Fatty acids, C14-22, 2-ethylhexyl esters, epoxidized

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 February 2022 to 27 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry, and Fisheries (JMAFF) Test Data for Registration of Agricultural Chemicals, 30 Shouan No. 6278, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

An exception is test item characterization as these tests were performed in accordance with the ISO 9001 quality standard.

Limit test:

no

Test material

Test material information

1

Reference substance name:

Fatty acids, C14-22, 2-ethylhexyl esters, epoxidized

EC Number:

305-962-8

EC Name:

Fatty acids, C14-22, 2-ethylhexyl esters, epoxidized

Cas Number:

95370-96-0

Molecular formula:

Not necessary, substance is a UVCB

IUPAC Name:

Fatty acids, C14-22, 2ethylhexylesters, epoxidized

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): Sponsor
- Lot/batch number of test material: WO61290
- Purity: 100% (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in a tightly closed container, room temperature, 15 to 25ºC.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: Stability was evaluated between 10 and 750 mg/mL (Labcorp Study 8484276). Stable for up to 24 hours at room temperature; up to 8 days at 2 to 8°C; and up to 8 days at -60 to -80°C. All formulations were considered homogeneous (within 70 -120% of the target concentration).
- Solubility and stability of the test material in the solvent/vehicle: Soluble in corn oil and stable (for up to 24 hours at room temperature; up to 8 days at 2 to 8°C; and up to 8 days at -60 to -80°C).
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): None

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: None

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD[SD]

Details on species / strain selection:

Rats have been historically used in safety evaluation studies as recommended and accepted by appropriate regulatory agencies. Sprague Dawley (Crl:CD[SD]) strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague Dawley (Crl:CD[SD]) strain from Charles River Laboratories, Raleigh, North Carolina, USA.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 8 weeks old,
- Weight at study initiation: 216 to 323 g (males) and 163 to 243 g (females).
- Fasting period before study: Overnight for scheduled blood and urine collections and prior to terminal procedures (on day 92).
- Housing: Group-housed (up to three animals of the same sex/cage) in polycarbonate
cages with hardwood chip bedding. Housed individually in stainless steel cages for study-related procedures.
- Diet (ad libitum): Certified Rodent Diet #2014C (Envigo RMS, Inc.)
- Water (ad libitum)
- Acclimation period: At least 16 days.

DETAILS OF FOOD AND WATER QUALITY: Diet routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Water samples routinely analyzed for specified microorganisms and environmental contaminants. Results are retained at Labcorp.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26°C,
- Humidity (%): 30 to 70%,
- Air changes (per hr): Minimum of 10.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle (interrupted where required for study-related activities).

IN-LIFE DATES: From: To: 3 February 2022 to (XXX - to be added)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was chosen as it is the most likely route of human exposure. A previous 7-day repeat dose study (Labcorp Study 8484277; Labcorp, 2021) was well tolerated, therefore dose levels of 0, 100, 300, and 1000 mg/kg/day were selected for the present 90-day study.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were prepared once daily on Days 1 to 14 (females) or 16 (males) of dosing, then prior to day of dosing for Days 15 to 21(females) or 17 to 23 (males), and then once weekly and apportioned for daily use. Dose concentrations (10, 20 & 100 mg/ml) were based on the test item as supplied. Formulations were stored at 2 to 8°C, protected from light until removed for dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil is an appropriate vehicle as specified within OECD 408.
- Amount of vehicle (if gavage): Dosed at 10 mL/kg.
- Lot/batch no. (if required): MKCP9878 & MKCQ0649
- Purity: Not specified.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analyzed by Labcorp using a validated HPLC method (Method 12795).

Duplicate samples (1 mL each) were taken from the middle of the vehicle control item. Two sets of triplicate samples (1 mL each) were taken from the middle of each test item formulation. Samples were collected from vehicle control item and test item formulations prepared for administration on Days 1, 42, and 91 of the dosing phase. For samples collected at the same interval as homogeneity, the mean result from the homogeneity analysis was also used for concentration verification.
All formulations were within 70 -120% of the target concentrations (means ranging from 83.5% to 97.3% of theoretical). No test item detected in any vehicle control item formulation. All formulations met the acceptance criteria and animals were administered the intended dose.

Duration of treatment / exposure:

13 weeks (90-days)

Frequency of treatment:

Once daily via oral gavage.

Doses / concentrations

Doses / concentrations 1

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High

No. of animals per sex per dose:

40 males & 40 females

Control: 10 males & 10 females
Low dose (100 mg/kg day): 10 males & 10 females
Mid dose (300 mg/kg/day): 10 males & 10 females
High dose (1000 mg/kg day): 10 males & 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels of 0, 100, 300 & 1000 mg/kg/day were selected based on tolerability of dose levels in a previous 7-day repeat dose study (Labcorp Study 8484277; Labcorp, 2021).
- Rationale for animal assignment: Assigned using a computerized procedure.
- Fasting period before blood sampling for clinical biochemistry: Fasted overnight
- Other:

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the dosing phase for each animal, once daily.
- Post dose cage side observations: Conducted for each animal once daily (week 1), twice weekly (weeks 2 to 4), or once weekly (weeks 5 to 13) at ca. 2 hours postdose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Checked twice daily (a.m. and p.m.) and once on days of arrival/sacrifice for mortality, abnormalities, and signs of pain or distress.

BODY WEIGHT: Yes
- Time schedule for examinations: Once during the predose phase, once weekly prior to dosing (based on Day 1) to Week 13, and on Day 91 of the dosing phase.

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumed by each cage of animals was recorded once for a 1-week interval during the predose phase, weekly to Week 13, and from Days 85 to 91 of the dosing phase.

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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Conducted once for each animal during the predose phase
and once during week 12 of the dosing phase.
- Dose groups that were examined: Control and all test item groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Dosing phase, on day 92.
- Anaesthetic used for blood collection: Yes, anesthetized with isoflurane
- Animals fasted: Yes, fasted overnight.
- How many animals: All control and test item group animals.
- Parameters checked in table [5.1 (summary) and 6.1 individual)] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Dosing phase, on Day 92.
- Animals fasted: Yes, fasted overnight.
- How many animals: All control and test item group animals.
- Parameters checked in table [5.2 (summary) & 6.2 (individual) No.?] were examined.

THYROID HORMONE & SERUM BILE ACIDS: Yes
- Time of blood sample collection: Collected from fasted animals on Day 92 of the dosing phase.
- Animals fasted: Yes
- How many animals: All control and test item group animals.

URINALYSIS: Yes
- Time schedule for collection of urine: Collected overnight before blood collection from animals fasted overnight. Urine samples were collected on Day 92 of the dosing phase.
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked in table [5.3 (summary), 6.3 (individual)] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the predose phase and once during Week 12 of
the dosing phase. All examinations were completed at the same time of day.
- Dose groups that were examined: All control and test item group animals.
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Elicited behaviour, Open field observations, Neurobehavioral observations - predose & dosing phase.

IMMUNOLOGY: No
Immunoglobulin A, G & M evaluated under clinical chemistry parameters.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On day 92 of the dosing phase, all animals (fasted overnight) were anesthetized with isoflurane inhalation, exsanguinated, and necropsied. An examination of the external features of the carcass; external body orifices; abdominal, thoracic, cranial, pelvic, and oral cavities; organs and tissues

HISTOPATHOLOGY: Yes
From all scheduled sacrifices.
Specified tissues (in the Necropsy, Organ Weights, and Macroscopic Observations section of the report) (unless noted as missing) from each animal were embedded in paraffin and sectioned at a nominal 5 μm, and slides prepared and stained with hematoxylin and eosin.
Microscopic Observations
Specified tissues (in the Necropsy, Organ Weights, and Macroscopic Observations section of the report) from the following animals were examined microscopically by the Principal Investigator for Anatomic Pathology.

- Animals in control and high-dose groups.
- Macroscopic lesions from animals in low- and mid-dose groups.

Statistics:

Means and SD were calculated for: absolute bw, bw change, quantitative food consumption, continuous clinical pathology values, terminal bw, absolute organ weight, organ:bw percentage, and organ:brain weight percentage & FOB evaluations.

Pairwise comparisons were Gp 1 vs Gps 2, 3 & 4.
Prior to the ANOVA, Levene’s test was performed:
- Where Levene’s test was significant (P ≤0.05), a rank transformation was applied before ANOVA.
- Where Levene’s test was not significant (P >0.05), ANOVA was conducted.

For comparisons to a single control or combined identical controls:
- If the group effect of the ANOVA was significant (P ≤0.05), Dunnett’s test was used.
- If the ANOVA was not significant (P >0.05), the Dunnett’s test results were not applicable.

For other comparisons (i.e., not against a single control or combined identical controls):
- If the group effect of the ANOVA is significant (P ≤0.05), t-tests were used.
- If not significant (P >0.05), the t-test results were not applicable.
Where only two groups were available, a two-sample t-test was performed.
Values above/below the LOQ were not analyzed.
All statistical tests were evaluated at the 5% probability level.

Locomotor Activity Assessment Data:
Results reported as significant at 5%.
Basic movements, xy ambulation, fine movement, rearing activity were analyzed using a repeated measures analysis of variance (Winer, 1971). The variance-covariance structure used were the Autoregressive Moving Average (1,1). T-tests were used for group comparisons of treatment groups with control.

Male Reproductive Assessment:
Male reproductive parameters were analyzed using a Kruskal-Wallis nonparametric ANOVA. If significant (P ≤0.05), pairwise comparisons of each test item-treated group with the control group were made using the Wilcoxon Rank Sum Test. If not significant (P > 0.05), no further analyses were conducted.
Where only two groups are available for analysis, a Wilcoxon Rank Sum Test was
performed.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test-item related clinical observations were noted.

Mortality:

no mortality observed

Description (incidence):

No test-item-related mortality occurred. All animals survived to scheduled sacrifice.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test-item alterations in body weight or body weight gain were noted up to 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Effects in a single male (aqueous fibrin clot, anterior chamber, left eye, day 84) at 100 mg/kg/day was not test-item related.

Haematological findings:

no effects observed

Description (incidence and severity):

No test-item related effects in haematology or coagulation test results.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test-item related effects consisted of minimally higher alkaline phosphatase activity in two males at 100 mg/kg/day, two males at 300 mg/kg/day, and males at 1000 mg/kg/day. Minimally higher serum bile acids were noted in one male at 300 mg/kg/day and males and two females at 1000 mg/kg/day and was likely associated with higher liver weights. Minimally higher T4 was noted in one female at 100 mg/kg/day, one female and three males at 300 mg/kg/day, and three males and four females at 1000 mg/kg/day. All other differences in clinical chemistry test results, statistically significant or not, between control and test-item treated animals were consistent with normal variation and considered incidental. Most or all of the following characterized these differences: of small magnitude, lack of dose relationship, inconsistency between sexes, and/or the absence of correlative findings.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Minimally higher T4 was noted in one female at 100 mg/kg/day, one female and three males at 300 mg/kg/day, and three males and four females at 1000 mg/kg/day. This increase was not considered treatment related as it was of a small magnitude with an absence of any clear dose relationship, as well as inconsistent between sexes with an absence of correlative findings.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test-item related effects in urinalysis were identified.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Mildly decreased activity were observed for 2/10 females at 1000 mg/kg/day. Decreased activity did not correlate to changes in LMA (no alterations observed). The low incidence (2/10 animals), confined to females only, with no corroborating additional observations, and mild severity, the
decreased activity was considered neither biologically impactful nor test-item related.
Other neurobehavioral findings included increased auditory startle response, decreased
extensor thrust, walking on toes, abnormal grasping loss, increased pain response, absent pinna response, increased reactivity to handling, elevated tail, and mild vocalization. These were considered not test-item related as incidences were comparable with controls, present during the predose collection, or lacked a dose response.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute and relative liver weights increased in animals at 1000 mg/kg/day, with mean liver-to-body weight ratios achieving statistical significance. Liver weight increases in animals at 1000 mg/kg/day were considered test item related. No macroscopic or microscopic correlates to the increased liver weights were observed, however, serum chemistry correlates were noted.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related microscopic findings were noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Details on results:

Male Reproductive Assessment: Decreased sperm concentration was noted for all test item treated males but was only statistically significant for males at 300 mg/kg/day, compared with controls. This decrease was not considered to be treatment related due to the lack of a dose response, as well as the absence of any organ weight effects, or histopathological correlates.
An increase in abnormal sperm morphology in males at 1000 mg/kg/day was observed, compared with controls. This increase was primarily due to a high number of abnormal sperm in two males and was not considered to be related to the test material, due to the lack of a dose response, as well as the absence of organ weight effects, or histopathological correlates.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Results of Homogeneity and Concentration Verification Analyses

Interval	Location	Replicate	Lankroflex ED6 in Corn Oil
			0 mg/mL		10 mg/mL		30 mg/mL		100 mg/mL
			Actual	% of Target	Actual	% of Target	Actual	% of Target	Actual	% of Target
Day 1	Top	1	-	-	9.3	93.3	-	-	93.2	93.2
TA 1		2	-	-	9.5	95.3	-	-	94.6	94.6
	Middle	1	DNS	-	9.4	94.4	27.3	90.9	92.1	92.1
		2	-	-	9.6	96.2	27.4	91.3	95.1	95.1
		3	-	-	-	-	27.7	92.2	-	-
	Bottom	1	-	-	9.6	96.1	-	-	95.2	95.2
		2	-	-	9.6	96.4	-	-	94.5	94.5
	Overall	Mean (n=6)	-	-	-	95.3	-	91.5	-	94.5
		RSD (%)	-	-	-	1.3	-	0.8	-	1.3
Day	Top	1	-	-	8.26	82.6	-	-	87.7	87.7
TA 3		2	-	-	8.22	82.3	-	-	88.0	88.0
	Middle	1	DNS	-	8.39	83.9	26.0	86.6	89.2	89.2
		2	-	-	8.29	82.9	26.3	87.8	88.6	88.6
		3		-	-	-	26.1	87.0	-	-
	Bottom	1	-	-	8.51	85.1	-	-	87.0	87.0
		2	-	-	8.45	84.5	-	-	88.9	88.9
	Overall	Mean (n=6)	-	-	-	83.5	-	87.1	-	88.2
		RSD (%)	-	-	-	1.4	-	0.7	-	0.9
Day	Top	1	-	-	9.02	90.2	-	-	91.6	91.6
TA 24		2	-	-	9.19	91.9	-	-	87.6	87.6
	Middle	1	DNS	-	9.23	92.3	-	-	91.8	91.8
		2	-	-	9.27	92.7	-	-	92.3	92.3
	Bottom	1	-	-	9.32	93.2	-	-	92.4	92.4
		2	-	-	9.79	97.9	-	-	90.5	90.5
	Overall	Mean (n=6)	-	-	-	93.1	-	-	-	91.0
		RSD (%)	-	-	-	2.8	-	-	-	2.0
Day	Top	1	-	-	9.33	93.3	-	-	90.1	90.1
TA 26		2	-	-	9.53	95.3	-	-	95.4	95.4
	Middle	1	DNS	-	10.0	100.1	26.8	89.3	96.0	96.0
		2	-	-	9.75	97.5	28.2	94.1	96.0	96.0
		3		-	-	-	28.5	95.0	-	-
	Bottom	1	-	-	9.84	98.4	-	-	98.2	98.2
		2	-	-	9.92	99.2	-	-	93.9	93.9
	Overall	Mean (n=6)	-	-	-	97.3	-	92.8	-	95.0
		RSD (%)	-	-	-	2.6	-	3.3	-	2.9
- = Not applicable DNS = Detection not significant RSD = Relative standard deviation

Results of Homogeneity and Concentration Verification Analyses - Continued

Interval	Location	Replicate	Lankroflex ED6 in Corn Oil
			0 mg/mL		10 mg/mL		30 mg/mL		100 mg/mL
			Actual	% of Target	Actual	% of Target	Actual	% of Target	Actual	% of Target
TA 33	Middle	1	DNS	-	9.52	95.2	27.7	92.3	89.7	89.7
		2	-	-	9.63	96.3	28.0	93.2	88.6	88.6
		3	-	-	9.72	97.2	28.4	94.6	92.4	92.4
		Mean	-	-	-	96.2	-	93.4	-	90.2
		RSD (%)	-	-	-	1.0%	-	1.2%	-	2.1%
- = Not applicable DNS = Detection not significant RSD = Relative standard deviation

Summary of Clinical Observations - Dosing Phase

Test Item	(dosage)	1	2	3	4	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000	0	100	300	1000
Category	Gp/Sex:	1/M	2/M	3/M	4/M	1/F	2/F	3/F	4/F
Observation	No. in gp.	10	10	10	10	10	10	10	10
Normal
No remarkable observations		10	10	10	10	10	10	10	10
Behaviour - other
aggression to handler		1	1	0	0	0	0	0	0
sensitivity to touch, increased		1	1	0	0	0	0	1	0
struggled excessively during sample collection		0	0	0	0	0	0	1	0
Discharge
source known, nose, red		1	0	0	0	0	0	0	0
Involuntary movements
convulsion, whole body, <1 minute		0	0	0	0	0	0	1	0

 

Test Item	(dosage)	1	2	3	4	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000	0	100	300	1000
Category	Gp/Sex:	1/M	2/M	3/M	4/M	1/F	2/F	3/F	4/F
Observation	No. in gp.	10	10	10	10	10	10	10	10
Pelage
abnormal color, ventral, brown		0	0	1	0	0	0	0	0
hair loss, feet, front both		0	0	0	0	0	0	0	1
hair loss, head		0	1	0	0	0	0	0	0
hair loss, head, right		0	1	0	0	0	0	0	0
thinning, dorsal		0	1	0	0	0	0	0	0
thinning, dorsal cervical		0	0	0	0	1	0	0	0
thinning, feet, front both		1	1	1	0	0	0	1	0
thinning, head		0	1	0	0	0	1	0	0
thinning, head, left		0	0	0	2	0	0	0	0
thinning, head, right		1	0	0	1	0	0	0	0
thinning, lateral, left		0	1	0	0	0	0	0	0
thinning, shoulders, both		0	0	0	0	1	0	0	0
Skin
scab, digit(s)		0	0	0	0	1	0	0	0
scab, dorsal thoracic		1	0	0	0	0	0	0	0
scab, foot, front right		0	0	0	0	0	1	0	0
scab, head, left		1	0	0	2	0	0	0	0
scab, head, right		1	0	0	0	0	0	0	1
scab, ventral		0	1	0	0	0	0	0	0
Tail
bent, tail, distal		0	0	0	0	0	0	0	1
Teeth
abnormal color, teeth, lower, white		0	0	0	0	1	0	0	0

 

Summary of Ophthalmic Observations - Dosing Phase - Indirect Ophthalmoscope

Test Item	(dosage)	1	2	3	4	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000	0	100	300	1000
Category	Gp/Sex:	1/M	2/M	3/M	4/M	1/F	2/F	3/F	4/F
Observation	No. in gp.	10	10	10	10	10	10	10	10
Anterior chamber   aqueous
fibrin clot, left eye		0	1	0	0	0	0	0	0
No visible lesions
no visible lesions, eyes		10	9	10	10	10	10	10	10
no visible lesions, right eye		0	1	0	0	0	0	0	0

 

Summary of Body Weight Dosing Phase

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g"
Gp/sex		Day 1	8	15	22	29	36
1/M	Mean	261	312	358	395	436	465
	SD	23.8	26.9	29.6	35.0	38.5	40.4
	No.	10	10	10	10	10	10
2/M	Mean	257	310	358	398	439	468
	SD	23.1	23.8	23.7	25.8	26.0	31.1
	No.	10	10	10	10	10	10
3/M	Mean	262	313	357	396	433	462
	SD	23.8	27.8	34.9	42.9	46.8	50.2
	No.	10	10	10	10	10	10
4/M	Mean	261	317	367	407	448	475
	SD	31.1	37.5	39.9	46.4	51.2	56.8
	No.	10	10	10	10	10	10
	Statistics	A	A	A		A	A

A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g"
Gp/sex		Day 43	50	57	64	71	78
1/M	Mean	488	510	534	561	580	600
	SD	44.0	46.6	48.8	57.0	64.6	67.3
	No.	10	10	10	10	10	10
2/M	Mean	492	512	536	560	581	600
	SD	39.5	43.3	45.4	49.0	56.7	62.4
	No.	10	10	10	10	10	10
3/M	Mean	487	509	533	559	579	593
	SD	54.8	58.2	59.5	62.2	64.3	66.2
	No.	10	10	10	10	10	10
4/M	Mean	502	526	549	573	598	616
	SD	62.9	70.2	76.3	77.8	81.3	85.4
	No.	10	10	10	10	10	10
	Statistics	A	A	A	A	A	A

A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g"
Gp/sex		Day 85	91
1/M	Mean	610	628
	SD	71.2	70.2
	No.	10	10
2/M	Mean	608	627
	SD	60.2	65.3
	No.	10	10
3/M	Mean	603	619
	SD	70.6	73.0
	No.	10	10
4/M	Mean	631	650
	SD	90.0	95.4
	No.	10	10
	Statistics	A	A

A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g"
Gp/sex		Day 1	8	15	22	29	36
1/F	Mean	197	228	248	264	274	285
	SD	9.1	9.5	13.1	15.5	19.2	18.5
	No.	10	10	10	10	10	10
2/F	Mean	202	223	246	262	277	286
	SD	21.8	26.4	28.4	29.8	32.3	35.2
	No.	10	10	10	10	10	10
3/F	Mean	204	226	243	265	279	289
	SD	14.6	14.7	18.6	20.3	22.9	23.4
	No.	10	10	10	10	10	10
4/F	Mean	199	222	239	254	267	277
	SD	15.2	17.7	20.0	18.3	20.0	21.1
	No.	10	10	10	10	10	10
	Statistics	A	A	A	A	A	A

A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g"
Gp/sex		Day 43	50	57	64	71	78
1/F	Mean	296	302	307	316	320	321
	SD	19.6	20.0	24.8	24.4	28.0	29.0
	No.	10	10	10	10	10	10
2/F	Mean	296	305	314	320	327	332
	SD	36.9	41.2	40.8	44.0	45.3	47.7
	No.	10	10	10	10	10	10
3/F	Mean	294	306	316	322	319	332
	SD	25.5	27.8	28.2	30.9	33.5	34.8
	No.	10	10	10	10	10	10
4/F	Mean	285	291	299	301	303	315
	SD	22.2	20.5	23.6	25.9	31.8	27.8
	No.	10	10	10	10	10	10
	Statistics	A	AT	A	A	A	A

A = ANOVA and Dunnett's
T = Rank transformed data

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g"
Gp/sex		Day 85	91
1/F	Mean	323	325
	SD	27.8	26.9
	No.	10	10
2/F	Mean	334	335
	SD	49.4	49.2
	No.	10	10
3/F	Mean	336	338
	SD	35.9	40.1
	No.	10	10
4/F	Mean	320	318
	SD	29.3	27.3
	No.	10	10
	Statistics	A	A

A = ANOVA and Dunnett's

 

Summary of Food Consumption - Dosing Phase

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g/animal/day"
Gp/sex		Day 1-8	8-15	15-22	22-29	29-36	36-43
1/M	Mean	27	25	23	24	23	21
	SD	1.1	1.5	1.1	0.7	1.5	1.4
	No.	4	4	4	4	4	4
2/M	Mean	27	26	24	24	24	22
	SD	1.8	2.4	2.5	2.2	2.9	4.4
	No.	4	4	4	4	4	4
3/M	Mean	27	24	23	23	23	22
	SD	1.6	2.8	3.3	3.1	1.9	2.2
	No.	4	4	4	3	4	4
4/M	Mean	28	27	25	25	24	22
	SD	2.2	2.1	2.4	1.5	2.2	1.8
	No.	4	4	4	4	4	4
	Statistics	A	A	A	A	A	A

ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g/animal/day"
Gp/sex		Day 43-50	50-57	57-64	64-71	71-78	78-85
1/M	Mean	20	20	20	22	19	17
	SD	1.1	0.8	1.3	7.0	2.0	1.1
	No.	4	4	4	4	4	4
2/M	Mean	21	23	20	17	19	22
	SD	3.9	6.1	2.6	6.4	4.4	10.2
	No.	4	4	4	4	4	4
3/M	Mean	20	20	20	19	18	16
	SD	1.6	0.9	1.8	1.0	1.0	1.1
	No.	4	4	4	4	4	4
4/M	Mean	22	21	21	20	20	19
	SD	1.4	1.6	1.8	0.9	1.3	0.8
	No.	4	4	3	4	4	4
	Statistics	A	AT	A	A	A	AT

A = ANOVA and Dunnett's
T = Rank transformed data

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g/animal/day"
Gp/sex		Day 85-91
1/M	Mean	24
	SD	12.5
	No.	4
2/M	Mean	18
	SD	4.2
	No.	3
3/M	Mean	18
	SD	1.1
	No.	4
4/M	Mean	19
	SD	0.9
	No.	3
	Statistics	AT

A = ANOVA and Dunnett's
T = Rank‑transformed data

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g/animal/day"
Gp/sex		Day 1-8	8-15	15-22	22-29	29-36	36-43
1/F	Mean	17	16	16	15	21	13
	SD	1.4	2.1	1.9	2.3	13.6	1.5
	No.	4	4	4	4	4	4
2/F	Mean	17	17	18	15	15	14
	SD	1.7	1.0	4.0	1.0	1.4	0.9
	No.	4	4	4	3	4	4
3/F	Mean	16	16	17	16	18	15
	SD	1.4	1.6	1.2	0.9	5.7	0.2
	No.	4	4	4	4	4	3
4/F	Mean	16	16	16	16	16	15
	SD	2.0	1.3	1.0	1.6	1.5	0.9
	No.	4	4	4	4	4	4
	Statistics	A	A	A	A	AT	A

A = ANOVA and Dunnett's
T = Rank transformed data

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g/animal/day"
Gp/sex		Day 43-50	50-57	57-64	64-71	71-78	78-85
1/F	Mean	13	13	13	11	11	11
	SD	1.6	1.4	2.1	1.4	1.4	1.8
	No.	4	4	4	4	4	4
2/F	Mean	15	14	18	15	13	11
	SD	1.4	1.1	7.1	5.1	1.8	0.8
	No.	4	4	4	4	4	4
3/F	Mean	16	17	13	13	13	11
	SD	2.4	6.2	0.9	1.2	1.1	0.6
	No.	4	4	4	4	4	4
4/F	Mean	15	15	13	18	14	13
	SD	1.1	1.8	2.3	6.9	2.2	1.8
	No.	4	4	4	4	4	4
	Statistics	A	AT	AT	A	A	A

A = ANOVA and Dunnett's
T = Rank transformed data

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
Data Presented in "g/animal/day"
Gp/sex		Day 85-91
1/F	Mean	11
	SD	1.3
	No.	4
2/F	Mean	12
	SD	0.7
	No.	4
3/F	Mean	11
	SD	0.7
	No.	4
4/F	Mean	13*
	SD	0.9
	No.	4
	Statistics	A

* P<=0.05
A = ANOVA and Dunnett's

 

Summary of Elicited Behaviour

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		FORE1 g		FORE2 g		FGSA2 g
	Phase	predose	dosing	predose	dosing	predose	dosing
Gp/sex		Day 10	79	10	79	10	79
1/M	Mean	758	2044	783	1953	770	1998
	SD	64.2	639.0	161.5	566.4	100.5	588.4
	No.	10	10	10	10	10	10
2/M	Mean	796	2199	819	2024	807	2112
	SD	176.9	273.4	174.1	239.2	158.5	223.4
	No.	10	10	10	10	10	10
3/M	Mean	908	2136	852	2064	880	2100
	SD	123.7	581.6	134.4	527.4	114.5	542.9
	No.	10	10	10	10	10	10
4/M	Mean	794	2338	780	2218	787	2278
	SD	132.8	236.4	135.6	372.2	113.9	283.8
	No.	10	10	10	10	10	10
	Statistics	X1	AT	X1	AT	X1	AT

X1 = No analysis required
A = ANOVA and Dunnett's
T = Rank transformed data

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		HIND1 g		HIND2 g		HGSA2 g
	Phase	predose	dosing	predose	dosing	predose	dosing
Gp/sex		Day 10	79	10	79	10	79
1/M	Mean	528	814	549	765	538	789
	SD	109.4	220.0	158.6	225.3	107.1	211.8
	No.	10	10	10	10	10	10
2/M	Mean	491	818	535	870	513	844
	SD	63.3	239.1	110.2	225.1	41.4	209.7
	No.	10	10	10	10	10	10
3/M	Mean	530	767	513	822	521	795
	SD	133.9	236.8	95.4	246.9	94.9	235.9
	No.	10	10	10	10	10	10
4/M	Mean	478	755	457	718	468	737
	SD	112.2	141.4	124.9	119.4	104.4	121.1
	No.	10	10	10	10	10	10
	Statistics	X1	A	X1	A	X1	A

X1 = No analysis required
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		FOOT1 cm		FOOT2 cm		FSA2 cm
	Phase	predose	dosing	predose	dosing	predose	dosing
Gp/sex		Day 10	79	10	79	10	79
1/M	Mean	8.6	12.3	7.7	12.1	8.1	12.2
	SD	2.09	1.87	1.56	2.08	1.68	1.91
	No.	10	10	10	10	10	10
2/M	Mean	9.4	12.9	8.5	12.5	8.9	12.7
	SD	1.45	2.70	1.40	2.42	1.28	2.39
	No.	10	10	10	10	10	10
3/M	Mean	9.0	13.5	7.6	13.2	8.3	13.3
	SD	1.96	1.30	1.54	1.84	1.44	1.50
	No.	10	10	10	10	10	10
4/M	Mean	8.4	13.1	8.5	12.8	8.4	12.9
	SD	1.78	1.85	1.99	1.62	1.65	1.64
	No.	10	10	10	10	10	10
	Statistics	X1	A	X1	A	X1	A

X1 = No analysis required
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		BODYTEMP °C		RBW g
	Phase	predose	dosing	predose	dosing
Gp/sex		Day 10	79	10	79
1/M	Mean	37.6	38.9	202	602
	SD	0.62	0.33	22.5	67.6
	No.	10	10	10	10
2/M	Mean	37.7	38.5	200	600
	SD	0.42	0.47	21.4	59.6
	No.	10	10	10	10
3/M	Mean	37.4	38.5	204	593
	SD	0.34	0.43	23.1	65.9
	No.	10	10	10	10
4/M	Mean	37.5	38.7	203	617
	SD	0.41	0.37	26.2	84.5
	No.	10	10	10	10
	Statistics	X1	A	X1	A

X1 = No analysis required
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		FORE1 g		FORE2 g		FGSA2 g
	Phase	predose	dosing	predose	dosing	predose	dosing
Gp/sex		Day 11	78	11	78	11	78
1/F	Mean	804	1689	803	1580	803	1634
	SD	98.8	258.1	103.9	263.1	93.3	214.0
	No.	10	10	10	10	10	10
2/F	Mean	775	1562	814	1595	795	1579
	SD	149.0	305.1	192.7	330.5	156.9	290.1
	No.	10	10	10	10	10	10
3/F	Mean	828	1653	834	1636	831	1644
	SD	81.2	290.7	118.5	281.0	94.4	263.7
	No.	10	10	10	10	10	10
4/F	Mean	830	1593	866	1606	848	1600
	SD	119.7	263.0	94.6	314.7	103.5	276.2
	No.	10	10	10	10	10	10
	Statistics	X1	A	X1	A	X1	A

X1 = No analysis required
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		HIND1 G		HIND2 g		HGSA2 g
	Phase	predose	dosing	predose	dosing	predose	dosing
Gp/sex		Day 11	78	11	78	11	78
1/F	Mean	527	702	498	698	513	700
	SD	122.0	134.5	126.2	154.3	109.3	133.8
	No.	10	10	10	10	10	10
2/F	Mean	479	631	484	609	481	620
	SD	100.9	188.8	109.0	185.3	92.5	181.1
	No.	10	10	10	10	10	10
3/F	Mean	528	591	486	582	507	586
	SD	173.3	97.4	80.1	144.6	118.5	97.3
	No.	10	10	10	10	10	10
4/F	Mean	513	593	554	622	533	608
	SD	103.1	214.0	76.9	207.6	78.9	204.7
	No.	10	10	10	10	10	10
	Statistics	X1	A	X1	A	X1	A

X1 = No analysis required
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		FORE1 g		FORE2 g		FGSA2 g
	Phase	predose	dosing	predose	dosing	predose	dosing
Gp/sex		Day 11	78	11	78	11	78
1/F	Mean	7.9	10.2	7.2	10.2	7.5	10.2
	SD	2.61	1.71	1.36	1.84	1.72	1.73
	No.	2.61	1.71	1.36	1.84	1.72	1.73
2/F	Mean	8.2	11.4	7.2	11.4	7.7	11.4
	SD	1.34	1.67	1.14	1.68	0.97	1.65
	No.	10	10	10	10	10	10
3/F	Mean	8.3	11.4	8.1	11.5	8.2	11.4
	SD	1.53	1.62	1.51	1.67	1.38	1.56
	No.	10	10	10	10	10	10
4/F	Mean	9.7	11.0	8.5	10.7	9.1	10.9
	SD	1.69	1.11	1.49	0.68	1.49	0.86
	No.	10	10	10	10	10	10
	Statistics	X1	A	X1	A	X1	A

X1 = No analysis required
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		BODYTEMP °C		RBW g
	Phase	predose	dosing	predose	dosing
Gp/sex		Day 11	78	11	78
1/F	Mean	37.8	38.5	169	324
	SD	0.45	0.58	8.5	28.4
	No.	10	10	10	10
2/F	Mean	37.7	38.7	167	333
	SD	0.39	0.47	9.2	35.9
	No.	10	10	10	10
3/F	Mean	37.9	38.9	171	334
	SD	0.39	0.47	9.2	35.9
	No.	10	10	10	10
4/F	Mean	37.9	38.8	168	316
	SD	0.51	0.67	10.4	27.4
	No.	10	10	10	10
	Statistics	X1	A	X1	A

X1 = No analysis required
A = ANOVA and Dunnett's

 

Summary of Open Field Observations

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		LAT sec		REAR		URIPOL
	Phase	predose	dosing	predose	dosing	predose	dosing
Gp/sex		Day 10	79	10	79	10	79
1/M	Mean	0	1	11	11	1	0
	SD	0.0	1.2	6.1	7.6	1.3	0.7
	No.	10	10	10	10	10	10
2/M	Mean	0	1	13	8	0	0
	SD	0.0	0.5	5.1	4.4	0.9	0.7
	No.	10	10	10	10	10	10
3/M	Mean	0	1	12	9	0	0
	SD	0.0	0.7	4.3	5.8	0.0	0.3
	No.	10	10	10	10	10	10
4/M	Mean	0	1	10	8	1	0
	SD	0.0	1.0	4.5	3.9	1.6	0.5
	No.	10	10	10	10	10	10

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		FECBOL
	Phase	predose	dosing
Gp/sex		Day 10	79
1/M	Mean	1	1
	SD	1.3	1.3
	No.	10	10
2/M	Mean	1	1
	SD	1.3	1.6
	No.	1.3	1.6
3/M	Mean	0	2
	SD	1.3	2.0
	No.	10	10
4/M	Mean	1	2
	SD	1.6	1.9
	No.	10	10

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		LAT sec		REAR		URIPOL
	Phase	predose	dosing	predose	dosing	predose	dosing
Gp/sex		Day 11	78	11	78	11	78
1/F	Mean	1	1	12	15	0	0
	SD	1.9	0.7	5.9	5.9	0.6	0.5
	No.	10	10	10	10	10	10
2/F	Mean	0	0	8	14	0	0
	SD	0.0	0.5	6.1	2.7	0.9	0.0
	No.	10	10	10	10	10	10
3/F	Mean	1	1	8	11	0	0
	SD	2.2	0.6	3.1	5.5	0.0	0.6
	No.	10	10	10	10	10	10
4/F	Mean	0	1	8	12	0	0
	SD	0.9	1.1	4.8	6.4	0.3	0.3
	No.	10	10	10	10	10	10

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
		FECBOL
	Phase	predose	dosing
Gp/sex		Day 11	78
1/F	Mean	0	0
	SD	0.0	0.0
	No.	10	10
2/F	Mean	1	0
	SD	1.6	0.0
	No.	10	10
3/F	Mean	0	0
	SD	0.3	0.6
	No.	10	10
4/F	Mean	0	0
	SD	0.0	0.7
	No.	10	10

 

Summary of Neurobehavioral Observations - Dosing Phase

Test Item	(dosage)	1	2	3	4	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000	0	100	300	1000
Category Observation	Gp/Sex:	1/M	2/M	3/M	4/M	1/F	2/F	3/F	4/F
	No. in gp.	10	10	10	10	10	10	10	10
Activity
decreased, mild		0	0	0	0	0	0	0	2
normal		10	10	10	10	10	10	10	8
Aggression to cage mate
normal		10	10	10	10	10	10	10	10
Alertness
normal		10	10	10	10	10	10	10	10
Approach response
increased		1	0	0	0	0	0	0	0
normal		9	10	10	10	10	10	10	10
Auditory startle response
increased		0	1	1	0	1	0	1	2
normal		10	9	9	10	9	10	9	8
Bar test
normal		10	10	10	10	10	10	10	10
Behaviour - other
normal		10	10	10	10	10	10	10	10
Behaviour -  stereotype
normal		10	10	10	10	10	10	10	10
Body tone
normal		10	10	10	10	10	10	10	10

Test Item	(dosage)	1	2	3	4	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000	0	100	300	1000
Category Observation	Gp/Sex:	1/M	2/M	3/M	4/M	1/F	2/F	3/F	4/F
	No. in gp.	10	10	10	10	10	10	10	10
Discharge
normal		10	10	10	10	10	10	10	10
Excretion
normal, group house observation		10	10	10	10	10	10	10	10
Extensor thrust
decreased		1	0	0	1	0	0	0	0
normal		9	10	10	9	10	10	10	10
Eye closure
normal		10	10	10	10	10	10	10	10
Eyes
normal		10	10	10	10	10	10	10	10
Gait
normal		10	10	10	10	10	9	10	10
walking on toes		0	0	0	0	0	1	0	0
Gasping loss
abnormal, mild		0	0	1	0	0	0	0	0
normal		10	10	9	10	10	10	10	10
Grip strength
normal		10	10	10	10	10	10	10	10
Involuntary movements
normal		10	10	10	10	10	10	10	10

 

Test Item	(dosage)	1	2	3	4	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000	0	100	300	1000
Category Observation	Gp/Sex:	1/M	2/M	3/M	4/M	1/F	2/F	3/F	4/F
	No. in gp.	10	10	10	10	10	10	10	10
Lacrimation
normal		10	10	10	10	10	10	10	10
Pain response
decreased		0	0	0	0	1	0	0	0
increased		0	0	0	1	0	0	0	0
normal		10	10	10	9	9	10	10	10
Palpebral reflex
normal		10	10	10	10	10	10	10	10
Pelage
normal		10	10	10	10	10	10	10	10
Pinna response
absent		0	0	0	0	0	0	0	1
normal		10	10	10	10	10	10	10	9
Posture
normal		10	10	10	10	10	10	10	10
Propreoception (right & hind)
normal		10	10	10	10	10	10	10	10
Pupil status
normal		10	10	10	10	10	10	10	10
Pupillary response
normal		10	10	10	10	10	10	10	10

 

Test Item	(dosage)	1	2	3	4	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000	0	100	300	1000
Category Observation	Gp/Sex:	1/M	2/M	3/M	4/M	1/F	2/F	3/F	4/F
	No. in gp.	10	10	10	10	10	10	10	10
Reactivity to handling
abnormal, increased		0	1	0	0	0	0	0	0
normal		10	9	10	10	10	10	10	10
Respiration
normal		10	10	10	10	10	10	10	10
Restlessness
normal		10	10	10	10	10	10	10	10
Righting reflex
normal		10	10	10	10	10	10	10	10
Salivation
normal		10	10	10	10	10	10	10	10
Skin colour
normal		10	10	10	10	10	10	10	10
Tail
elevated, mild		0	0	1	0	0	0	0	0
normal		10	10	9	10	10	10	10	10
Visual response
normal		10	10	10	10	10	10	10	10
Vocalization
normal		10	9	10	10	9	10	10	9
present, mild		0	1	0	0	1	0	0	1
Waxy rigidity
normal		10	10	10	10	10	10	10	10

 

Summary of Hematology

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	RBC E6/uL	HGB g/dL	Hct %	MCV fL	MCH pg	MCHC g/dL
Gp/sex		Day 92	92	92	92	92	92
1/M	Mean	8.05	13.9	43.2	53.6	17.2	32.1
	SD	0.342	0.56	1.99	1.29	0.60	0.55
	No.	9	9	9	9	9	9
2/M	Mean	8.33	14.4	44.3	53.3	17.2	32.4
	SD	0.401	0.55	1.88	2.15	0.73	0.37
	No.	7	7	7	7	7	7
3/M	Mean	8.35	14.1	43.8	52.4	16.8	32.1
	SD	0.252	0.33	1.06	0.55	0.39	0.63
	No.	7	7	7	7	7	7
4/M	Mean	8.20	14.1	43.8	53.4	17.3	32.3
	SD	0.192	0.62	0.94	1.35	0.78	1.07
	No.	7	7	7	7	7	7
	Statistics	A	A	A	AT	A	A

A = ANOVA and Dunnett's
T = Rank transformed data

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	RBC E6/uL	HGB g/dL	Hct %	MCV fL	MCH pg	MCHC g/dL
Gp/sex		Day 92	92	92	92	92	92
1/F	Mean	7.20	13.0	39.7	55.2	18.1	32.8
	SD	0.294	0.36	1.16	2.16	0.77	0.44
	No.	10	10	10	10	10	10
2/F	Mean	7.31	12.9	39.5	54.0	17.7	32.7
	SD	0.282	0.67	1.99	1.66	0.62	0.44
	No.	10	10	10	10	10	10
3/F	Mean	7.15	12.9	39.3	55.0	18.0	32.8
	SD	0.122	0.39	1.03	1.13	0.48	0.60
	No.	10	10	10	10	10	10
4/F	Mean	7.38	13.1	40.1	54.4	17.8	32.7
	SD	0.244	0.38	1.03	1.59	0.62	0.45
	No.	10	10	10	10	10	10
	Statistics	A	A	A	A	A	A

A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	RDW %	RETIC E3/uL	PLT E3/uL	WBC E3/uL	NEUT E3/uL	LYM E3/uL
Gp/sex		Day 92	92	92	92	92	92
1/M	Mean	12.4	183.5	997	7.96	1.21	6.42
	SD	0.34	27.32	102.0	2.182	0.327	1.855
	No.	9	9	9	9	9	9
2/M	Mean	12.6	178.0	1028	9.37	1.44	7.50
	SD	0.25	21.70	95.5	1.407	0.318	1.188
	No.	7	7	7	7	7	7
3/M	Mean	12.3	169.2	1064	8.58	1.39	6.78
	SD	0.42	15.52	108.7	2.024	0.550	1.684
	No.	7	7	7	7	7	7
4/M	Mean	12.9	205.8	1032	8.78	1.36	6.94
	SD	0.71	30.82	83.0	1.880	0.434	1.366
	No.	7	7	7	7	7	7
	Statistics	AT	A	A	A	A	A

A = ANOVA and Dunnett's
T = Rank transformed data

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	RDW %	RETIC E3/uL	PLT E3/uL	WBC E3/uL	NEUT E3/uL	LYM E3/uL
Gp/sex		Day 92	92	92	92	92	92
1/F	Mean	11.0	181.6	996	4.44	0.57	3.66
	SD	0.21	40.20	70.6	1.432	0.170	1.334
	No.	10	10	10	10	10	10
2/F	Mean	11.2	183.2	953	5.34	0.93	4.15
	SD	0.33	33.43	116.2	1.873	0.326	1.757
	No.	10	10	10	10	10	10
3/F	Mean	11.2	168.3	1059	4.96	0.77	3.95
	SD	0.25	33.94	125.7	1.533	0.219	1.322
	No.	10	10	10	10	10	10
4/F	Mean	11.2	177.8	995	4.75	0.78	3.73
	SD	0.29	31.74	116.8	1.132	0.371	0.886
	No.	10	10	10	10	10	10
	Statistics	AT	A	A	A	A	A

A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	MONO E3/uL	EOS E3/uL	BASO E3/uL	LUC E3/uL	PT sec	APTT sec
Gp/sex		Day 92	92	92	92	92	92
1/M	Mean	0.21	0.09	0.02	0.01	11.4	18.2
	SD	0.083	0.051	0.008	0.009	0.39	2.37
	No.	9	9	9	9	10	10
2/M	Mean	0.29	0.07	0.03	0.05*	11.6	18.3
	SD	0.063	0.019	0.005	0.030	0.80	2.21
	No.	7	7	7	7	10	10
3/M	Mean	0.27	0.07	0.02	0.04	11.7	17.4
	SD	0.106	0.023	0.008	0.034	0.20	1.34
	No.	7	7	7	7	9	9
4/M	Mean	0.34	0.07	0.03	0.04	11.1	18.5
	SD	0.193	0.025	0.013	0.037	0.73	2.86
	No.	7	7	7	7	10	10
	Statistics	AT	A	X2	AT	AT	A

* P<=0.05
A = ANOVA and Dunnett's
T = Rank transformed data
X2 = Not analyzed (too few distinct values)

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	MONO E3/uL	EOS E3/uL	BASO E3/uL	LUC E3/uL	PT sec	APTT sec
Gp/sex		Day 92	92	92	92	92	92
1/F	Mean	0.13	0.06	0.01	0.02	10.1	16.1
	SD	0.050	0.022	0.005	0.007	0.41	0.99
	No.	10	10	10	10	10	10
2/F	Mean	0.17	0.07	0.01	0.01	9.9	16.7
	SD	0.061	0.016	0.008	0.011	0.51	1.04
	No.	10	10	10	10	10	10
3/F	Mean	0.15	0.07	0.01	0.02	10.0	16.6
	SD	0.092	0.031	0.007	0.010	0.33	1.17
	No.	10	10	10	10	10	10
4/F	Mean	0.15	0.07	0.01	0.01	9.9	15.9
	SD	0.040	0.023	0.005	0.008	0.51	1.00
	No.	10	10	10	10	10	10
	Statistics	A	A	X2	X2	A	A

A = ANOVA and Dunnett's
X2 = Not analyzed (too few distinct values)

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	FIB mg/dL
Gp/sex		Day 92
1/M	Mean	155
	SD	8.4
	No.	10
2/M	Mean	163
	SD	7.8
	No.	10
3/M	Mean	157
	SD	15.9
	No.	10
4/M	Mean	161
	SD	5.0
	No.	10
	Statistics	A

A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	FIB mg/dL
Gp/sex		Day 92
1/F	Mean	132
	SD	9.1
	No.	10
2/F	Mean	128
	SD	11.1
	No.	10
3/F	Mean	127
	SD	6.3
	No.	10
4/F	Mean	132
	SD	9.3
	No.	10
	Statistics	A

A = ANOVA and Dunnett's

 

Summary of Clinical Chemistry

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	GLU mg/dL	UN mg/dL	CREAT mg/dL	TP g/dL	ALB g/dL	GLOB g/dL
Gp/sex		Day 92	92	92	92	92	92
1/M	Mean	166	8	0.3	5.4	4.0	1.4
	SD	24.9	1.1	0.04	0.21	0.18	0.19
	No.	10	10	10	10	10	10
2/M	Mean	168	8	0.3	5.6	4.2	1.4
	SD	19.8	1.0	0.05	0.26	0.22	0.17
	No.	10	10	10	10	10	10
3/M	Mean	174	8	0.3	5.4	4.0	1.4
	SD	10.0	1.7	0.05	0.28	0.17	0.18
	No.	10	10	10	10	10	10
4/M	Mean	173	9	0.3	5.5	4.1	1.4
	SD	22.6	1.1	0.04	0.28	0.20	0.18
	No.	10	10	10	10	10	10
	Statistics	AT	A	X2	A	A	A

A = ANOVA and Dunnett's
T = Rank transformed data
X2 = Not analyzed (too few distinct values)

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	GLU mg/dL	UN mg/dL	CREAT mg/dL	TP g/dL	ALB g/dL	GLOB g/dL
Gp/sex		Day 92	92	92	92	92	92
1/F	Mean	148	10	0.3	6.2	5.0	1.2
	SD	16.7	2.2	0.05	0.41	0.44	0.16
	No.	10	10	10	10	10	10
2/F	Mean	152	10	0.3	6.5	5.3	1.2
	SD	30.1	2.3	0.05	0.44	0.33	0.26
	No.	10	10	10	10	10	10
3/F	Mean	146	10	0.3	6.3	5.2	1.1
	SD	19.3	1.4	0.04	0.44	0.35	0.17
	No.	10	10	10	10	10	10
4/F	Mean	153	12	0.3	6.2	5.0	1.2
	SD	25.3	2.5	0.05	0.30	0.39	0.16
	No.	10	10	10	10	10	10
	Statistics	A	A	X2	A	A	A

A = ANOVA and Dunnett's
X2 = Not analyzed (too few distinct values)

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	A:G Ratio	CHOL mg/dL	HDL mg/dL	LDL mg/dL	TRIG mg/dL	TBIL mg/dL
Gp/sex		Day 92	92	92	92	92	92
1/M	Mean	2.9	48	35	<7	87	<0.1
	SD	0.46	5.7	5.9	0.9	14.3	0.00
	No.	10	10	10	10	10	10
2/M	Mean	3.0	48	35	<8	82	<0.1
	SD	0.35	8.4	6.4	2.3	26.9	0.00
	No.	10	10	10	10	10	10
3/M	Mean	2.9	50	35	<8	76	<0.1
	SD	0.33	4.5	4.4	2.1	22.1	0.00
	No.	10	10	10	10	10	10
4/M	Mean	3.0	62*	43*	<10	89	<0.1
	SD	0.41	11.9	8.1	4.2	28.8	0.01
	No.	10	10	10	10	10	10
	Statistics	A	AT	A	X5	A	X5

* P<=0.05
A = ANOVA and Dunnett's
T = Rank‑transformed data
X5 = Not analyzed (values above/below the limit of quantitation)

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	A:G Ratio	CHOL mg/dL	HDL mg/dL	LDL mg/dL	TRIG mg/dL	TBIL mg/dL
Gp/sex		Day 92	92	92	92	92	92
1/F	Mean	4.4	61	51	<6	47	<0.1
	SD	0.83	12.1	11.5	0.7	8.2	0.00
	No.	10	10	10	10	10	10
2/F	Mean	4.9	65	57	<6	58	<0.1
	SD	1.39	22.5	19.0	0.6	27.4	0.00
	No.	10	10	10	10	10	10
3/F	Mean	4.7	59	52	<6	55	<0.1
	SD	0.72	10.5	10.6	0.2	20.5	0.00
	No.	10	10	10	10	10	10
4/F	Mean	4.3	72	60	<6	51	<0.1
	SD	0.92	12.9	13.1	0.4	13.3	0.00
	No.	10	10	10	10	10	10
	Statistics	A	A	A	X5	A	X5

A = ANOVA and Dunnett's
X5 = Not analyzed (values above/below the limit of quantitation)

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	AST U/L	ALT U/L	ALP U/L	GGT U/L	SBA umol/L	CK U/L
Gp/sex		Day 92	92	92	92	92	92
1/M	Mean	69	30	96	<3	34	255
	SD	12.2	5.9	10.6	0.0	20.4	136.0
	No.	10	10	10	10	10	10
2/M	Mean	68	28	100	<3	35	283
	SD	15.2	5.6	26.2	0.0	28.3	213.4
	No.	10	10	10	10	10	10
3/M	Mean	61	29	109	<3	50	137
	SD	8.2	8.4	33.8	0.0	22.0	79.2
	No.	10	10	10	10	10	10
4/M	Mean	62	26	129	<3	65	185
	SD	10.0	5.5	48.9	0.0	43.6	162.9
	No.	10	10	10	10	10	10
	Statistics	A	A	AT	X5	AT	AT

A = ANOVA and Dunnett's
T = Rank transformed data
X5 = Not analyzed (values above/below the limit of quantitation)

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	AST U/L	ALT U/L	ALP U/L	GGT U/L	SBA umol/L	CK U/L
Gp/sex		Day 92	92	92	92	92	92
1/F	Mean	71	18	42	<3	26	332
	SD	18.9	3.6	19.3	0.0	22.4	227.5
	No.	10	10	10	10	10	10
2/F	Mean	79	21	43	<3	15	384
	SD	16.9	7.5	29.5	0.0	13.6	280.1
	No.	10	10	10	10	10	10
3/F	Mean	65	19	39	<3	32	233
	SD	12.5	2.3	12.8	0.0	23.9	142.8
	No.	10	10	10	10	10	10
4/F	Mean	77	23	46	<3	47	298
	SD	22.0	7.0	15.4	0.0	68.4	224.4
	No.	10	10	10	10	10	10
	Statistics	A	A	A	X5	AT	A

A = ANOVA and Dunnett's
X5 = Not analyzed (values above/below the limit of quantitation)
T = Rank‑transformed data

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	Ca mg/dL	PHOS mg/dL	Na mmol/L	K mmol/L	Cl mmol/L	TSH uIU/mL
Gp/sex		Day 92	92	92	92	92	92
1/M	Mean	10.1	7.0	141	4.5	102	1.61
	SD	0.29	0.47	1.3	0.19	1.0	1.291
	No.	10	10	10	10	10	10
2/M	Mean	10.2	7.0	140	4.6	101	1.77
	SD	0.26	0.39	1.0	0.23	1.1	1.295
	No.	10	10	10	10	10	10
3/M	Mean	10.3	7.2	140	4.5	101	3.59
	SD	0.18	0.58	1.1	0.29	0.7	3.063
	No.	10	10	10	10	10	10
4/M	Mean	10.5	7.2	140	4.5	100*	2.10
	SD	0.61	0.59	0.8	0.24	1.5	0.983
	No.	10	10	10	10	10	10
	Statistics	AT	A	A	A	A	AT

* P<=0.05
A = ANOVA and Dunnett's
T = Rank transformed data

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	Ca mg/dL	PHOS mg/dL	Na mmol/L	K mmol/L	Cl mmol/L	TSH uIU/mL
Gp/sex		Day 92	92	92	92	92	92
1/F	Mean	10.3	5.8	139	4.2	102	0.84
	SD	0.42	0.43	0.7	0.24	1.9	0.471
	No.	10	10	10	10	10	10
2/F	Mean	10.5	6.2	139	4.1	101	0.94
	SD	0.43	0.48	1.4	0.33	1.7	0.437
	No.	10	10	10	10	10	10
3/F	Mean	10.5	6.0	139	4.1	102	1.64
	SD	0.45	0.56	1.4	0.19	1.1	0.965
	No.	10	10	10	10	10	10
4/F	Mean	10.5	6.4	139	4.2	101	1.75
	SD	0.61	0.65	1.1	0.22	1.6	1.455
	No.	10	10	10	10	10	10
	Statistics	A	A	A	A	A	A

A = ANOVA and Dunnett's

 

A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	T3 ng/dL	T4 ug/dL
Gp/sex		Day 92	92
1/M	Mean	101	4.8
	SD	19.0	0.68
	No.	10	10
2/M	Mean	101	5.2
	SD	15.5	0.84
	No.	10	10
3/M	Mean	112	6.5*
	SD	21.6	1.01
	No.	10	10
4/M	Mean	100	6.9*
	SD	20.5	1.62
	No.	10	10
	Statistics	A	A

* P<=0.05
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	T3 ng/dL	T4 ug/dL
Gp/sex		Day 92	92
1/F	Mean	139	4.5
	SD	24.0	1.16
	No.	10	10
2/F	Mean	138	5.2
	SD	17.0	1.19
	No.	10	10
3/F	Mean	136	5.5
	SD	31.3	1.00
	No.	10	10
4/F	Mean	120	6.5*
	SD	20.9	1.48
	No.	10	10
	Statistics	A	A

* P<=0.05
A = ANOVA and Dunnett's

 

Summary of Urinalysis

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	UVOL mL	SPGR	UpH
Gp/sex		Day 92	92	92
1/M	Mean	6.1	1.047	5.6
	SD	8.63	0.0216	0.50
	No.	10	10	10
2/M	Mean	4.8	1.043	5.7
	SD	2.72	0.0137	0.35
	No.	10	10	10
3/M	Mean	5.7	1.046	5.6
	SD	4.83	0.0157	0.39
	No.	10	10	10
4/M	Mean	6.2	1.055	5.5
	SD	1.62	0.0101	0.00
	No.	10	10	10
	Statistics	A	A	X2

A = ANOVA and Dunnett's
X2 = Not analyzed (too few distinct values)

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg/day	0	100	300	1000
	Phase- Dosing	UVOL mL	SPGR	UpH
Gp/sex		Day 92	92	92
1/F	Mean	1.8	1.042	5.9
	SD	0.71	0.0097	0.34
	No.	10	10	10
2/F	Mean	1.7	1.057*	5.6
	SD	0.96	0.0138	0.39
	No.	10	10	10
3/F	Mean	4.1	1.041	5.9
	SD	3.12	0.0154	0.34
	No.	10	10	10
4/F	Mean	3.3*	1.055	5.3
	SD	1.25	0.0123	0.26
	No.	10	10	10
	Statistics	AT	A	X2

* P<=0.05
A = ANOVA and Dunnett's
T = Rank transformed data
X2 = Not analyzed (too few distinct values)

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

Male and female rats were administered corn oil (control) or 100, 300 or 1000 mg/kg/day Lankroflex ED6 once daily via oral gavage for 13 weeks. Clinical pathology changes were minimal in magnitude and included higher alkaline phosphatase activity in two males at 100 mg/kg/day, two males at 300 mg/kg/day, and males at 1000 mg/kg/day. Higher serum bile acid was noted in one male at 300 mg/kg/day and males and two females at 1000 mg/kg/day and was likely associated with higher liver weights. Higher T4 was noted in one female at 100 mg/kg/day, three males and one female at 300 mg/kg/day, and three males and four females at 1000 mg/kg/day. All effects were considered non-adverse due to the mild severity of observed findings. The no observed adverse effect level (NOAEL) is 1000 mg/kg/day.

Executive summary:

The toxicity of Lankroflex ED6 (Fatty acids, C14-22, 2-ethylhexyl esters, epoxidised) was evaluated when administered once daily via oral gavage to rats for at least 13 weeks. Forty male and 40 female (Sprague Dawley) rats were assigned to four groups, 0(vehicle control, corn oil), 100 mg/kg/day, 300 mg/kg/day and 1000 mg/kg/day. Animals were dosed via oral gavage once daily at 10 mL/kg. Assessment of toxicity was based on mortality, clinical observations, body weights, food consumption, ophthalmic observations, functional observational battery (FOB),
locomotor activity (LMA), estrous phase determination, and clinical and anatomic pathology. Blood samples were collected for thyroid hormone and serum bile acids evaluation.
There were no test item-related mortality, clinical observations, differences in body weights, food consumption, FOB or LMA findings. Clinical chemistry results were minimal in magnitude and consisted of higher alkaline phosphatase activity in in two males at 100 mg/kg/day, two males at 300 mg/kg/day and most males at 1000 mg/kg/day. Higher serum bile acids were noted in one male at 300 mg/kg/day and males and two females at 1000 mg/kg/day and we considered likely associated with higher liver weights. Higher T4 was noted one female at 100 mg/kg/day, three males and one female at 300 mg/kg/day and three males and four females at 1000 mg/kg/day. No test item related effects in hematology, coagulation, or urinalysis were identified.

Test item-related mean absolute and relative liver weights were increased in animals at 1000 mg/kg/day, without macroscopic or microscopic correlates. No test item-related macroscopic or microscopic findings were noted.
Decreased sperm concentration was noted for all test item treated males and was statistically significant for males at 300 mg/kg/day compared with controls.
An increase in abnormal sperm morphology was noted in males at 1000 mg/kg/day, compared with controls. These changes were considered not related to the test material, due to a lack of organ weight effects or histopathological correlates. No test item related effect on motility was noted at any dose level.

All effects were considered non-adverse due to the mild severity of observed findings. The no observed adverse effect level (NOAEL) is 1000 mg/kg/day.