosion and oedema. The changes observed in stomach were considered as local effects due to irritant nature of test item.

Hence, the evaluated No Observed Adverse Effect Level (NOAEL) is considered to be 300 mg/kg/day following oral gavage administration for 28 consecutive days to wistar rats under the test conditions and doses employed due to the changes in clinical chemistry and the stomach leisions at 600 mg/kg/day


Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
histopathology: non-neoplastic
Critical effects observed:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes

Table 1: Summary of Body Weights of Males

Doses

[mg/kg /day]

 

Body weight of males [g]; n = 5

1d

8d

15d

22d

28d

35d

42d

For animals sacrificed after treatment

0

Mean

210.086

244.690

278.944

304.458

325.160

 

 

S.D.

9.040

6.990

7.331

9.180

8.688

 

 

100

Mean

206.544

238.548

269.034

294.628

311.564

 

 

S.D.

16.276

12.731

13.420

14.384

14.208

 

 

300

Mean

209.104

246.334

277.158

304.352

324.370

 

 

S.D.

15.113

24.273

30.076

34.161

37.899

 

 

1000

Mean

207.660

233.268

261.274

287.538

300.544

 

 

S.D.

9.802

6.067

10.664

18.403

20.509

 

 

For animals sacrificed after treatment and recovery

0

Mean

203.746

238.310

262.396

285.550

299.930

319.584

333.862

S.D.

10.285

11.942

16.784

20.510

23.024

22.900

20.744

1000

Mean

207.292

229.712

254.906

276.442

286.496

311.366

331.774

S.D.

12.468

17.238

25.379

28.855

30.442

32.510

33.752

Table 2: Summary of Body Weights of Females

Doses

[mg/kg /day]

 

Body weight of females [g]; n = 5

1d

8d

15d

22d

28d

35d

42d

For animals sacrificed after treatment

0

Mean

156.008

166.978

180.464

191.322

198.392

 

 

S.D.

10.830

12.744

14.790

16.694

18.497

 

 

100

Mean

156.496

167.038

179.084

188.520

195.388

 

 

S.D.

9.629

7.727

8.159

8.562

9.869

 

 

300

Mean

150.210

166.214

181.098

190.954

195.932

 

 

S.D.

6.191

8.571

8.039

13.386

10.753

 

 

1000

Mean

154.164

168.024

184.602

193.736

198.534

 

 

S.D.

8.164

9.515

6.922

6.160

6.642

 

 

For animals sacrificed after treatment and recovery

0

Mean

155.786

171.910

183.540

193.056

200.874

208.528

214.148

 

S.D.

12.153

10.266

11.923

13.253

9.332

12.148

14.511

1000

Mean

154.428

166.490

179.690

191.356

198.790

202.364

208.266

 

S.D.

7.126

9.692

10.421

9.719

13.503

12.603

11.752

Table 3: Summary of Clinical Chemistry of Males; Values with meaningful changes

Doses

[mg/kg /day]

 

Hematology of Males; n = 5

CK

[U/L]]

AST

[U/L]

ALT

[U/L]

For animals sacrificed after treatment

0

Mean

291

98

34

S.D.

59

9

7

100

Mean

336

100

37

S.D.

88

6

6

300

Mean

423

101

37

S.D.

130

10

5

1000

Mean

452*

120*

92*

S.D.

52

15

26

For animals sacrificed after treatment and recovery

0

Mean

268

104

46

 

S.D.

32

20

7

1000

Mean

319

95

35*

 

S.D.

66

12

4

Table 4: Summary of Clinical Chemistry of Females; Values with meaningful changes

Doses

[mg/kg /day]

 

Hematology of Females; n = 5

CK

[U/L]]

AST

[U/L]

ALT

[U/L]

For animals sacrificed after treatment

0

Mean

310

98

23

S.D.

30

13

4

100

Mean

340

100

29

S.D.

93

10

4

300

Mean

323

103

40*

S.D.

40

13

12

1000

Mean

514*

118

77*

S.D.

221

16

28

For animals sacrificed after treatment and recovery

0

Mean

270

94

34

 

S.D.

76

7

7

1000

Mean

258

107

28

 

S.D.

54

35

4

Table 5: Histopathology

Sex

MALES

FEMALES

Group

After treatment

After treatment and recovery

After treatment

After treatment and recovery

Dose (mg/kg/day)

0

100

300

600

0

600

0

100

300

600

0

600

No. of rats

5

5

5

5

5

5

5

5

5

5

5

5

STOMACH;

(5)

(5)

(5)

(5)

(5)

(5)

(5)

(5)

(5)

(5)

(5)

(5)

Epithelial hyperplasia-non glandular

0

0

1

4

0

0

0

0

0

5

0

0

minimal

1

mild

2

1

moderate

2

4

Erosion-non glandular

0

0

1

3

0

0

0

0

0

4

0

0

minimal

1

3

4

Oedema; non glandular; submucosa

0

0

1

4

0

0

0

0

0

5

0

0

minimal

1

1

3

mild

2

2

moderate

1

Conclusions:
The repeated dose toxicity of the registration substance was investigated according to the Guideline OECD 407.The animals treated at dose of 600 mg/kg bw/day exhibited changes in clinical chemistry (AST, ALT in males and females). In stomach, focal to multifocal thickening of non glandular mucosa was found that was associated with epithelial hyperplasia, erosion and oedema. The NOAEL of 300 mg/kg bw/day was obtained.
Executive summary:

The repeated dose toxicity of the registration substance 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C18(unsatd) acyl derivs., hydroxides, inner salt (C18unsat-AAPB) was investigated according to the Guideline OECD 407.

Five male and five female rats per dose group were treated per gavage once daily for 28 days at doses of 0, 100, 300 and 600 mg/kg bw/day. These animals were sacrificed on day 29.In addition, five male and five female rats were treated in the same manner at doses of 0 and 600 mg/kg bw/day and allowed to recover for 14 days.

No effect was found for animals treated at 100 and 300 mg/kg bw.

The animals treated at dose of 600 mg/kg bw/day exhibited changes in clinical chemistry (AST, ALT and CK in males and females). In stomach, focal to multifocal thickening of non glandular mucosa was found that was associated with epithelial hyperplasia, erosion and oedema.

The NOAEL of 300 mg/kg bw/day was obtained.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
14-day treatment, reduced number of animals
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
The test material corresponded to the approximately 45% of the registration substance. The given dose refers to the amount of the registration substance.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Age at study initiation: 7 weeks.
- Weight at study initiation: Males: 172 to 224g and Females: 137 to 164g
- Housing: Rats were housed in a group of two animals of same sex per cage in sterilized suspended polysulfone cages with solid floor (size: L 425 x B 266 x H 185 mm) with stainless steel top grill having facilities for providing pelletted food and drinking water in polycarbonate bottle with stainless steel sipper tubes. Polycarbonate rat huts was 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 Harlan Laboratories (Envigo), P.O. Box 44220, Madison, Wi 53744-4220 was provided ad libitum to the animals.
- 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: ive days before start of the treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature maintained during the experiment was between 21 and 24°C.
- Humidity (%): The relative humidity was between 58 to 68 %.
- Air changes (per hr): Adequate fresh air supply of 12 - 15 air changes/hour was maintained in the experimental room.
- Photoperiod (hrs dark / hrs light): The photoperiod was 12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From: 23 June 2016 To: 06 July 2016
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
Carboxymethylcellulose Sodium salt
Details on oral exposure:
VEHICLE PREPARATION
About 5.0 g of Sodium carboxy methyl cellulose was added to about 900 mL of Milli-Q® water in a 1000 mL pre-marked beaker* and stirred on a magnetic stirrer. The vehicle was made up to the mark with Milli-Q® water. Once a uniform suspension is obtained, the suspension was stored at room temperature in the experimental room.

This vehicle was used for the preparation of the dose formulations and administration to the rats in vehicle control group.
*Pre-marking of beaker: The magnetic stir bar was placed in a clean beaker, measured the purified water in a graduated cylinder to the final volume of 1000 mL. The water was transferred into the beaker containing magnetic bar and the upper meniscus of the water was marked. Once marking was done, water was discarded and the beaker and stir bar was allowed to dry.

The weights and volumes of the above reagents were varied proportionately depending on the volume of the vehicle prepared during the course of the study.

Lot No.: SLBF3845V
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 of the treatment and analysed in-house. Samples were drawn from top, middle and bottom layers of each preparation for treatment groups in duplicate sets and from middle layers of preparation for control group (three samples corresponding to each treatment group).

The analysis was done as per the method validated under Advinus Study Nos.: G11354. 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:
14 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
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:
4 Males and 4 Females per group
Control animals:
yes
Details on study design:
The dose levels of 100, 300 and 1000 mg/kg bwt/day were selected based on the results acute oral toxicity study (Study No.G11352) in consultation with the Sponsor.

The dose levels were corrected for the purity (45% in water) of the test material, so that the given dose levels refers to the doses of registration substance.


Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Morning
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: day 1, 4, 8, 11 and 14

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment
- Anaesthetic used for blood collection: Yes (identity)
- Animals fasted: Yes
- How many animals: 16 animals
- Parameters checked in table were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment
- Animals fasted: Yes
- How many animals: 16 animals
- Parameters checked in table were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
Statistics:
Data captured using ProvantisTM: Parameters such as body weight, laboratory investigations - Haematology (Coagulation tests data-PT and APTT was entered retrospectively in ProvantisTM) and Clinical Chemistry, terminal fasting body weight and organ weights data was analysed using ProvantisTM built-in statistical tests.
Derived data such as net body weight change, food consumption and organ weight ratios were analyzed using built-in statistical tests.
All analyses and comparisons were evaluated at the 5% (P<0.05) level. Statistically significant differences (P<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
+/-: Significantly higher/lower than the vehicle control group
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bwt/day dose, the mean body weights (9.1 to 13.3%) and net body weight gains were apparently lower in males, statistically not significant, when compared to vehicle control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bwt/day dose, the food intake was significantly lower during Days 1-4 of the treatment period in both sexes.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
14 days through oral route to Wistar rats showed increase in WBC, neutrophil, and monocyte count in both the sexes at 1000 mg/kg bwt/day dose group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry analysis revealed higher creatinine, ALT, AST and lower total protein, albumin, globulin levels in males and higher levels of ALT and AST n females.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Decreased terminal fasting body weight was noted in male rats. Grossly white foci and mucosal thickening was observed in non-glandular stomach of both the sexes at the high dose and solitary incidence of mucosal thickening at mid dose
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no clinical signs or mortalities observed at 100 and 300 mg/kg bwt/day doses in both sexes. At 1000 mg/kg bwt/day dose, salivation was observed in 3/6 male rats on Days 13 and 14 during post-dose observation. In females, there were no clinical signs or mortalities observed at 1000 mg/kg bwt/day dose.

BODY WEIGHT AND WEIGHT GAIN: Treatment did not affect the mean body weights and net body weight gains at 100 and 300 mg/kg bwt/day doses in males and at all the doses tested in females. At 1000 mg/kg bwt/day dose, the mean body weights (9.1 to 13.3%) and net body weight gains were apparently lower in males, statistically not significant, when compared to vehicle control group.

FOOD CONSUMPTION : Treatment did not affect the food consumption at 100 and 300 mg/kg bwt/day doses in males and at all the doses tested in females. At 1000 mg/kg bwt/day dose, the food intake was significantly lower during Days 1-4 of the treatment period in both sexes.

HAEMATOLOGY: The administration of 1-propanaminium, 3-amino-N- (carboxymethyl)- N,N-dimethyl-, N-C18(unsaturated) acyl derivs.,hydroxides, inner salts for 14 days through oral route to Wistar rats showed increase in WBC, neutrophil, and monocyte count in both the sexes at 1000 mg/kg bwt/day dose group.

CLINICAL CHEMISTRY: Clinical chemistry analysis revealed higher creatinine, ALT, AST and lower total protein, albumin, globulin levels in males and higher levels of ALT and AST n females.


ORGAN WEIGHTS: Decreased terminal fasting body weight was noted in male rats.

GROSS PATHOLOGY: Grossly white foci and mucosal thickening was observed in non-glandular stomach of both the sexes.

At 300 mg/kg bwt/day, solitary incidence of mucosal thickening of non-glandular stomach was present grossly in a female rat.

At 100 mg/kg bwt/day, no test item-related changes were observed in both the sexes.

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
Key result
Critical effects observed:
not specified
System:
other: no histopathological examination performed

Table 1: Summary of Body Weights of Males

Doses

[mg/kg /day]

 

Body weight of males [g]; n = 4

1d

4d

8d

11d

14d

0

Mean

198.408

212.655

229.713

240.563

251.695

S.D.

17.734

18.166

19.506

22.196

20.651

100

Mean

202.918

213.478

231.865

246.318

258.090

S.D.

5.160

8.376

5.856

5.090

4.250

300

Mean

196.723

211.520

228.653

243.930

250.993

S.D.

7.238

6.012

9.020

13.101

11.355

1000

Mean

196.835

193.243

204.365

208.670

221.308

S.D.

19.121

19.081

26.961

28.385

25.472

Table 2: Summary of Body Weights of Females

Doses

[mg/kg /day]

 

Body weight of males [g]; n = 4

1d

4d

8d

11d

14d

0

Mean

156.215

162.715

171.170

180.468

186.318

S.D.

7.379

9.863

9.594

11.828

9.247

100

Mean

153.985

164.540

174.330

181.345

188.645

S.D.

12.331

10.961

9.915

7.207

7.915

300

Mean

154.228 

160.475 

167.293 

177.178 

177.980

S.D.

8.402   

7.876   

6.809   

5.833   

6.642

1000

Mean

153.538 

157.140 

166.920 

174.953 

181.513

S.D.

6.217   

6.116   

4.328   

3.208   

5.810

Table 3: Summary of Hematology of Males; Only values presented with potentially meaningful changes (statistical or dose-response relation)

Doses

[mg/kg /day]

 

Hematology of Males; n = 4

Retic A

[10^12/L]

WBC

[10^9/L]

Neut A

[10^9/L]

Lymp A

[10^9/L]

Eosi A

[10^9/L]

0

Mean

0.180

7.60

0.95

6.41

0.09

S.D.

0.039

0.74

0.13

0.85

0.01

100

Mean

0.236

8.41

0.91

7.26

0.08

S.D.

0.020

0.57

0.05

0.53

0.02

300

Mean

0.216

7.69

0.81

6.68

0.07

S.D.

0.064

0.12

0.14

0.07

0.01

1000

Mean

0.131

9.29

1.70*

7.17

0.13

S.D.

0.068

3.09

0.73

2.21

0.12

Table 4: Summary of Hematology of Females; Only values presented with potentially meaningful changes (statistical or dose-response relation)

Doses

[mg/kg /day]

 

Hematology of Females; n = 4

Retic A

[10^12/L]

WBC

[10^9/L]

Neut A

[10^9/L]

Lymp A

[10^9/L]

Eosi A

[10^9/L]

0

Mean

0.169

6.97

0.69

6.05

0.08

S.D.

0.060

3.06

0.30

2.69

0.02

100

Mean

0.174

5.20

0.58

4.47

0.04

S.D.

0.036

1.27

0.13

1.13

0.01

300

Mean

0.157

5.66

1.09

4.33

0.11

S.D.

0.040

0.83

0.46

0.57

0.9

1000

Mean

0.151

8.83

1.57*

6.90

0.12

S.D.

0.042

1.62

0.14

1.49

0.02

Table 5: Summary of Clinical Chemistry of Males; Only values presented with potentially meaningful changes (statistical or dose-response relation)

Doses

[mg/kg /day]

 

Hematology of Males; n = 4

T.Bil

[µmol/L]

Creat

[µmol/l]

AST

[U/L]

ALT

[U/L]

T. Pro

[g/L]

Alb

[g/L]

Glob

[g/L]

0

Mean

2.73

26

77

55

66.5

33.3

33.3

S.D.

0.07

4

10

6

1.7

1.3

1.3

100

Mean

3.21

33

95

65

65.8

33.0

32.8

S.D.

0.31

4

7

10

3.2

1.5

1.8

300

Mean

1.85

29

95

71

67.5

33.7

33.9

S.D.

0.73

1

20

13

0.6

1.0

0.9

1000

Mean

1.72

38*

132*

169*

58.8*

30.2*

28.6*

S.D.

0.79

8

21

56

2.5

1.2

1.4

Table 6: Summary of Clinical Chemistry of Females; Only values presented with potentially meaningful changes (statistical or tendency of dose-response relation)

Doses

[mg/kg /day]

 

Hematology of Females; n = 4

T.Bil

[µmol/L]

Creat

[µmol/l]

AST

[U/L]

ALT

[U/L]

T. Pro

[g/L]

Alb

[g/L]

Glob

[g/L]

0

Mean

3.32

43

98

56

63.7

33.1

30.5

S.D.

0.70

9

24

6

2.3

1.5

0.9

100

Mean

1.85

35

90

56

64.7

33.4

31.4

S.D.

0.88

4

10

4

1.6

1.1

0.7

300

Mean

1.92

29*

83

55

66.6

34.3

32.3

S.D.

1.08

2

5

5

3.3

1.8

1.7

1000

Mean

1.90

38

134

152*

60.6

32.1

28.5

S.D.

0.74

2

28

76

3.6

2.4

1.3

Conclusions:
The 14-day toxicity of the registration substance to rats were investigated using the study design similar to the Guideline OECD 407. At dose of 1000 mg/kg bw/day, reduced body weight in males and changes of hematological and clinical chemistry were found for males and females and gross pathological effects in stomach. The NOAEL of 300 mg/kg bw/day was obtained.
Executive summary:

For the purpose of doses determination for a 28 -day toxicity study, rats were treated per gavage once daily for 14 days at doses of 0, 100, 300 and 1000 mg/kg bw/day. At dose of 1000 mg/kg bw/day, reduced body weight in males and changes of hematological and clinical chemistry were found for males and females and gross pathological effects in stomach. The NOAEL of 300 mg/kg bw/day was obtained.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One 14-day repeated dose toxicity study and one 28-day repeated dose toxicity study (OECD 407) in rats. In both studies consistent results were obtained.
Organ:
stomach

Additional information

Justification for classification or non-classification

The repeated dose toxicity of the registration substance in rats was investigated in two repeated dose toxicity studies (14 -day and 28 -day). In both studies the NOAEL of 300 mg/kg bw/day was obtained.

No classification is justified.