1,1'-[[3-(dimethylamino)propyl]imino]bispropan-2-ol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-12-28 to 2021-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-[[3-(dimethylamino)propyl]imino]bispropan-2-ol

EC Number:

264-261-4

EC Name:

1,1'-[[3-(dimethylamino)propyl]imino]bispropan-2-ol

Cas Number:

63469-23-8

Molecular formula:

C11H26N2O2

IUPAC Name:

1-{[3-(dimethylamino)propyl](2-hydroxypropyl)amino}propan-2-ol

Test material form:

liquid

Details on test material:

Details described in study specific records

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): JEFFCAT® DPA catalyst
- lot/batch number: PFW190123
- Physical appearance: Liquid
- Manufactured date: 04.03.2019
- Retest date: 03.06.2022
- pH: 11,7
- Purity: This product is not specified by purity/assay but total titratable amine content. Amine content: 9.0 meq/g. Typical purity: 88.74 A%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25ºC) Store under inert atmosphere, at temperature no higher than 22ºC.
- Stability and homogeneity of the test material in the vehicle under test conditions and during storage: The stability of the test item in the vehicle was established at 1 and 100 mg/mL under Eurofins Advinus Study No. G21131. Based on the interim results, the test item was stable in the vehicle up to to 24 hours when stored at room temperature and 5 days at refrigerated condition.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not specified
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): not specified

FORM AS APPLIED IN THE TEST: liquid

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: 40 male and 40 female Wistar rats, Hylasco Biotechnology (India)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at start of treatment: 6-7 weeks
- Weight at start of treatment: Males : 0.195 to 0.246 kg, Females: 0.153 to 0.192 kg
- Fasting period before study: not specified
- 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. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area (SC-15). Steam sterilized corn cob was used as bedding and changed along with the cage at least twice a week.
- Diet: ad libitum, Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany
- Water: 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 to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: five days before start of the treatment

DETAILS OF FOOD AND WATER QUALITY: The feed and water provided to the rats was tested for contaminants. Based on the latest analytical certificate/s available, there were no known contaminants in the food, water and bedding that are expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 24°C
- Humidity (%): 49 to 66 %
- Air changes (per hr): 12.5-14.3 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2021-01-05 To: 2021-05-05

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (+/-3 hours) each day for 90 consecutive days.

Vehicle:

water

Remarks:

Milli-Q water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- pre-calibration of the beaker to desired volume: Milli-Q® water was measured in a graduated cylinder to the final volume of 60 mL. The measured water was transferred into a clean beaker and upper and lower meniscus of water was marked on the beaker using a marker. The water was discarded and the beaker was dried.
- Required quantities of the test item was weighed in a beaker (previously calibrated to a desired volume*) for each dose levels separately and a small volume of vehicle (Milli-Q® water) was added and stirred using a glass rod till a uniform solution was obtained. The volume was made up to the mark using the vehicle to get the final desired concentration of 7.5, 15 and 45 mg/mL for the G2, G3 and G4 groups, respectively. The solutions were mixed well by stirring using a magnetic stirrer.
- the dose formulations were prepared once daily before start of each day of dosing
- the volume of dose formulation prepared was varied depending on the requirement and/or body weights of the rats during experimental period.

VEHICLE
- Concentration in vehicle: 7.5, 15 and 45 mg/mL for the G2, G3 and G4 groups, respectively.
- Amount of vehicle (if gavage): 10 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd (Day 35) and 3rd (Day 71) month of the treatment period and analysed in-house. For each set, composite sample was drawn from each preparation and in case of control, duplicate composite sample was drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G21131. One set of samples were analyzed and other set (second set) of samples were stored at room temperature for possible reanalysis as a backup and these samples were discarded, as the analysis results of first set of samples were within the acceptable limits.
Formulations were considered acceptable as overall mean results were within ± 10.0 % of the claimed concentration and relative standard deviation (% RSD) was less than 10.0 %.

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

Daily at approximately the same time (± 3 hours)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

G1, vehicle control

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw (total dose)

Remarks:

G2, Low dose

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw (total dose)

Remarks:

G3, Mid dose

Doses / concentrations 4

Dose / conc.:

450 mg/kg bw (total dose)

Remarks:

G4, High dose

No. of animals per sex per dose:

Each group in the experiment was comprised of ten male and ten female rats.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 75 (G2), 150 (G3) and 450 (G4) mg/kg/day were selected for this study based on the results of an available OECD 422 study conducted by sponsor where the NOAEL was considered to be > 500 mg/kg bw/day (nominal dose received) for male and systemic toxicity and maternal systemic toxicity, and greater than 500 mg/kg bw/day for embryo-fetal toxicity in consultation with the Sponsor. In addition to the test doses, vehicle control group were included.

- Route of administration and justification: Route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of possible exposure in humans.

- Justification for Selection of Vehicle: The Formulation Analytical Method Validation and Stability was established in Milli-Q water under Eurofins Advinus study (G21131). Hence, Milli-Q water was used as vehicle for dose formulation preparation.

Examinations

Observations and examinations performed and frequency:

MORBIDITY and MORTALITY: Yes
- all rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon except during holidays wherein the observation was done once daily as there were no clinical signs observed.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each rat was observed for checking general clinical signs twice daily during treatment period. On the days of scheduled detailed clinical examination, clinical signs were included as a part of detailed clinical observations except on day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examination was done prior to the test item administration on Day 1 and at weekly intervals thereafter (± 1 days) during treatment period.
- During detailed clinical examination, all rats were observed cage-inside/outside for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size,
unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. selfmutilation, walking backwards).

FUNCTIONAL OBSERVATION BATTERY TESTS
- Time schedule: performed during the 13th week (day 85) of treatment 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 group. During this observation period, rat was evaluated as it moves about freely/unperturbed and the following observations were made and observations were recorded using core/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. The Opto-Varimex 5 data was analyzed in 10 minutes intervals 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. Observations: 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 onto a
horizontal surface of the tabletop from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were 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 SOP format was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values is 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. Averages 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

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 period. Fasting body weight was recorded prior to sacrifice for all surviving toxicity group animals.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption was measured at weekly intervals (± 1 day) during treatment period. The cage wise average food consumption (g/rat/day) was calculated


WATER CONSUMPTION AND COMPOUND INTAKE : No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment and at the end of the treatment period. Before examination, mydriasis was induced using a 1 % solution of Tropicamide.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, approximately 4.0 mL of blood was collected.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, fasted overnight (water allowed)
- blood was collected with a fine capillary tube, by retro-orbital sinus puncture (anti-coagulant: K2 EDTA for hematology; tridosium citrate for coagulation)
- How many animals: 40
- parameters: red blood corpuscles, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes count, white blood corpuscles, differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelets, red blood cell morphology (red cell distribution width, haemoglobin distribution width, hyperchromic cells, hypochromic cells, macrocytes, microcytes, RBC fragments, RBC ghosts)
- Coagulation: Blood samples collected for coagulation analysis were centrifuged at 2500 times gravity (xg) for 10 minutes 15ºC for separation of plasma and analysed for the following parameters in plasma sample using STart Max coagulation analyzer (Diagnostica stago, 92600 Asnieres, France)
- parameters: prothrombin time, activated partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, approximately 4.0 mL of blood was collected.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes , fasted overnight (water allowed)
- blood was collected with a fine capillary tube, by retro-orbital sinus puncture (anti-coagulant: lithium heparin)
- How many animals: 40
- Parameters: alanine aminotransferase, alkaline phosphatase, albumin, albumin/globulin ratio (calculated value), aspartate aminotransferase, blood urea nitrogen, chloride, creatinine, calcium, glucose, globulin (calculated value), HDL cholesterol, inorganic phosphorus, LDL cholesterol, potassium, sodium, total cholesterol, total plasma protein, triglycerides, total bilirubin

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: At the end of the treatment period, approximately 4.0 mL of blood was collected.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes , fasted overnight (water allowed)
- blood was collected with a fine capillary tube, by retro-orbital sinus puncture
- How many animals: 40
- blood samples collected in plain labeled tubes were kept on bench top for approximately 60 minutes before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 5 °C. The serum samples were placed in labeled plastic tubes and stored at +/- -70 °C until they were analyzed. Thyroid hormones were estimated by ELISA using Bio-RAD microplate washer and BIO-RAD model 680 readers.
- Hormones estimated: rodent thyroid stimulating hormone (TSH), rodent thyroxine (T4), rodent triiodothyronine (T3)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all rats at the end of the treatment period in the urine collection tube.
- Metabolism cages used for collection of urine: Yes, each rat was placed overnight in a specially fabricated cage (water allowed) and the next morning the collected urine was sent for analysis.
- Animals fasted: yes, water allowed
- Parameters: specific gravity, nitrite, pH, proteins, glucose, ketone bodies, urobilinogen, bilirubin, appearance (colour and clarity), volume (approximate)
- microscopic examination: crystals, epithelial cells, casts

NEUROBEHAVIOURAL EXAMINATION: Yes
- functional battery tested, as described above

IMMUNOLOGY: No

OTHER:
- Oestrous Cycle Evaluation: Vaginal smear was examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All rats at the end of treatment period were subjected to detailed necropsy and findings were recorded. The necropsy included examination of external surfaces of the body, all orifices, cranial, thoracic and abdominal cavities and their contents). Terminal body weights were recorded for all animals immediately
prior to sacrifice. All rats were fasted overnight (water allowed), euthanized with isoflurane anesthesia, exsanguinated and subjected for gross examination.
- tissue collection, weighing and preservation: On completion of gross pathology examination, the tissues and organs were collected and weighed from all rats. (see table in 'additional information'-field . The tissues were preserved in 10% Neutral Buffered Formalin (NBF) except for the testes
and eyes.

ORGAN WEIGHTS:
- The organ weight ratios (organ to body weight and organ to brain weight) as percentage of fasting body weight and brain weight were determined and presented in the report. The paired organs were weighed together, and combined weight was presented.

HISTOPATHOLOGY: Yes
- Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose group animals (G4).
- Histopathological examination of the testes included a qualitative assessment of stages of
spermatogenesis.
- all gross lesions from all the animals were examined microscopically. Liver, lungs, spleen and mandibular lymph nodes were examined in lower dose (G2 and G3) group rats as test item-related changes were noted in these organs at high dose (G4).
- The tissues were processed for routine paraffin embedding and 4-5-micron sections were stained with Haematoxylin and Eosin stain. In addition, testes were sectioned at 3-4 μm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.

Statistics:

Data was captured using the ProvantisTM laboratory information management system (LIMS). Parameters such as body weight, body weight change, body temperature, hindlimbs footsplay, grip performance, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry and transferred (motor activity, thyroid profile) data was evaluated using the Levene
Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of non-normal distribution, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data captured outside of ProvantisTM: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0. Descriptive statistics Mean, SD, Percentages & Numbers were presented by Treatment group and Day.
All hypothesis testing was carried out at the 5% (2-sided) significance level unless otherwise specified. Significant differences were designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

At 450 mg/kg/day, transient clinical sign of slight salivation was observed soon after the dose administration in all animals from treatment Day 21. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal.
There were no clinical signs or mortalities observed during the treatment at 75 and 150 mg/kg bwt/day dose group in either sex.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment did not affect the mean body weights in all the tested doses in either sex during the treatment period.
Significantly lower absolute body weight gain during days 8-15 at all the tested doses, during days 79-85 at mid and high doses and significantly higher absolute weight gain during days 64-71 in mid and high dose males was observed. In females, significantly higher absolute weight gain during days 57-64 and 71-79, significantly lower absolute body weight gain during days 64-71 at high dose was observed. These significant differences were toxicologically not significant as total absolute gains during day 1-90 were comparable to vehicle control. at 450 mg/kg/day, increase in the weight of liver was associated with vacuolation of centrilobular hepatocytes in both sexes.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males, the food consumption was significantly lower during Days 22-29, 29-36, 36-43, 50-57, 57-64, 71-79 and 79-85 was observed at mid dose. In females, significantly higher food consumption during Days 1-8 at low dose, during Days 1-8 and 85-90 at mid dose, during Days 79-85 at high dose and significantly lower during Days 43-50 at high dose was observed.
These significant differences were not considered toxicologically relevant as the body weights were not altered by the treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmological examination was carried out with an ophthalmoscope prior to start of treatment and at the end of the treatment period did not reveal any abnormalities in the eyes of the experimental rats.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg/day, increase in the total leukocyte, neutrophil, lymphocyte and monocyte counts
were observed in females and correlated with inflammation observed in lungs histologically.
There were no test item-related changes in coagulation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg/day in females, increase in total cholesterol, HDL cholesterol and triglyceride were correlated to the centrilobular vacuolation in liver microscopically. Decrease in total protein, albumin in both sexes; A/G ratio in females were observed without microscopic correlates.
At 75 and 150 mg/kg/day, increase in total cholesterol, HDL cholesterol in females were considered as test item-related changes without microscopic correlates.

Endocrine findings:

no effects observed

Description (incidence and severity):

The administration of the test item for 90 consecutive days through oral gavage to Wistar rats at 75, 150 and 450 mg/kg/day did not show any test item-related changes in thyroid profile.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The administration of the test item for 90 consecutive days through oral gavage to Wistar rats at 75, 150 and 450 mg/kg/day did not show any test item-related changes in urinalysis parameters.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Home cage and Handling observations: No treatment-related abnormalities were observed in any of the tested dose groups in either sex.
- Open field observations: No treatment-related abnormalities were observed in any of the doses tested in either sex.
- Sensory observations: No treatment-related abnormalities were observed in any of the doses tested in either sex.
- Motor Activity: There were no significant differences observed at all the tested doses in both sexes. Incidence of significantly lower distance travelled during interval 2 was observed at all treated groups in females. This significant difference was considered toxicologically not relevant as the total distance travelled was comparable the vehicle control.
- Neuromuscular observation:
- Landing hind limb footsplay: Significantly lower hindlimb footsplay was observed in high dose males. This significant difference was considered to toxicologically not significant as there were no changes observed in the home cage or open field observations. Further, there were no clinical signs observed during daily clinical observation.
- Grip strength: Significantly lower hindlimb grip strength at high dose in males and forelimb grip strength at mid dose in females was observed. These significant differences were considered toxicologically not significant as the animals were normal in the home cage and open field observations.
- Physiological observation:
- Body temperature: Incidence of significantly lower body temperature was observed in males at mid dose. This change was considered to be toxicologically not significant as there was no dose dependency.
- Body weight: There were no significant differences observed in the body weights, measured at the end of neurological observations at any of the doses tested in both sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg/day, increase in the weight of liver was associated with vacuolation of centrilobular
hepatocytes in both sexes.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross pathological findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings were observed in liver (centrilobular vacuolation of hepatocytes), lungs (increased alveolar macrophages/chronic inflammation), spleen (vacuolated macrophage in red pulp) and mandibular lymph nodes (vacuolated macrophage) in males and/or females.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Oestrous Cycle Evaluation: The vaginal smear was examined for all the animals prior to necropsy and following is the details of various stages observed. Most of the rats showed diestrous and proestrous to necropsy

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (nominal)

System:

immune system

Organ:

liver

lungs

lymph node

spleen

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Dose Formulation Analysis of the Test Item
The dose formulations were considered acceptable, as the percent agreement of the analyzed concentrations were in the range, 90% to 110% of the claimed concentrations and the relative standard deviation (RSD) was equal to or less than 10.0%.

 

Data tables

Table 1: Details of Experimental Design, Treatment Regime, Clinical Pathology Investigations, Sacrifice and Pathology Schedule.

 

				Clinical pathology investigations				Pathology
Group No.	Dose (mg/kg/ day)	No. of rats per Group	Treatment Period (Days 1-90)	Haema -tology	Coagula -tion	Clinical Chemistry	Urina -lysis	Gross Pathology	Organ Weights	Histopathology	Sacrificed on Day
G1	0	M:10 F: 10	M: + F: +	+	+	+	+	+	+	+	91
G2	75	M:10 F: 10	M: + F: +	+	+	+	+	+	+	X	91
G3	150	M:10 F: 10	M: + F: +	+	+	+	+	+	+	X	91
G4	450	M:10 F: 10	M: + F: +	+	+	+	+	+	+	+	91

+: Performed
M: Male
F: Female
X: Gross lesions

 

 

 

 

 

 

Table 2: Total leukocyte, neutrophil, lymphocyte and monocyte counts in males and females (percentage alterations).

 

Sex	Male			Female
Group	G2	G3	G4	G2	G3	G4
Dose (mg/kg/day)	75	150	450	75	150	450
No. of rats	10	10	10	10	10	10
WBC	―	―	―	―	―	↑(24)
Neut A	―	―	―	―	―	↑(55)*
Lymp A	―	―	―	―	―	↑(13)
Mono A	―	―	―	―	―	↑(46)

 

 

 

 

 

 

 

 

↑: Increased
*: Statistically significant
─: Not statistically/ biologically significant

Values in parenthesis indicate percentage change, when compared to vehicle control

 

 

 

 

 

 

 

 

Table 3:  Total cholesterol, HDL cholesterol, triglyceride, protein and albumin counts, and A/G ratio in males and females (percentage alterations).

 

Sex	Male			Female
Group	G2	G3	G4	G2	G3	G4
Dose (mg/kg/day)	75	150	450	75	150	450
No. of rats	10	10	10	10	10	10
T.Chol	―	―	―	↑(21)*	↑(20)*	↑(28)*
HDL Chol	―	―	―	↑(19)*	↑(15)*	↑(25)*
Trig	―	―	―	―	―	↑(129)*
T.Pro	―	―	↓(6)*	―	―	↓(9)*
ALB	―	―	↓(7)*	―	―	↓(23)*
A/G ratio	―	―	―	―	―	↓(27)*

 

 

 

 

 

 

 

 

 

 

↑: Increased ↓: Decrease *: Statistically significant

─: Not statistically/ biologically significant

Values in parenthesis indicate percentage change, when compared to vehicle control

 

 

Table 4: Increase in the weight of liver was associated with vacuolation of centrilobular hepatocytes in males and females (percentage alterations).

 

Sex	Male			Female
Group	G2	G3	G4	G2	G3	G4
Dose (mg/kg/day)	75	150	450	75	150	450
No. of rats	10	10	10	10	10	10
Liver     –Absolute	―	―	↑(6)	―	―	↑(27)*
–Relative to Bwt	―	―	↑(10)*	―	―	↑(28)*
–Relative to Brain wt	―	―	↑(8)	―	―	↑(29)*

↑: Increased *: Statistically significant ─: Not statistically/ biologically significant

Values in parenthesis indicate percentage change, when compared to vehicle control

 

 

Table 5: Treatment related microscopic findings observed in liver, lungs, spleen and mandibular lymph nodes in males and females.

 

Sex	Males				Females
Group	G1	G2	G3	G4	G1	G2	G3	G4
Dose (mg/kg/day)	0	75	150	450	0	75	150	450
No. of rats	10	10	10	10	10	10	10	10
LIVER; Vacuolation; hepatocyte; centrilobular minimal mild moderate	(10) 0 − − −	(10) 0 − − −	(10) 0 − − −	(10) 9 6 3 −	(10) 0 − − −	(10) 0 − − −	(10) 0 − − −	(10) 9 4 3 2
LUNGS (WITH BRONCHI AND BRONCHIOLES); Alveolar macrophages, increased minimal mild Inflammation; chronic; bronchioloalveolar; multi-focal minimal mild	(10) 0 − − 0 − −	(10) 4 4 − 0 − −	(10) 3 3 − 0 − −	(10) 2 2 − 0 − −	(10) 2 2 − 0 − −	(10) 2 2 − 0 − −	(10) 2 2 − 0 − −	(10) 10 6 4 5 2 3
SPLEEN; Vacuolation; macrophage; red pulp minimal mild	(10) 0 − −	(10) 0 − −	(10) 0 − −	(10) 9 8 1	(10) 0 − −	(10) 0 − −	(10) 0 − −	(10) 10 1 9
LYMPH NODES, MANDIBULAR; Vacuolation; macrophage; unilateral/bilateral minimal	(10) 0 −	(10) 0 −	(10) 0 −	(10) 1 1	(10) 0 −	(10) 0 −	(10) 0 −	(10) 9 9

 

Applicant's summary and conclusion

Conclusions:

Considering the changes observed in haematology, clinical chemistry, organ weights and microscopic changes at 450 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the test item is considered to be 150 mg/kg/day under the test conditions and doses employed.