N-[3-(dimethylamino)propyl]stearamide monoacetate

Endpoint summary

Description of key information

One valid repeated dose toxicity studies are available for the test item NDPSa (Cas n.: 13282-70-7,other identifier: H-31339). 

 

Repeated Dose Oral 28 days, EU B.7, K1; NOAEL= 5 mg/kg body weight/day, LOAEL=25 mg/kg; Hoban D.B.A. (2015)

Supporting Study:

Repeated Dose Oral 14 days, EU B.7, S1; (NOAEL) 100 mg/kg/day in male rats and 250 mg/kg/day in female rats; Hoban D.B.A. (2015)

 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2015-02-04 to 2015-08-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at DuPont Haskell.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Laboratories International, Inc., Raleigh, North Carolina, U.S.A.
- Females (if applicable) nulliparous and non-pregnant: [yes] 55
- Age at study initiation: 7 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing:housed in pairs in solid-bottom caging with bedding and appropriate species- specific enrichment.
- Diet (e.g. ad libitum): PMI® Nutrition International LLC Certified Rodent LabDiet® 5002 ad libitum, except during the neurobehavioral evaluations.
- Water (e.g. ad libitum):tap water ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
-Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants.
-Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-26°C
- Humidity (%):30-70%
- Air changes (per hr): n/a
- Photoperiod (hrs dark / hrs light):12-hour light/dark cycle

IN-LIFE DATES: From: 19 February 2015 To: 20 March 2015

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

0.1% Tween 80 in deionized water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):n/a
- Mixing appropriate amounts with (Type of food):n/a
- Storage temperature of food:n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a
- Concentration in vehicle: 0.1% tween 80 in deionised water
- Amount of vehicle (if gavage): n/a
- Lot/batch no. (if required): n/a
- Purity:n/a

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical results show that the test substance was at the targeted concentrations for all dose levels and stable in the gavage vehicle when stored at room temperature in the concentration range from 1 to 50 mg/mL for up to 16 days.
The test substance was not detected in the gavage vehicle.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Control

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Remarks:

group 2

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

group 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

group 4

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

group 5

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale: n/a
- Rationale for animal assignment (if not random):n/a
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups: n/a
- Post-exposure recovery period in satellite groups: n/a
- Section schedule rationale (if not random):n/a
- Dose range finding studies:n/a
- Other:n/a

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily. An additional cage-site evaluation was conducted daily at approximately 2 hours (± 1 hour) post- dosing to detect acute clinical signs of systemic toxicity. Exceptions to the time range did not impact the integrity of the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: day 1 and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 and weekly thereafter.

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; Food consumption was statistically significantly increased in the 25 and 100 mg/kg/day female groups. These increases were observed during weeks 1, 2, 3 (test days 1-8, 8-15, 15-22) and overall (test days 1-28).

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
250 mg/kg/day female group during week 4 (test days 22-28)

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes; This difference correlated with lower body weight gains during the same period; however, overall food consumption was similar to the control and not statistically significant. Therefore, lower food consumption in females may be test substance-related but not adverse.
Overall food consumption (days 1-28) in females dosed with 5, 25, 100 or 250 mg/kg/day was 104, 108, 107, and 95% of the control group, respectively.

Food efficiency was statistically significantly lower in males dosed with 250 mg/kg/day during week 4 and overall. Overall food efficiency (days 1-28) in males dosed with 5, 25, 100 or 250 mg/kg/day was 100, 92, 84, and 68% of the control group, respectively.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:n/a

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-test and approximately week 4.
- Dose groups that were examined: all groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:end of the study: end of the study
- Anaesthetic used for blood collection: Yes (identity), isoflurane.
- Animals fasted: Yes, for 15 hours with access to water.
- How many animals:all animal.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 1 day before the end of the study.
- Animals fasted: Yes for 15 hours.
- How many animals:all

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: 1 day before the end of the study.
- Animals fasted: Yes, for 15 hours.
- How many animals: all.

URINALYSIS: Yes
- Time schedule for collection of urine: 1 day before the end of the study.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, 15 hours.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:during acclimatisation period and on week 4, on all surviving animals.
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Yes
- Time schedule for examinations: a day before the end of the study.
- How many animals: all
- Dose groups that were examined: all

OTHER: n/a

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
Rats sacrificed by design (scheduled sacrifice) were fasted at least 15 hours.

HISTOPATHOLOGY: Yes (see table)

Statistics:

Significance was judged at p < 0.05. Separate analyses were performed on the data collected for each sex.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance–related clinical signs observed in the 100 or 250 mg/kg/day male and female groups included labored breathing, lung noise, diarrhea, gasping, brown/red skin discoloration, swollen abdomen, and fast breathing. Other clinical signs were either typical for the animals age and strain or did not occur in a dose-related manner and were not considered test substance- related.

Mortality:

mortality observed, treatment-related

Description (incidence):

Only one male died.
One male in the 250 mg/kg/day group (#509) was euthanized on test day 12. Gross and microscopic evaluation indicated lesions compatible with exposure to the test compound (gastrointestinal distension with gas).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Adverse, test substance-related effects on body weight and body weight gain were observed at 100 and 250 mg/kg/day in male rats. Statistically significant differences on group mean body weight in these male groups were noted at test days 8 (not significant at 100 mg/kg/day), 15, 22, and 28. At test day 28, body weight differences were 10 and 17% below the control for male rats in the 100 or 250 mg/kg/day groups, respectively. There were no statistically significant differences on body weight in females at any dose level.
Variable statistically significant differences on mean body weight gain were observed in male rats at 100 and 250 mg/kg/day throughout the study. Overall body weight gains (days 1-28) in males dosed with 5, 25, 100 or 250 mg/kg/day were 97, 90, 80, and 60% of the control group (statistically significant at 100 and 250 mg/kg/day). There were no adverse effects on body weight gain in females at any dose level. A statistically significant decrease in body weight gain was observed in females at 250 mg/kg/day during week 4. This lower body weight gain was related to decreased food consumption and may have been test substance-related but not adverse as there were no significant changes in body weight. Overall body weight gains (days 1-28) in females dosed with 5, 25, 100 or 250 mg/kg/day were 109, 116, 111, and 88% of the control group, not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was statistically significantly increased in the 25 and 100 mg/kg/day female groups. These increases were observed during weeks 1, 2, 3 (test days 1-8, 8-15, 15-22) and overall (test days 1-28). Statistically significantly lower food consumption was observed in the 250 mg/kg/day female group during week 4 (test days 22-28). This difference correlated with lower body weight gains during the same period; however, overall food consumption was similar to the control and not statistically significant. Therefore, lower food consumption in females may be test substance-related but not adverse. Overall food consumption (days 1-28) in females dosed with 5, 25, 100 or 250 mg/kg/day was 104, 108, 107, and 95% of the control group, respectively.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Adverse, test substance-related effects on food consumption and food efficiency were observed at 250 mg/kg/day in male rats.
Food efficiency was statistically significantly lower in males dosed with 250 mg/kg/day during week 4 and overall. Overall food efficiency (days 1-28) in males dosed with 5, 25, 100 or 250 mg/kg/day was 100, 92, 84, and 68% of the control group, respectively.
There were no statistically significant differences in food efficiency in females at any dose level. Overall food efficiency (days 1-28) in females dosed with 5, 25, 100 or 250 mg/kg/day was 104, 107, 104, and 92% of the control group, respectively.

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test substance-related ophthalmological lesions were observed. At the end of the study, retinal degeneration was observed in one male at 5 mg/kg/day and one female in each 25 and 250 mg/kg/day groups and retinal hemorrhage in one female at 25 mg/kg/day. This observation was considered to be spurious, as the finding only occurred in one animal, and there was no dose response relationship. During the baseline ophthalmology evaluation, one female rat was observed with ophthalmology abnormalities and was not placed on study.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Absolute neutrophil count (ANEU) was statistically significantly higher in males and females at 250 mg/kg/day (76% and 146% above control, respectively). These changes were considered to be test substance related and were likely a response to adverse changes—reflux rhinitis— observed in the nose of male and female rats at this dose level.
Red blood cell mass parameters (red blood cell [RBC], hemoglobin [HGB], and hematocrit [HCT]) were minimally higher in male rats exposed to 250 mg/kg/day. Decrements in body weight were also observed in this group, and these minimally higher red cell mass parameters were likely the result of hemoconcentration (dehydration), a common secondary effect associated with body weight decrements in rats.
All other statistically significant hematology changes were considered unrelated to exposure and/or non-adverse

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no adverse effects on clinical chemistry parameters in male or female animals. The statistically significant differences in mean clinical chemistry parameters were not adverse or not related to exposure to the test substance.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related or adverse effects on urinalysis parameters in male or female animals. Statistically significant differences were limited to higher urine volume and lower urine specific gravity in the 25 mg/kg/day male group, and were considered to be unrelated to treatment and non-adverse because they did not occur in a dose-related pattern.

Immunological findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight effects considered secondary to stress associated with reflux rhinitis (see below under Microscopic Findings) were observed at 250 mg/kg/day in male and female rats. These included increased adrenal weights and decreased weights of lymphoid organs (thymus and spleen). Decreases in weight parameters for the thymus (both sexes) and the spleen (males only) correlated with histological observations of lymphoid depletion in both organs.
All other individual and mean organ weight differences were not associated with any test substance-related gross or microscopic findings

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no adverse changes in clinical pathology parameters in male or female rats administered the test substance at doses of ≤ 100 mg/kg/day.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Test substance-related decreases in duration of movement and number of ambulatory movements were observed in both sexes at 250 mg/kg/day and in males at 100 mg/kg/day; the motor activity decreases were considered secondary to systemic toxicity. There were no test substance-related effects on motor activity in males at ≤25 mg/kg/day or in females at ≤100 mg/kg/day. There were no test substance related effects on any other neurobehavioral parameter evaluated in males or females at any dose level.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Animals 509, 510, 551 and 556 had correlative histological lesions within their gastrointestinal tracts, including crypt hyperplasia within the jejunum and/or duodenum. Animal 509 also had vesicle formation with inflammation within the forestomach. The small thymus from animal 560 had histologically evident lymphoid depletion. All of the above lesions were test substance-associated.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

LOAEL

Effect level:

25 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Table: Haematology, urinalysis and clinical chemistry parameters (checked (X) examined at end of study period)

Laboratory parameters determined
Haematology:
	Red blood cells		White blood cells		Clotting Potential
x	Erythrocyte count (RBC)		Total leukocyte count		Prothrombine time
x	Hemoglobin (Hb)	x	Neutrophils (differential)		Thrombocyte count
x	Hematocrit (Ht)		Eosinophils (differential)	x	Thromboplastin time (PT)
x	Mean corp. volume (MCV)		Basophils (differential)		Clotting time
x	Mean corp. hemoglobin (MCH)		Lymphocytes (differential)	x	Activated partial thromboplastin time (APTT)
x	Mean corp. Hb. conc. (MCHC)		Monocytes  (differential)
	Reticulocytes
x	Platelets
Clinical chemistry:
	Electrolytes		Metabolites and Proteins		Enzymes:		Hormones:
x	Calcium	x	Albumin	x	Alanine aminotransferase (ALAT)		T3
x	Chloride (Cl)		A/G ratio	x	Aspartate aminotransferase (ASAT)		T4
x	Phosphorus (inorganic)	x	Bilirubin (total)	x	Alkaline phosphatase (ALP)		TSH
x	Potassium (K)		Cholesterol (total)	x	g-glutamyl transpeptidase (g-GT)		Other hormones^a
x	Sodium (Na)	x	Creatinine		Cholinesterase (ChE)
	Magnesium		Globulins	x	Creatine phosphokinase
		x	Glucose		Lactic acid dehydrogenase (LDH)
			Protein (total)		Sorbitol dehydrogenase
		x	Triglycerides
			Urea nitrogen
			Serum protein electrophoresis
Urinalysis:
	Quantitative parameters:		Semiquantitative parameters
x	Urine volume		Bilirubin		Leukocytes
x	Relative density		Ocult blood		Protein
			Color and appearance	x	pH-value
			Glucose	x	Urobilinogen
			Ketones		Sediment (microscopical exam.)

 

Table: Gross necropsy and tissue preparation (checked (X) collected (column C), weighed (column W) and 
examined for histopathology (column H with X = all groups, # = control, 0 mg/kg/day and 5250 mg/kg bw/day animals)

Pathology:
C	W	H		C	W	H		C	W	H
x	x	x	Adrenal glands	x	x	x	Liver (2 lobes)	x			Stomach
x			Aorta	x			Lung (2 lobes)	x	x	x	Testis
			Bile duct	x			Lymph nodes^#	x	x	x	Thymus
x	x	x	Brain* (3 levels)	x			Female mammary gland	x			Thyroid/parathyroid
x			Bone marrow (FEMUR and STERNUM)				Male mammary gland				Tongue
x			Caecum	x			Muscle, skeletal(biceps femoris knee joint sternum)	x			Trachea
x			Colon	x			Nasal cavity (Level I – IV)	x			Urinary bladder
x			Duodenum	x			Nerve, peripheral [sciatic n. optic n. spinl cords (3 levels)]	x	x	x	Uterus
			Epididymides	x	x	x	Ovary and oviduct	x			Vagina
x			Esophagus	x			Pancreas				Others:skin
x			Eyes (with optic nerve)	x			Pituitary	x		x	Body (exsanguinated)
x			Femur (with joint)	x			Pharynx		n.a.		Vaginal smears
			Gall bladder (mouse)	x	x	x	Prostate
			Gross lesions/masses	x			Rectum
x	x		Heart	x			Salivary glands (mandibular, sublingual)
x			Ileum	x	x	x	Seminal vesicles (with coagulating glands and fluids)
x			Jejunum	x			Skin/subcutis
x	x	x	Kidneys	x		x	Spinal cord (3 levels cervical mid-torachic lumbar)
			Lachrymal glands	x	x	x	Spleen
x			Larynx (Level I – III)	x			Sternum

 

 

 

 

Incidence and Severity of Stomach and Small Intestine Lesions in Rats (n=10)

Sex	Males					Females
Dose (mg/kg/day)	0	5	25	100	250	0	5	25	100	250
				Stomach
Vesicles, mucosal epithelium	0	0	0	0	2	0	0	0	0	2
Minimal	-	-	-	-	-	-	-	-	-	2
mild	-	-	-	-	1	-	-	-	-	-
Moderate	-	-	-	-	1	-	-	-	-	-
Hyperplasia, mucosal epithelial	0	0	0	0	2	0	0	0	0	0
Mild	-	-	-	-	2	-	-	-	-	-
				Duodenum
Crypt hyperplasia	0	0	0	0	5	0	0	0	0	5
Minimal	-	-	-	-	5	-	-	-	-	4
Mild	-	-	-	-	-	-	-	-	-	1
				Jejunum
Crypt hyperplasia	0	0	0	0	4	0	0	0	0	0
minimal	-	-	-	-	4	-	-	-	-	-

 

Incidence and Severity of Reflux Rhinitis-Associated Lesions in Rats (N=10)

Sex	Males					Females
Dose (mg/kg/day)	0	5	25	100	250	0	5	25	100	250
Rhinitis, Reflux	0	0	4	7	7	0	0	4	6	8
Minimal	-	-	-	3	1	-	-	3	1	1
Mild	-	-	4	4	2	-	-	1	4	1
Moderate	-	-	-	-	3	-	-	-	1	2
Severe	-	-	-	-	1	-	-	-	-	4
Adhesion	0	0	0	1	3	0	0	0	1	6
present	-	-	-	1	3	-	-	-	1	6

 

Summary of Clinical Observations and Mortality in Male Rats

Day numbers relative to Start Date
	0 mg/kg/day	5 mg/kg/day	25 mg/kg/day	100 mg/kg/day	250 mg/kay/day
Scheduled sacrifice
Number of Observation	10	10	10	10	9
Number of Animals	10	10	10	10	9
Days from – to	29  29	29  29	29  29	29  29	29  29
Unscheduled Sacrifice
Number of Observation	-	-	-	-	1
Number of Animals	-	-	-	-	1
Days from – to	-	-	-	-	12  12
Breathing – labored
Number of Observation	-	-	-	1	-
Number of Animals	-	-	-	1	-
Days from – to	-	-	-	6  6	-
Breathing – lung noise
Number of Observation	-	-	-	1	6
Number of Animals	-	-	-	1	3
Days from – to	-	-	-	28  28	10  28
Diarrhoea
Number of Observation	-	-	-	-	1
Number of Animals	-	-	-	-	1
Days from – to	-	-	-	-	12  12
Discharge – red
Number of Observation	-	-	-	1	-
Number of Animals	-	-	-	1	-
Days from – to	-	-	-	6  6	-
Gasping
Number of Observation	-	-	-	-	4
Number of Animals	-	-	-	-	2
Days from – to	-	-	-	-	10  28
Hair loss
Number of Observation	1	1	-	1	-
Number of Animals	1	1	-	1	-
Days from – to	28  28	28  28	-	28  28	-
Scab
Number of Observation	1	-	-	1	-
Number of Animals	1	-	-	1	-
Days from – to	28  28	-	-	28  28	-
Discoloured skin – brown
Number of Observation	-	-	-	-	1
Number of Animals	-	-	-	-	1
Days from – to	-	-	-	-	28  28
Discoloured skin – red
Number of Observation	-	-	-	-	1
Number of Animals	-	-	-	-	1
Days from – to	-	-	-	-	12  12
Swollen – abdomen
Number of Observation	-	-	-	-	2
Number of Animals	-	-	-	-	2
Days from – to	-	-	-	-	12  28
Wound - superficial
Number of Observation	-	2	-	-	-
Number of Animals	-	1	-	-	-
Days from – to	-	22  28	-	-	-
Retinal Degeneration
Number of Observation	-	1	-	-	-
Number of Animals	-	1	-	-	-
Days from – to	-	23  23	-	-	-

 

Summary of Clinical Observations and Mortality in Female Rats

Day numbers relative to Start Date
	0 mg/kg/day	5 mg/kg/day	25 mg/kg/day	100 mg/kg/day	250 mg/kay/day
Scheduled sacrifice
Number of Observation	9	10	10	10	10
Number of Animals	9	10	10	10	10
Days from – to	30  30	30  30	30  30	30  30	30  30
Unscheduled Sacrifice
Number of Observation	1	-	-	-	-
Number of Animals	1	-	-	-	-
Days from – to	8  8	-	-	-	-
Abnormal gait
Number of Observation	2	-	-	-	-
Number of Animals	1	-	-	-	-
Days from – to	7  8	-	-	-	-
Breathing – fast
Number of Observation	-	-	-	-	3
Number of Animals	-	-	-	-	1
Days from – to	-	-	-	-	15  28
Breathing – lung noise
Number of Observation	-	-	-	1	1
Number of Animals	-	-	-	1	1
Days from – to	-	-	-	28  28	28   28
Discharge - red
Number of Observation	1	-	-	-	-
Number of Animals	1	-	-	-	-
Days from – to	8  8	-	-	-	-
Hair loss
Number of Observation	-	-	-	1	-
Number of Animals	-	-	-	1	-
Days from – to	-	-	-	28  28	-
Swollen shoulder left
Number of Observation	2	-	-	-	-
Number of Animals	1	-	-	-	-
Days from – to	7  8	-	-	-	-
Retinal Degeneration
Number of Observation	-	-	1	-	1
Number of Animals	-	-	1	-	1
Days from – to	-	-	23  23	-	23  23
Retinal Hemorrhage
Number of Observation	-	-	1	-	-
Number of Animals	-	-	1	-	-
Days from – to	-	-	23  23	-	-

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for H-31339 was 5 mg/kg body weight/day for male and female rats based on the adverse anatomic pathology findings (rhinitis due to reflux of the test substance into the nasal cavity) at 25 mg/kg body weight/day and above and test substance-related, adverse microscopic findings in the stomach, duodenum and jejunum (jejunum males only) of male and female rats administered 250 mg/kg body weight /day. Adverse, test substance-related effects on body weight and body weight gain were observed in males dosed with 100 and 250 mg/kg/day and on nutritional parameters in males at 250 mg/kg/day. Additional findings occurring in clinical pathology parameters included increases in absolute neutrophil count which were present in males and females administered 250 mg/kg/day and red cell mass parameters indicative of secondary hemoconcentration were also observed in the 250 mg/kg/day male group.

Executive summary:

The objective of this study was to assess the potential repeated-dose oral toxicity of H-31339 in male and female rats. Five groups of young adult male and female Crl:CD(SD) rats (10/sex/group) were dosed by oral gavage for 28 days. Rats were dosed at 0, 5, 25, 100, or
250 mg/kg body weight/day H-31339. The analytical results showed that the test substance was at the target concentrations for all analyzed levels in the gavage vehicle. All rats received evaluations of weekly body weight and nutritional parameters, daily and weekly clinical observations, baseline and end of study ophthalmology and neurobehavioral parameters. Clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis) and anatomic pathology parameters (organ weights, gross and microscopic pathology) were evaluated at the end of the study.

One test substance-related death occurred in a male administered 250 mg/kg/day. One control group female was euthanized on test day 8 due to a dosing accident. All other animals survived to the scheduled necropsy. Test substance–related clinical signs observed in the 100 or
250 mg/kg/day male and female groups included labored breathing, lung noise, diarrhea, gasping, brown/red skin discoloration, swollen abdomen, and fast breathing.

Adverse, test substance-related reductions on body weight and body weight gain were observed in males dosed with 100 and 250 mg/kg/day H-31339. Adverse, test substance-related reductions in nutritional parameters were observed in males at 250 mg/kg/day. No adverse effects on body weight, body weight gain, food consumption or food efficiency were observed in females at any dose level.

Test substance-related decreases in duration of movement and number of ambulatory movements were observed in both sexes at 250 mg/kg/day and in males at 100 mg/kg/day; the motor activity decreases were considered secondary to systemic toxicity. There were no test substance-related effects on motor activity in males at ≤25 mg/kg/day or in females at ≤100 mg/kg/day. There were no test substance related effects on any other neurobehavioral parameter evaluated in males or females at any dose level.

Adverse, test substance-related increases in absolute neutrophil count were present in males and females administered 250 mg/kg/day of the test substance. These changes are indicative of an inflammatory response and were likely correlative to reflux rhinitis observed microscopically. Minimal increases in red cell mass parameters indicative of secondary hemoconcentration were also observed in the 250 mg/kg/day male group. There were no adverse changes in clinical pathology parameters in male or female rats administered the test substance at doses of

≤ 100 mg/kg/day.

There were no adverse effects on ophthalmology in male or female rats at any dose level.

Test substance-related and adverse changes were present in the nose of male and female rats administered 25 mg/kg/day and above and were characteristic of rhinitis due to reflux of test substance into the nasal cavity. Laryngeal lesions indicative of reflux were also present in some male and female rats administered 250 mg/kg/day. Test substance-related and adverse

microscopic findings were also present in the stomach, duodenum and jejunum (jejunum in males only) of male and female rats administered 250 mg/kg/day and included mucosal epithelial vesicle formation with associated mucosal hyperplasia and inflammation (forestomach) and crypt hyperplasia (duodenum and jejunum). Mesenteric lymph nodes from male and female rats administered 250 mg/kg/day had histiocytosis. In addition, changes in lymphoid organs, largely considered stress-related findings occurring secondary to severe nasal lesions, were observed in males and females at 250 mg/kg/day and in males at 100 mg/kg/day. Gross lesions consistent with the microscopic changes observed in the stomach, small intestine and thymus were present in animals administered 250 mg/kg/day. Organ weight changes (adrenals and lymphoid organs) considered secondary to stress associated with reflux rhinitis occurred in males and females administered 250 mg/kg/day. No test substance-related microscopic changes were present in males or females administered 5 mg/kg/day.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for H-31339 was 5 mg/kg body weight/day for male and female rats based on the adverse anatomic pathology findings (rhinitis due to reflux of the test substance into the nasal cavity) at 25 mg/kg body weight/day and above and test substance-related, adverse microscopic findings in the stomach, duodenum and jejunum (jejunum males only) of male and female rats administered 250 mg/kg body weight /day. Adverse, test substance-related effects on body weight and body weight gain were observed in males dosed with 100 and 250 mg/kg/day and on nutritional parameters in males at 250 mg/kg/day. Additional findings in clinical pathology parameters included increases in absolute neutrophil count, in males and females administered 250 mg/kg/day and increased red cell mass parameters indicative of secondary hemoconcentration in the 250 mg/kg/day male group.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The materials/methods and results are described in detail and are sufficient for evaluation.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

 

 

 

Additional information

In the 28-days, K1 study, the substance was formulated with 0.1% of Tween 80 in deionized water vehicle, and adiministrated at doses by daily oral gavage from 0 (control), 5, 25, 100, to 250 mg/kg/day to 10 females and 10 males for each group. The animals were observed for clinical signs, mortality, through the monitoring period of 28 days. 

One test substance-related death occurred in a male administered 250 mg/kg/day.

One control group female was euthanized on test day 8 due to a dosing accident. (gavage administration lesions)

The NDPSa showed diverse adverse effect, reductions on body weight and body weight gain were observed in males dosed with 100 and 250 mg/kg/day H-31339. Adverse, test substance-related reductions in nutritional parameters were observed in males at 250 mg/kg/day.

Other decreases in duration of movement and number of ambulatory movements were observed in both sexes at 250 mg/kg/day and in males at 100 mg/kg/day; the motor activity decreases were considered secondary to systemic toxicity.

Adverse, test substance-related increases in absolute neutrophil count were present in males and females administered 250 mg/kg/day of the test substance. These changes are indicative of an inflammatory response and were likely correlative to reflux rhinitis observed microscopically. Minimal increases in red cell mass parameters indicative of secondary hemoconcentration were also observed in the 250 mg/kg/day male group. There were no adverse changes in clinical pathology parameters in male or female rats administered the test substance at doses of ≤ 100 mg/kg/day.

There were no adverse effects on ophthalmology in male or female rats at any dose level.

No-observed-adverse-effect level (NOAEL) for H-31339 was 5 mg/kg body weight/day for male and female rats and Lowest-Observed-Adverse-Effect-Level (LOAEL) is 25 mg/kg based on the evaluation of the adverse effects observed in the anatomic pathology results.

 

The 14 days study, can be used as supporting data.

It was assessed the potential repeated-dose oral toxicity of H-31339 in male and female rats at established concentrations for a subsequent repeat-dose toxicity study. Five groups of young adult male and female Crl:CD(SD) rats (5/sex/group) were dosed by oral gavage for 14 days. Rats were dosed at 0, 20, 100, 250, or 500 mg/kg/day H-31339.

Adverse test substance-related effects on in-life parameters were observed in male and female rats at 500 mg/kg/day. Four rats in the 500 mg/kg/day group were euthanized prior to the scheduled sacrifice due to severe weight loss, decreased food consumption and clinical signs. Adverse test substance-related effects were observed on body weight, body weight gain, food consumption, and food efficiency in male and female rats fed 500 mg/kg/day H-31339.

No adverse changes were noted in clinical pathology parameters in males or females at any dose level.

Microscopic pathology findings included adverse lesions in the nose at 250 and 500 mg/kg/day and in the stomach at 500 mg/kg/day; and test substance-related changes in the thymus, pancreas and salivary glands at 250 and/or 500 mg/kg/day. Minimal to severe reflux rhinitis in the nose occurred in animals dosed at 500 and 250 mg/kg/day. The dose-related increase in severity grades and incidence of reflux rhinitis suggest that the highest dose of test substance both increased the frequency of reflux/regurgitation and was directly irritating to the nasal epithelium upon contact. Stomach findings (hyperplasia, inflammation, degeneration, erosion/ulcer) were probably a result of irritation from contact with the test substance.

(NOAEL) for H-31339 was 100 mg/kg/day in male rats and 250 mg/kg/day in female rats.

 

Justification for selection of repeated dose toxicity – oral  route endpoint -
one key 1 study used

 

 

 

 

Justification for classification or non-classification

Based on the results of the 28-days sub-acute oral toxicity study, the substance does not meet the criteria for STOT-RE classification in accordance with GHS and CLP.