Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 September 2016 to 11 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.

Deviations:

yes

Remarks:

See "Any other information" for details

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 407: "Repeated Dose 28-day Oral Toxicity Study in Rodents", Paris, 03 October 2008.

Deviations:

yes

Remarks:

See "Any other information" for details

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OTS: 870.3050 (Repeated dose 28-day oral toxicity study in rodents)

Version / remarks:

United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, July 2000.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

EC Number:

701-003-6

Cas Number:

1454803-04-3

Molecular formula:

C20H39NO3 to C26H51NO3

IUPAC Name:

Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Test material form:

liquid

Details on test material:

Identification: MLA-3202
Appearance: Clear amber-red liquid
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until 17 February 2019 (expiry date)
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)
CAS Number: 1454803-04-3
Test item handling No specific handling conditions required

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system Rat: Crl:WI(Han) (outbred, SPF-Quality).Source: Charles River Deutschland, Sulzfeld, Germany.Total number of animals: 20 males, 20 females (females were nulliparous and non-pregnant).Age at start of treatment: Approximately 6 weeks.Identification: Earmark and tattoo.Randomization: By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.Acclimatization period: At least 5 days before the start of treatment under laboratory conditions.Health inspection: Upon receipt of the animals.Conditions: Environmental controls for the animal room were set to maintain 18 to 24 °C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.The light/dark cycle was interrupted for study related activities.Any variations to these conditions were evaluated and maintained in the raw data.Accommodation: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® pezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.Water: Free access to tap water.Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube.Formulations were placed on a magnetic stirrer during dosing.The dose control system (DCS) was used to verify the dosing procedure.

Vehicle:

water

Details on oral exposure:

Vehicle InformationVehicle: Water (Elix, Millipore S.A.S., Molsheim, France).Rationale for vehicle: Based on trial formulations performed at Charles River Den Bosch and on information from the Sponsor.Test Item PreparationMethod of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.Storage conditions: At room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method (Test Facility Study No. 514869). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

At least 28 days. Animals were dosed up to the day prior to necropsy.

Frequency of treatment:

Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 other: mL/kg body weight.

Remarks:

Actual dose volumes were calculated weekly according to the latest body weight.

Doses / concentrations 2

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per dose (5/sex)

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the results of a 5-day range finding study (Test Facility Study No. 514938), the dose levels for this 28-day oral gavage study were selected to be 0, 100,300 and 1000 mg/kg.The study should provide a rational basis for toxicological risk assessment in man. The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.

Positive control:

Postive control not required for this study.

Examinations

Observations and examinations performed and frequency:

Mortality / Viability: At least twice daily.Clinical signs: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately (0-15 minutes) after dosing (no peak effect of occurrence of clinical signs was observed in the dose range finding study (Test Facility Study No. 514938)).Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena (collected under Test Facility Study No. 514939 for logistic reasons and reported under Test Facility Study No. 514867).The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades are reported, as well as the percentage of animals affected in summary tables.Functional Observations: During Week 4 of treatment, functional observation tests were performed on all animals. Tests were performed after dosing at no specific time point, but within a similar time period after dosing (based on the absence of a peak effect in occurrence of clinical signs in the dose range finding study (Test Facility Study No. 514938)).The following tests were performed (abbreviations mentioned in the respective tables indicated between brackets):-Hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) (Score 0 =normal/present, score 1 = abnormal/absent).-Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).-Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA).Total movements and ambulations are reported.Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.Body weights: Weekly.Food consumption: Weekly.Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.Clinical Laboratory InvestigationsBlood samples were collected under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m. at the end of the treatment. Animals were deprived of food overnight (for a maximum of 24 hours), but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids. The following parameters were determined: Reported in table form – see “Any other information” for details.

Sacrifice and pathology:

PathologyNecropsyOn the scheduled day of necropsy, animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination. Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands): Reported in table form – see “Any other information for details”. Organ WeightsThe following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:Adrenal glands SpleenBrain TestesEpididymides ThymusHeart Uterus (including cervix)Kidneys ProstateLiver Seminal vesicles including coagulating glandsOvaries Thyroid including parathyroidHistotechnologyAll organ and tissue samples, as defined under Histopathology (following), were processed, embedded in paraffin wax (Klinipath, Duiven, The Netherlands), cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).HistopathologyThe following slides were examined by a pathologist:- all tissues collected at the scheduled sacrifice from all Group 1 and 4 animals,- spleens of all male animals of Groups 2 and 3 and livers of female animals of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4,- all gross lesions.All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.Histopathology was subjected to a peer review.

Other examinations:

No further examinations specified in the study report.

Statistics:

The following statistical methods were used to analyse the data:-If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.-The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.-The Fisher Exact-test (Ref. 3) was applied to frequency data.-The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test (Ref. 5) was applied to compare the treated groups to the control group.All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. As a result, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation was noted after dosing for the test item treated animals showing a dose related trend but was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend.At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight and body weight gain were statistically significantly reduced in 1000 mg/kg males from Day 8 onwards but the change was only slight and without a clear dose response.In 1000 mg/kg females, body weight gain was statistically significantly increased on an intermittent basis throughout the study and 300 mg/kg females show an increased body weight gain after 2 weeks of treatment only.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Slightly reduced absolute and relative food consumption, was seen in males treated at 1000 mg/kg without a clear dose response.In females, absolute and relative food consumption before or after correction for body weight remained similar to the control level over the study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in haematological parameters.Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Alanine aminotransferase (ALAT) and cholesterol levels were statistically significantly increased at 1000 mg/kg in both sexes.Calcium levels were significantly increased at 1000 mg/kg in males but in the absence of corroborative findings were considered not toxicologically relevant also taking into account the slight nature of the change.Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.There was a slight tendency for reduced hindgrip strength and increased foregrip strength in high dose animals, however this effect is not statistically significant and was not supported by clinical observations or other functional observation changes, and had no supportive neuromorphological correlates. Therefore it was considered not toxicologically relevant.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related lower spleen weights (absolute -24% compared to control) were noted in the 1000 mg/kg males and higher liver weights (absolute 24%; relative 19% compared to control) were noted in the 1000 mg/kg females.For males, the other organ weights were in line with the decrease in body weight.There were no other test item-related organ weight changes.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the spleen of the 1000 mg/kg group males and the liver of the 1000 mg/kg females.In the spleen, the normally occurring extramedullary hematopoiesis was not detectable in males treated at 1000 mg/kg.Hepatocellular hypertrophy of the liver was present in females treated at 1000 mg/kg at minimal degree.The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There were no test item-related alterations in the prevalence, severity, or histological character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

No adverse treatment-related effects were seen in any of the dose levels tested. In addition, there were no toxicologically significant changes observed in parameters including clinical appearance, motor activity, functional observations, and macroscopic examination.Microscopic examination revealed absence of the normally occurring hematopoiesis observed in the spleens of 1000 mg/kg males, which correlated with reduced spleen weight. However, in the absence of any other indicator of toxicity (for example haematology parameters), these spleen findings were considered non-adverse. The minimal hepatocellular hypertrophy of the liver seen in 1000 mg/kg females was accompanied by increased liver weight, as well as changes in alanine aminotransferase levels and cholesterol, liver-related biochemical parameters. However, in the absence of any degenerative findings in the liver, these effects were considered to be non-adverse.Other test item-related findings include a slightly reduced bodyweight gain in 1000 mg/kg males together with a slight reduction in food intake in 1000 mg/kg males. In contrast females showed a slightly increased body weight gain. Without a clear dose response or any corroborative findings these changes small changes in body weights were considered not adverse.

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:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

CLINICAL SIGNS SUMMARY

MALES

		TREATMENT
SIGN (MAX. GRADE)	WEEK:	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	4	.	.	.	.	.	.
(LOCATION)	DAY:	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7
GROUP 1 (CONTROL) No clinical signs noted
GROUP 2 (100 MG/KG) Secretion / excretion                Salivation (3)	G: %:	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	1 2	1 2	1 2	. .	. .	. .	. .	. .
GROUP 3 (300 MG/KG) Secretion / excretion                Salivation (3)	G: %:	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A
GROUP 4 (1000 MG/KG) Secretion / excretion                Salivation (3)	G: %:	. .	1 4	1 8	1 8	1 8	1 8	1 8	1 8	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A

FEMALES

		TREATMENT
SIGN (MAX. GRADE)	WEEK:	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	4	.	.	.	.	.	.
(LOCATION)	DAY:	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7
GROUP 1 (CONTROL) No clinical signs noted
GROUP 2 (100 MG/KG) Skin / fur                Swelling (4)                (Tail apex)                Scabs (3)                (Tail)                Scabs (3)                (Tail apex)                Wound (3)                (Tail) Secretion / excretion                Salivation (3)	G: %: G: %: G: %: G: %:   G: %:	. . . . . . . .   . .	. . . . . . . .   . .	. . . . . . . .   . .	. . . . . . . .   . .	. . . . . . . .   . .	. . . . . . . .   . .	. . . . . . . .   . .	. . . . . . 1 2   . .	1 2 . . . . 1 2   . .	1 2 . . . . 1 2   . .	1 2 . . . . 1 2   . .	1 2 . . . . 1 2   . .	1 2 . . . . 1 2   . .	1 2 . . . . 1 2   . .	. . . . 1 2 . .   . .	. . 1 2 . . . .   . .	. . . . 1 2 . .   . .	. . . . 1 2 . .   . .	. . . . 1 2 . .   . .	. . . . 1 2 . .   . .	. . . . 1 2 . .   1 2	. . . . 1 2 . .   1 2	. . . . . . . .   1 2	. . . . . . . .   1 2	. . . . . . . .   1 2	. . . . . . . .   1 2	. . . . . . . .   1 2	. . . . . . . .   1 2
GROUP 3 (300 MG/KG) Skin / fur                Scabs (3)                (Shoulder left) Secretion / excretion                Salivation (3)	G: %:   G: %:	. .   . .	. .   . .	. .   . .	. .   . .	. .   . .	. .   . .	. .   . .	. .   . .	. .   1 2	. .   1 2	. .   1 2	. .   1 2	. .   1 2	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	1 2   1 A	. .   1 A
GROUP 4 (1000 MG/KG) Secretion / excretion                Salivation (3)	G: %:	. .	1 4	1 8	1 8	1 8	1 8	1 8	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A

G: Median value of the highest individual daily grades

%: Percent of affected animals (0=less than 5%, 1=between 5% and 15%,…, A=more than 95%)

.: Observation performed, sign not present

 

FUNCTIONAL OBSERVATIONS SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
AT WEEK 4
HEARING SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
PUPIL L SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
PUPIL R SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
STATIC R SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
GRIP FORE GRAM	MEAN ST. DEV N	882 152 5	858 296 5	877 184 5	943 214 5
GRIP HIND GRAM	MEAN ST. DEV N	453 107 5	407 30 5	394 53 5	356 57 5

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
AT WEEK 4
HEARING SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
PUPIL L SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
PUPIL R SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
STATIC R SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
GRIP FORE GRAM	MEAN ST. DEV N	547 116 5	766 226 5	700 280 5	724 120 5
GRIP HIND GRAM	MEAN ST. DEV N	334 47 5	343 48 5	296 48 5	295 46 5

*/* Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

+/++ Steel-test significant at 5% (+) or 1% (++) level

 

MOTOR ACTIVITY TEST SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
AT WEEK 4
Total Movements	MEAN^1 ST. DEV N	4405 1533 5	6404 2007 5	3706 891 5	3970 821 5
Ambulations	MEAN^1 ST. DEV N	938 484 5	1406 548 5	924 204 5	1299 330 5

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
AT WEEK 4
Total Movements	MEAN^1 ST. DEV N	5820 984 5	6215 1920 5	5807 1445 5	4785 301 5
Ambulations	MEAN^1 ST. DEV N	2065 466 5	2356 921 5	1780 454 5	1667 182 5

*/** Wilcoxon test significant at 5% (*) or 1% (**) level

¹Group mean of all intervals combined

 

BODY WEIGHTS (GRAM) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT
DAY 1 WEEK 1	MEAN ST. DEV N	181 4.9 5	175 8.0 5	181 7.3 5	180 8.8 5
DAY 8 WEEK 2	MEAN ST. DEV N	226 9.7 5	214 12.4 5	226 6.6 5	217 14.3 5
DAY 15 WEEK 3	MEAN ST. DEV N	266 12.7 5	244 17.5 5	265 8.5 5	249 18.9 5
DAY 22 WEEK 4	MEAN ST. DEV N	297 17.3 5	268 23.6 5	294 8.3 5	269 21.7 5
DAY 28 WEEK 4	MEAN ST. DEV N	319 19.0 5	285* 25.6 5	311 10.1 5	280* 20.0 5

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT
DAY 1 WEEK 1	MEAN ST. DEV N	138 6.0 5	136 6.2 5	138 5.8 5	137 6.8 5
DAY 8 WEEK 2	MEAN ST. DEV N	154 6.4 5	153 6.7 5	159 5.7 5	160 9.9 5
DAY 15 WEEK 3	MEAN ST. DEV N	168 5.9 5	170 5.8 5	175 8.6 5	179 11.1 5
DAY 22 WEEK 4	MEAN ST. DEV N	184 7.6 5	179 11.1 5	194 7.0 5	192 13.9 5
DAY 28 WEEK 4	MEAN ST. DEV N	190 9.9 5	188 11.0 5	198 7.6 5	203 13.4 5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

BODY WEIGHT GAIN (%) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT
DAY 1 WEEK 1	MEAN ST. DEV N	0 0.0 5	0 0.0 5	0 0.0 5	0 0.0 5
DAY 8 WEEK 2	MEAN ST. DEV N	25 2.1 5	22 2.8 5	25 2.6 5	20* 2.6 5
DAY 15 WEEK 3	MEAN ST. DEV N	47 3.6 5	39 6.1 5	46 4.5 5	38* 5.1 5
DAY 22 WEEK 4	MEAN ST. DEV N	64 5.8 5	53 10.1 5	62 6.3 5	49* 7.2 5
DAY 28 WEEK 4	MEAN ST. DEV N	76 6.7 5	63 11.3 5	72 7.0 5	55** 6.3 5

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT
DAY 1 WEEK 1	MEAN ST. DEV N	0 0.0 5	0 0.0 5	0 0.0 5	0 0.0 5
DAY 8 WEEK 2	MEAN ST. DEV N	11 5.3 5	13 5.7 5	16 2.4 5	17 2.7 5
DAY 15 WEEK 3	MEAN ST. DEV N	21 2.1 5	25 4.7 5	27* 3.2 5	31** 4.2 5
DAY 22 WEEK 4	MEAN ST. DEV N	33 5.6 5	32 5.5 5	41 2.5 5	40 4.8 5
DAY 28 WEEK 4	MEAN ST. DEV N	37 7.3 5	39 8.6 5	44 4.0 5	48* 3.5 5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

HAEMATOLOGY SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
END OF TREATMENT
WBC 10E9/L	MEAN ST. DEV N	9.4 2.4 5	8.5 0.6 5	9.4 1.3 5	10.6 2.0 5
Neutrophils %WBC	MEAN ST. DEV N	12.2 2.5 5	12.8 2.0 5	15.2 3.8 5	11.0 0.8 5
Lymphocytes %WBC	MEAN ST. DEV N	85.8 2.5 5	84.6 2.4 5	81.7 4.2 5	86.1 1.3 5
Monocytes %WBC	MEAN ST. DEV N	1.4 0.9 5	1.6 0.5 5	1.9 0.6 5	2.1 0.7 5
Eosinophils %WBC	MEAN ST. DEV N	0.7 0.4 5	0.9 0.2 5	1.1 0.5 5	0.6 0.1 5
Basophils %WBC	MEAN ST. DEV N	0.1 0.1 5	0.2 0.1 5	0.1 0.1 5	0.2 0.1 5
Red blood cells 10E12/L	MEAN ST. DEV N	8.77 0.43 5	8.72 0.13 5	8.97 0.32 5	8.95 0.27 5
Reticulocytes %RBC	MEAN ST. DEV N	2.9 0.2 5	2.7 0.3 5	2.3 0.4 5	2.3 0.4 5
RDW %	MEAN ST. DEV N	11.6 0.6 5	11.5 0.3 5	11.1 0.8 5	11.0 0.5 5
Haemoglobin mmol/L	MEAN ST. DEV N	9.8 0.2 5	10.0 0.4 5	10.4* 0.2 5	10.2 0.3 5
Haematocrit L/L	MEAN ST. DEV N	0.479 0.016 5	0.485 0.016 5	0.501 0.010 5	0.491 0.011 5
MCV fL	MEAN ST. DEV N	54.7 1.1 5	55.6 1.7 5	55.9 1.4 5	54.8 1.1 5
MCH fmol	MEAN ST. DEV N	1.12 0.03 5	1.14 0.04 5	1.16 0.04 5	1.14 0.04 5
MCHC mmol/L	MEAN ST. DEV N	20.50 0.24 5	20.57 0.21 5	20.69 0.35 5	20.75 0.40 5
Platelets 10E9/L	MEAN ST. DEV N	813 106 5	736 41 5	850 85 5	874 81 5
PT s	MEAN ST. DEV N	16.4 1.3 5	18.7* 1.6 5	17.9 1.1 5	17.1 1.3 5
APTT s	MEAN ST. DEV N	17.4 1.3 4	20.7* 1.5 5	20.0 2.0 5	18.9 1.9 5

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
END OF TREATMENT
WBC 10E9/L	MEAN ST. DEV N	5.3 1.1 5	5.0 1.6 5	7.1 1.9 5	5.9 1.7 5
Neutrophils %WBC	MEAN ST. DEV N	7.2 3.1 5	13.7+ 3.5 5	10.2 2.7 5	13.3+ 1.5 5
Lymphocytes %WBC	MEAN ST. DEV N	89.6 4.3 5	82.7+ 3.7 5	86.4 2.8 5	83.1+ 2.2 5
Monocytes %WBC	MEAN ST. DEV N	1.3 0.8 5	1.6 0.5 5	1.8 0.3 5	1.9 0.7 5
Eosinophils %WBC	MEAN ST. DEV N	1.7 0.8 5	1.9 0.5 5	1.5 0.3 5	1.6 0.6 5
Basophils %WBC	MEAN ST. DEV N	0.1 0.1 5	0.1 0.0 5	0.1 0.0 5	0.1 0.1 5
Red blood cells 10E12/L	MEAN ST. DEV N	8.17 0.15 5	8.07 0.83 5	8.32 0.26 5	8.24 0.46 5
Reticulocytes %RBC	MEAN ST. DEV N	2.8 0.4 5	2.9 1.0 5	2.4 0.3 5	2.4 0.2 5
RDW %	MEAN ST. DEV N	11.1 0.7 5	10.9 1.1 5	10.8 0.4 5	11.0 0.5 5
Haemoglobin mmol/L	MEAN ST. DEV N	9.3 0.1 5	9.1 0.9 5	9.3 0.3 5	9.1 0.5 5
Haematocrit L/L	MEAN ST. DEV N	0.450 0.006 5	0.439 0.043 5	0.451 0.012 5	0.449 0.027 5
MCV fL	MEAN ST. DEV N	55.1 0.6 5	54.4 2.1 5	54.2 1.2 5	54.4 1.2 5
MCH fmol	MEAN ST. DEV N	1.14 0.01 5	1.13 0.04 5	1.12 0.04 5	1.11 0.02 5
MCHC mmol/L	MEAN ST. DEV N	20.61 0.13 5	20.74 0.32 5	20.63 0.30 5	20.38 0.19 5
Platelets 10E9/L	MEAN ST. DEV N	834 141 5	844 64 5	925 105 5	804 305 5
PT s	MEAN ST. DEV N	17.6 1.6 5	16.3 1.0 5	16.2 0.2 5	17.9 2.4 5
APTT s	MEAN ST. DEV N	19.3 1.0 5	18.3 2.4 5	19.1 1.2 5	19.1 2.4 5

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Applicant's summary and conclusion

Conclusions:

From the results presented, a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg was established for MLA-3202.

Executive summary:

Repeated dose 28-day oral toxicity study with MLA-3202 via daily gavage in the rat.

 

The study was based on the following guidelines:

-EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.

-OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.

-OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.

 

Rationale for dose levels

Based on the results of a 5-day range finding study (Test Facility Study No. 514938), the dose levels for this 28-day oral gavage study were selected to be 0, 100, 300 and 1000 mg/kg.

 

Study outline

The test item, formulated in water, was administered daily for 28 days via oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.

 

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

 

The following parameters were evaluated: clinical signs daily; functional observation tests in Week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

 

Results

Formulation analyses confirmed that formulations of test item in water were prepared accurately and homogenously.

 

No adverse treatment-related effects were seen in any of the dose levels tested. In addition, there were no toxicologically significant changes observed in parameters including clinical appearance, motor activity, functional observations, and macroscopic examination.

 

Microscopic examination revealed absence of the normally occurring hematopoiesis observed in the spleens of 1000 mg/kg males, which correlated with reduced spleen weight. However, in the absence of any other indicator of toxicity (for example haematology parameters), these spleen findings were considered non-adverse. The minimal hepatocellular hypertrophy of the liver seen in 1000 mg/kg females was accompanied by increased liver weight, as well as changes in alanine aminotransferase levels and cholesterol, liver-related biochemical parameters. However, in the absence of any degenerative findings in the liver, these effects were considered to be non-adverse.

 

Other test item-related findings include a slightly reduced bodyweight gain in 1000 mg/kg males supported by a slightly reduced food intake. In contrast females showed a slightly increased body weight gain. Without a clear dose response or any corroborative findings these changes small changes in body weights were considered not adverse.

 

Conclusion

From the results presented in this report, a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg was established for MLA-3202.