1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C18(unsaturated) acyl derivs., hydroxides, inner salts

Repeated dose toxicity: oral

Administrative data

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

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)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanaminium,3-amino-N-(carboxymethyl)-N,N-dimethyl-,N-C18(unsaturated) acyl derivs., hydroxides, inner salts

IUPAC Name:

1-Propanaminium,3-amino-N-(carboxymethyl)-N,N-dimethyl-,N-C18(unsaturated) acyl derivs., hydroxides, inner salts

Test material form:

liquid

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.

Test animals

Species:

rat

Strain:

Wistar

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
- 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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

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.

Examinations

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

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not 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

Effect levels

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

clinical biochemistry

haematology

histopathology: non-neoplastic

Target system / organ toxicity

1

Critical effects observed:

not specified

2

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

Any other information on results incl. tables

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	—	—	—	—	—	—	—	—

Applicant's summary and conclusion

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.