Methylphosphonic acid

Endpoint summary

Description of key information

The key study used to cover this endpoint is a 90-day oral gavage study with the test substance MPAAU. MPAAU data are used and considered acceptable for read-across to MPA since the target substance methylphosphonic acid is highly corrosive and cannot be applied to animals in a repeated dosing scheme. Therefore, the neutralized salt form of this chemical is used to conduct the test. Details on the read-across applied can be read in the read-across justification document attached to this dossier. MPAAU was tested up to 1000 mg/kg bw /day in rats in the 90-day oral gavage study and no adverse effects were observed. As MPAAU did not induce any changes that were considered to be of toxicological significance in this study, the no-observed-adverse-effect level (NOAEL) was placed at 1000 mg/kg body weight/day. Therefore, a NOAEL of 1000 mg/kg bw/day should be applied to MPA.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-03 until 2019-03-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

90 consecutive days

Dose / conc.:

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

Remarks:

measured concentrations

Dose / conc.:

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

Remarks:

measured concentrations

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

measured concentrations

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.
Food consumption was measured weekly during the treatment period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days. Inadvertently, clinical observations were not made on one day (25 February 2019, study day 74, 75, 76 or 77 respectively (staggered start)).
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examination using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period.

Functional Observations
Once before the first exposure and once in the last week multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests [14]. These tests were conducted in all animals.

Sacrifice and pathology:

Haematology
Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined:
haematocrit value (HCT)
haemoglobin content (HGB)
red blood cell count (RBC)
mean corpuscular volume (MCV)
mean corpuscular haemoglobin (MCH)
mean corpuscular haemoglobin concentration (MCHC)
reticulocytes (Ret)
platelet count (PLT)
white blood cells (WBC)
neutrophils (Neut)
lymphocytes (Lym)
monocytes (Mono)
eosinophils (Eos)
basophils (Baso)
large unstained cells (Luc)

Blood Coagulation
Coagulation parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes. The following coagulation parameters were examined:
prothrombin time (PT)
activated partial thromboplastin time (aPTT)

Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined:
alanine aminotransferase (ALAT)
aspartate-aminotransferase (ASAT)
alkaline phosphatase (AP)
creatinine (Crea)
total protein (TP)
albumin (Alb)
urea
total bilirubin (TBIL)
total bile acids (TBA)
total cholesterol (Chol)
low density lipoprotein (LDL)
high density lipoprotein (HDL)
triglycerides (TG)
glucose (Gluc)
sodium (Na)
potassium (K)
For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals were retained at the end of treatment (80 animals) and stored at < -15 °C. T3, T4 and TSH serum levels were determined of main study animals (80 animals).

Urinalysis
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. Additionally, urine colour / appearance were recorded. The following parameters (Table 7) were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL).
specific gravity
nitrite
pH-value (pH)
protein
glucose
ketone bodies
urobilinogen (UBG)
bilirubin (BIL)
erythroctes (Ery)
leukocytes (Leuc)

Pathology
Gross necropsy
One day after the last administration (study day 91) all surviving animals of the treatment period were sacrificed using anaesthesia (ketamine/xylazine) and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle.

Organ Weight
The wet weight of the organs (Table 8) was taken from sacrificed animals as soon as possible. Paired organs were weighed together. Weight of thyroid/parathyroid glands was measured after fixation. Organ weights of the animal euthanised on study day 8 for animal welfare reasons were not recorded:
liver, uterus with cervix
kidneys, thymus
adrenals, thyroid / parathyroid glands (were weighed after fixation)
testes, spleen
epididymides, brain
prostate, seminal vesicles and coagulating glands, pituitary gland
ovaries, heart
The following tissues from all animals were preserved in 4 % neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.
adrenal glands x x
all gross lesions x x
aorta x x
brain (incl. medulla/pons, cerebellar and cerebral
cortex) x x
caecum x x
colon x x
duodenum x x
epididymides x x
eyes with optic nerve and Harderian gland x --
femur with knee joint x --
heart x x
ileum (including Peyer´s patches) x x
jejunum x x
kidneys x x
liver x x
lungs x x
lymph nodes (mandibular) x --
lymph nodes (mesenteric and axillary) x x
mammary gland area (male and female) x x
oesophagus x x
ovaries x x
oviducts x --
pancreas x x
pituitary x x
prostate and seminal vesicles with coagulating glands as a whole x x
rectum x x
salivary glands (sublingual, submandibular, parotis) x x
sciatic nerve x x
skeletal muscle x x
skin x x
spinal cord (cervical, thoracic and lumbar segments) x x
spleen x x
sternum (with bone marrow) x x
stomach x x
testes x x
thymus x x
thyroid gland including parathyroid glands x x
tongue x --
trachea x x
ureters x --
urinary bladder x x
uterus with cervix and vagina x x
The animal intercurrently euthanised for animal welfare reasons was also subjected to a gross necropsy and the organs preserved for a histopathological examination.

Histopathology
The afore-listed organs (Table 9) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the groups 1 and 4 sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice.
These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in the high dose group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In the female HD group, prone position was noted on treatment days 68 to 73 for one animal and dehydration on day 15 for five animals. As these findings were observed in one single animal or on one single day, a toxicological effect of the test item is not considered.
The findings crust and/or hairless area at the cheek, head or forelimbs in several female animals of the control group or for one female LD group (hairless area at forelimbs, hindlimbs, lateral area, abdomen, legs) are not considered to be of biological or toxicological relevance.
Moving the bedding and/or slight to moderate salivation (in one HD female animal with a severe grade) were noted in 4 out of 10 male LD group animals and in all male and female MD and HD group animals. The clinical signs salivation and moving the bedding were observed in close timely relation to the dose administration and therefore considered to be signs of a local reaction after test item treatment rather than a systemic adverse effect.
Regarding detailed clinical observation, statistical significance was noted for males in week 3 for a slightly increased mean score of salivation in the HD group (15 % above control), in week 12 for animal sleeps (87.1 % below control) and animal moving in cage (305 % above control) in the MD group. In females, statistical significance was found in week 2 for an increase of 10% above control for response to handling in the LD group, for an increase of salivation in the HD group in week 3 (20 % above control), in week 4 (30 % above control), in week 5 (25 % above control), in week 6 (25 % above control), in week 8 (20 % above control), in week 10 (35 % above control) and additionally in week 11 (30 % above control) and in the HD group for an increased mean score of animal moving in cage in week 6 (100 % above control). The statistically significant effects on detailed clinical examination parameters were not considered to be test item-related as they were seen occasionally in single weeks. Significantly increased salivation, which was observed in several weeks in the female HD group, was assessed under consideration of the presence of salivation during daily clinical observation and therefore not considered to be a systemic adverse effect of the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control male animal (animal no. 4) was euthanized in moribund condition on study day 8. Clinical findings on day of sacrifice were prone position, severely reduced spontaneous activity, eyes closed and abnormal breathing. No clinical findings were seen on treatment days 1 to 7. Considering histopathological assessment, death of animal no. 4 was considered to be incidental. The cause of morbidity was not evident at the histopathological evaluation and is assumed to be related to gavage procedure. There were no mortalities in the remaining males and females of the control and all dose groups until the end of the treatment period.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects were found for all haematological and coagulation parameters of all male and female dose groups at the end of the treatment period.
A statistical significant decrease of 71.4 % below control was found for the mean value of eosinophils in the male MD dose group and for an increase of monocytes in the female MD dose group (51.1 % above control). The statistical significant changes from control group were found without dose dependency and therefore the single findings are not considered to be of toxicological relevance.
No statistical significances were noted for coagulation parameters in any male and female dose group when compared to control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with the test item had no toxicologically relevant effects on parameters of clinical biochemistry or hormone analysis of males and females in any of the test item-treated groups.
Statistical significance was found in the female HD group for a decrease of 64.87 % below control for total bile acid. Differences between test item-treated males or females and their respective controls showed no dose-dependency or consistency. Additionally, no test item- related effects were noted at histopathological assessment. Therefore, the statistical significance is considered to be of no biological or toxicological relevance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There was no adverse effect of the test item on urinary parameters measured at the end of the treatment period.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects were observed in any parameter of the functional observation battery at the end of the treatment period when compared to observations before treatment for the male and female groups.
The statistical significant mean scores (decrease of animal sleeps, increase of moving in cage) for the male LD group in the last week of treatment were not considered to be an effect of test item treatment as they were seen without dose dependency.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Treatment with the test item had no toxicologically relevant effects on absolute and relative organ weight of males and females in any of the test item-treated groups. No statistical significant organ weight changes were found for all male and female dose groups when compared to control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic finding of the male control animal sacrificed in moribund condition was a dark red coloured right caudal lobe of the lung. Considering histopathological assessment, the cause of morbidity was not evident at the histopathological evaluation. In one HD male animal, a cyst at the prostate gland and spotted harderian glands were observed at necropsy on study day 91. Regarding the female HD group, necropsy findings were few black foci on the stomach and red coloured axillary or mandibular lymph nodes. Uterus dilatation was found in one control and one MD animal and several HD animals. Considering histopathological evaluation, all gross lesions observed at necropsy for terminally sacrificed animals were deemed to be incidental or represented the normal animal physiology and therefore, considered to be not related to the treatment with the test item.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed during the histopathological evaluation. All findings recorded in decedent and surviving animals were deemed to be incidental or were within the range of background alterations that may be recorded in animals of this strain and age and in this study type.
Considering histopathological assessment, there were no organ weight changes, gross lesions or histological alterations that could be attributed to the treatment with the test item. Thus, the histopathological NOEL (no observed effect level) could be established at 1000 mg/kg bw.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no relevant effect found

Key result

Critical effects observed:

no

Clinical sign (male)

			C/F	LD/F	MD/F	HD/F
Day 1 -> 90	Animals examined	N	10	10	10	10
	Animals with signs	N	1	4	10	10
		%	10	40	100	100
	Digestive Tract	N	0	0	9	10
	Salivation increased	%	0	0	90	100
	Central Nervous System	N	1	4	10	10
		%	10	40	100	100
	Moving the bedding	N	0	4	10	10
		%	0	40	100	100
	Prone Position	N	1	0	0	0
		%	10	0	0	10
	Spontaneous activity reduced	N	1	0	0	0
		%	10	0	0	0
	Eyes / Mucous Membranes	N	1	0	0	0
	Eyes closed	%	10	0	0	0
	Respiratory / Pulmonary Tract	N	1	0	0	0
	Abnormal breathing	%	10	0	0	0
	Dead	N	1	0	0	0
	Sacrificed, moribund	%	10	0	0	0

 

Clinical signs female:

			C/F	LD/F	MD/F	HD/F
Day 1 -> 90	Animals examined	N	10	10	10	10
	Animals with signs	N	4	1	10	10
		%	40	10	100	100
	Digestive Tract	N	0	0	5	10
	Salivation increased	%	0	0	50	100
	Central Nervous System	N	0	0	10	10
		%	0	0	100	100
	Moving the bedding	N	0	0	10	10
		%	0	0	100	100
	Prone Position	N	0	0	0	1
		%	0	0	0	10
	Skin and Fur	N	4	1	0	5
		%	40	10	0	50
	Hairless area	N	3	1	0	0
		%	30	10	0	0
	Crust	N	2	0	0	0
		%	20	0	0	0
	Dehydration	N	0	0	0	5
		%	0	0	0	50
	Dead	N	0	0	0	0
		%	0	0	0	0

 

 Body weight (Male) in-life phase

		C/M	LD / M	MD / M	HD / M
day 1	Mean	233.1a	235	241.2	237.9
	S.d.	9	10.5	18.7	10.3
	N	10	10	10	10
	Deviation Vs Control [%]		0.8	3.5	2.1
day 8	Mean	266.1 k	264.7	267.9	270.1
	S.d.	14.6	11.4	12.7	12.5
	N	10	10	10	10
	Deviation Vs Control [%]		-0.5	0.7	1.5
day 15	Mean	291.2 a	288.9	292.2	296.9
	S.d.	15	13.6	11.1	16.7
	N	9	10	10	10
	Deviation Vs Control [%]		-0.8	0.3	1.9
day 22	Mean	311.6 a	308.3	314.5	317.9
	S.d.	18.7	16.4	16.2	21.2
	N	9	10	10	10
	Deviation Vs Control [%]		-1	0.9	2
day 29	Mean	330.7 a	322.7	334.3	335.1
	S.d.	21.2	16	17.8	25.2
	N	9	10	10	10
	Deviation Vs Control [%]		-2.4	1.1	1.3
day 36	Mean	346.8 a	340.2	350.8	348.3
	S.d.	22.4	17.5	20.3	28.6
	N	9	10	10	10
	Deviation Vs Control [%]		-1.9	1.2	0.4
day 43	Mean	360.0 a	350.9	362.8	362
	S.d.	25.3	19	22.7	28.5
	N	9	10	10	10
	Deviation Vs Control [%]		-2.5	0.8	0.6
day 50	Mean	368.8 a	360.9	370.2	371.8
	S.d.	26.2	22.2	22.4	28.2
	N	9	10	10	10
	Deviation Vs Control [%]		-2.1	0.4	0.8
day 57	Mean	379.1 a	371.2	381.7	382.3
	S.d.	28.6	23.6	23.7	31.3
	N	9	10	10	10
	Deviation Vs Control [%]		-2.1	0.7	0.8
day 64	Mean	390.2 a	381.7	391.8	389.8
	S.d.	29.7	23.4	24.2	32
	N	9	10	10	10
	Deviation Vs Control [%]		-2.2	0.4	-0.1
day 71	Mean	397.6 a	388.9	398.2	401.9
	S.d.	30.1	24.4	24.2	33.6
	N	9	10	10	10
	Deviation Vs Control [%]		-2.2	0.2	1.1
day 78	Mean	402.7 a	393.7	404.1	410.4
	S.d.	31.5	29.3	26.1	35
	N	9	10	10	10
	Deviation Vs Control [%]		-2.2	0.4	1.9
day 85	Mean	408.7 a	401.1	409.1	417.5
	S.d.	31.5	29.5	24.8	34.5
	N	9	10	10	10
	Deviation Vs Control [%]		-1.9	0.1	2.2
day 90	Mean	418.7 a	403.2	412.4	424.3
	S.d.	35.3	25.7	24.9	35.6
	N	9	10	10	10
	Deviation Vs Control [%]		-3.7	-1.5	1.3

Statistic Profile = Decision Tree, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, X = Group excluded from statistics
a=ANOVA; k=KRUSKALL-WALLIS. 

 

Body weight (female - in life)

		C/M	LD / M	MD / M	HD / M
day 1	Mean	173.0 k	177.7	175.8	172.4
	S.d.	7.3	11.6	8.9	7.2
	N	10	10	10	10
	Deviation Vs Control [%]		2.7	1.6	-0.3
day 8	Mean	187.8 a	192.7	188.3	187.1
	S.d.	5.4	10.2	9.1	7.1
	N	10	10	10	10
	Deviation Vs Control [%]		2.6	0.3	-0.4
day 15	Mean	199.6 a	206.2	198.1	193
	S.d.	7.2	14.3	9.1	12
	N	10	10	10	10
	Deviation Vs Control [%]		3.3	-0.8	-3.3
day 22	Mean	207.3 a	213.5	207.3	206.1
	S.d.	9.6	14.8	9.2	7
	N	10	10	10	10
	Deviation Vs Control [%]		3	0	-0.6
day 29	Mean	212.0 a	218.7	214.1	215
	S.d.	9	13.7	8.8	9.6
	N	10	10	10	10
	Deviation Vs Control [%]		3.2	1	1.4
day 36	Mean	219.9 a	226.8	222.7	223
	S.d.	9.1	13.4	10	9.9
	N	10	10	10	10
	Deviation Vs Control [%]		3.1	1.3	1.4
day 43	Mean	222.7 a	230.3	226.1	224.4
	S.d.	10	14.8	12.1	9.5
	N	10	10	10	10
	Deviation Vs Control [%]		3.4	1.5	0.8
day 50	Mean	227.7 k	236	230.2	229.4
	S.d.	10.5	17.5	11.4	9.8
	N	10	10	10	10
	Deviation Vs Control [%]		3.6	1.1	0.7
day 57	Mean	231.7 k	242	232.1	236.2
	S.d.	10.4	16.2	11.3	13.3
	N	10	10	10	10
	Deviation Vs Control [%]		4.4	0.2	1.9
day 64	Mean	233.9 a	242.5	234.7	237.3
	S.d.	9.6	15.9	13	9.6
	N	10	10	10	10
	Deviation Vs Control [%]		3.7	0.3	1.5
day 71	Mean	234.0 a	242.3	237.8	238.9
	S.d.	11.4	16.4	13.8	10.1
	N	10	10	10	10
	Deviation Vs Control [%]		3.5	1.6	2.1
day 78	Mean	233.6 a	249.2	242.4	241.3
	S.d.	14.7	20.1	13.8	8.9
	N	10	10	10	10
	Deviation Vs Control [%]		6.7	3.8	3.3
day 85	Mean	238.7 k	249.8	243.2	243.8
	S.d.	10.9	17.4	14.5	11.4
	N	10	10	10	10
	Deviation Vs Control [%]		4.7	1.9	2.1
day 90	Mean	239.3 a	249.1	243.4	243.5
	S.d.	11.7	17.4	13.4	11.1
	N	10	10	10	10
	Deviation Vs Control [%]		4.1	1.7	1.8

 

Body weight changes (Male, in-life)

		C / M	LD / M	MD / M	HD / M
d 1 -> 8	Mean	33.0 k	29.7	26.7	32.2
	S.d.	7.9	4.2	22.8	11.5
	N	10	10	10	10
d 8 -> 15	Mean	22.6 a	24.2	24.3	26.8
	S.d.	4.5	4.5	5.4	4.7
	N	9	10	10	10
d 15 -> 22	Mean	20.3 a	19.4	22.3	21
	S.d.	6.6	4.4	6.2	5.8
	N	9	10	10	10
d 22 -> 29	Mean	19.1 a	14.4	19.8	17.2
	S.d.	7.3	8.2	3.6	7.5
	N	9	10	10	10
d 29 -> 36	Mean	16.1 k	17.5	16.5	13.2
	S.d.	3.7	5.7	12.5	5.1
	N	9	10	10	10
d 36 -> 43	Mean	13.2 k	10.7	12	13.7
	S.d.	4.4	3.4	11.6	5.3
	N	9	10	10	10
d 43 -> 50	Mean	8.8 a	10	7.4	9.8
	S.d.	3.7	7.3	3.4	3.5
	N	9	10	10	10
d 50 -> 57	Mean	10.3 a	10.3	11.5	10.5
	S.d.	3.9	3.9	2.3	4.1
	N	9	10	10	10
d 57 -> 64	Mean	11.1 k	10.5	10.1	7.5
	S.d.	4.9	4.2	4.5	4.5
	N	9	10	10	10
d 64 -> 71	Mean	7.3 ad	7.2	6.4	12.1 *
	S.d.	3.5	2.9	2.9	3.9
	N	9	10	10	10
d 71 -> 78	Mean	5.1 k	4.8	5.9	8.5
	S.d.	3.3	7.2	3.8	2.9
	N	9	10	10	10
d 78 -> 85	Mean	6.0 a	7.4	5	7.1
	S.d.	3.3	4.6	6.1	2.8
	N	9	10	10	10
d 85 -> 90	Mean	10.0 k	2.1	3.3	6.8
	S.d.	17.1	5.2	4.3	3
	N	9	10	10	10
d 1 -> 90	Mean	184.0 k	168.2	171.2	186.4
	S.d.	31.6	20.4	31.7	30.5
	N	9	10	10	10

 

Body weight changes (Female; in-life)

		C / F	LD / F	MD / F	HD / F
d 1 -> 8	Mean	14.8 a	15	12.5	14.7
	S.d.	4.9	6	4.6	4.8
	N	10	10	10	10
d 8 -> 15	Mean	11.8 k	13.5	9.8	5.9
	S.d.	4	5.4	4.2	7.8
	N	10	10	10	10
d 15 -> 22	Mean	7.7 k	7.3	9.2	13.1
	S.d.	5.4	6	4.7	11
	N	10	10	10	10
d 22 -> 29	Mean	4.7 a	5.2	6.8	8.9
	S.d.	4.8	7	5.2	5.1
	N	10	10	10	10
d 29 -> 36	Mean	7.9 k	8.1	8.6	8
	S.d.	5.1	5.8	7.9	4
	N	10	10	10	10
d 36 -> 43	Mean	2.8 k	3.5	3.4	1.4
	S.d.	4.7	4.2	7.7	5.7
	N	10	10	10	10
d 43 -> 50	Mean	5.0 a	5.7	4.1	5
	S.d.	5.4	6.4	3.6	6.5
	N	10	10	10	10
d 50 -> 57	Mean	4.0 a	6	1.9	6.8
	S.d.	3.3	5.9	4.7	5.8
	N	10	10	10	10
d 57 -> 64	Mean	2.2 a	0.5	2.6	1.1
	S.d.	3.1	3.9	6.6	6.3
	N	10	10	10	10
d 64 -> 71	Mean	0.1 a	-0.2	3.1	1.6
	S.d.	5.5	6.4	4.8	2.1
	N	10	10	10	10
d 71 -> 78	Mean	-0.4 k	6.9	4.6	2.4
	S.d.	7.5	6.6	5.6	5.7
	N	10	10	10	10
d 78 -> 85	Mean	5.1 a	0.6	0.8	2.5
	S.d.	8.8	8.5	10.2	8.6
	N	10	10	10	10
d 85 -> 90	Mean	0.6 k	-0.7	0.2	-0.3
	S.d.	8.2	6.9	7.7	10.4
	N	10	10	10	10
d 1 -> 90	Mean	66.3 a	71.4	67.6	71.1
	S.d.	10.4	11.5	10.4	11.2
	N	10	10	10	10

 

Summary Haematology

		C / M	LD / M	MD / M	HD / M
HCT	Mean	48.97 a	49.96	49.74	49.27
[%]	S.d.	1.88	2.73	2.4	2.99
day 91	N	9	9	9	10
	Deviation Vs Control [%]		2.02	1.59	0.62
HGB	Mean	16.01 a	16.36	16.43	16.44
[g/dL]	S.d.	0.62	0.91	0.45	1.09
day 91	N	9	9	9	10
	Deviation Vs Control [%]		2.15	2.64	2.68
RBC	Mean	8.986 a	9.22	9.257	9.253
[1E12/L]	S.d.	0.255	0.598	0.361	0.501
day 91	N	9	9	9	10
	Deviation Vs Control [%]		2.609	3.017	2.976
MCV	Mean	54.51 a	54.22	53.73	53.25
[fL]	S.d.	1.96	1.6	1.53	1.27
day 91	N	9	9	9	10
	Deviation Vs Control [%]		-0.53	-1.43	-2.31
MCH	Mean	17.81 k	17.77	17.76	17.77
[pg]	S.d.	0.48	0.57	0.63	0.47
day 91	N	9	9	9	10
	Deviation Vs Control [%]		-0.25	-0.31	-0.23
MCHC	Mean	32.70 a	32.76	33.07	33.37
[g/dL]	S.d.	0.95	0.75	1.05	0.74
day 91	N	9	9	9	10
	Deviation Vs Control [%]		0.17	1.12	2.05
PLT	Mean	643.2 a	709.8	675	692
[1E9/L]	S.d.	75.7	68.5	89.1	101.1
day 91	N	9	9	9	10
	Deviation Vs Control [%]		10.3	4.9	7.6
WBC	Mean	3.957 a	3.632	3.46	4.127
[1E9/L]	S.d.	1.202	0.919	1.227	1.299
day 91	N	9	9	9	10
	Deviation Vs Control [%]		-8.2	-12.553	4.305
RET%	Mean	1.649 a	1.54	1.526	1.553
[%]	S.d.	0.233	0.235	0.221	0.164
day 91	N	9	9	9	10
	Deviation Vs Control [%]		-6.604	-7.48	-5.815
NEUT%	Mean	18.38 k	23.86	23.46	19.83
[%]	S.d.	4.97	6.07	12.01	4.22
day 91	N	9	9	9	10
	Deviation Vs Control [%]		29.81	27.63	7.9
LYM%	Mean	78.62 a	73.07	74.19	77.1
[%]	S.d.	5.46	6.93	12.44	4.63
day 91	N	9	9	9	10
	Deviation Vs Control [%]		-7.07	-5.64	-1.94
MONO%	Mean	1.69 a	1.88	1.8	2.28
[%]	S.d.	0.74	0.49	0.7	0.75
day 91	N	9	9	9	10
	Deviation Vs Control [%]		11.18	6.58	35
EOS%	Mean	1.011 u	0.911	0.289 *	0.38
[%]	S.d.	0.983	0.827	0.078	0.199
day 91	N	9	9	9	10
	Deviation Vs Control [%]		-9.89	-71.429	-62.418
BASO%	Mean	0.100 k	0.1	0.056	0.09
[%]	S.d.	0.05	0.1	0.053	0.074
day 91	N	9	9	9	10
	Deviation Vs Control [%]		0	-44.444	-10
LUC%	Mean	0.222 k	0.233	0.233	0.33
[%]	S.d.	0.192	0.194	0.212	0.226
day 91	N	9	9	9	10
	Deviation Vs Control [%]		5	5	48.5

		C / F	LD / F	MD / F	HD / F
HCT	Mean	45.50 a	46.1	46.09	45.75
[%]	S.d.	2.18	1.51	1.79	2.18
day 91	N	9	10	10	10
	Deviation Vs Control [%]		1.32	1.3	0.55
HGB	Mean	14.89 a	15.18	15.25	15.04
[g/dL]	S.d.	0.61	0.5	0.63	0.75
day 91	N	9	10	10	10
	Deviation Vs Control [%]		1.96	2.43	1.01
RBC	Mean	8.190 a	8.367	8.348	8.22
[1E12/L]	S.d.	0.398	0.257	0.316	0.492
day 91	N	9	10	10	10
	Deviation Vs Control [%]		2.161	1.929	0.366
MCV	Mean	55.59 k	55.11	55.21	55.71
[fL]	S.d.	1.56	1.01	0.93	2.13
day 91	N	9	10	10	10
	Deviation Vs Control [%]		-0.86	-0.68	0.22
MCH	Mean	18.19 a	18.13	18.27	18.33
[pg]	S.d.	0.53	0.47	0.28	0.75
day 91	N	9	10	10	10
	Deviation Vs Control [%]		-0.32	0.45	0.78
MCHC	Mean	32.72 a	32.93	33.1	32.89
[g/dL]	S.d.	0.46	0.77	0.39	0.4
day 91	N	9	10	10	10
	Deviation Vs Control [%]		0.63	1.15	0.51
PLT	Mean	754.8 a	694.7	745.6	660.2
[1E9/L]	S.d.	53.4	72.4	106.7	108.6
day 91	N	9	10	10	10
	Deviation Vs Control [%]		-8	-1.2	-12.5
WBC	Mean	1.582 a	1.557	1.77	1.787
[1E9/L]	S.d.	0.487	0.574	0.821	0.713
day 91	N	9	10	10	10
	Deviation Vs Control [%]		-1.594	11.868	12.942
RET%	Mean	1.889 a	1.833	1.875	1.809
[%]	S.d.	0.226	0.266	0.407	0.42
day 91	N	9	10	10	10
	Deviation Vs Control [%]		-2.959	-0.735	-4.229
NEUT%	Mean	14.60 k	16.72	20.74	20.79
[%]	S.d.	4.99	5.14	11.05	5.6
day 91	N	9	10	10	10
	Deviation Vs Control [%]		14.52	42.05	42.4
LYM%	Mean	83.21 k	80.86	76.46	77.25
[%]	S.d.	4.88	5.46	11.65	5.52
day 91	N	9	10	10	10
	Deviation Vs Control [%]		-2.83	-8.11	-7.16
MONO%	Mean	1.49 ad	1.63	2.25 *	1.29
[%]	S.d.	0.62	0.52	0.77	0.35
day 91	N	9	10	10	10
	Deviation Vs Control [%]		9.48	51.12	-13.36
EOS%	Mean	0.489 a	0.47	0.34	0.44
[%]	S.d.	0.431	0.472	0.207	0.259
day 91	N	9	10	10	10
	Deviation Vs Control [%]		-3.864	-30.455	-10
BASO%	Mean	0.100 k	0.17	0.04	0.06
[%]	S.d.	0.087	0.236	0.052	0.07
day 91	N	9	10	10	10
	Deviation Vs Control [%]		70	-60	-40
LUC%	Mean	0.167 k	0.19	0.18	0.18
[%]	S.d.	0.132	0.074	0.132	0.123
day 91	N	9	10	10	10
	Deviation Vs Control [%]		14	8	8

a=ANOVA; k=KRUSKALL-WALLIS; ad=ANOVA-DUNNETT; * = p < 0.05

 

SUMMARY CLINICAL BIOCHEMISTRY

		C/M	LD / M	MD / M	HD / M
ALAT	Mean	28.24 k	26.77	25.48	24.61
[U/L]	S.d.	9.63	7.32	9.55	8.6
day 91	N	9	10	10	9
	Deviation Vs Control [%]		-5.22	-9.79	-12.86
Gluc	Mean	8.007 a	8.434	8.302	8.01
[mmol/L]	S.d.	2.54	1.627	2.498	1.505
day 91	N	9	10	10	9
	Deviation Vs Control [%]		5.337	3.689	0.042
Crea	Mean	18.0 k	21.2	16.6	37.2
[µmol/L]	S.d.	4.6	8.2	4.7	40.9
day 91	N	9	10	9	9
	Deviation Vs Control [%]		17.8	-8	106.8
ASAT	Mean	61.07 a	73.75	73.07	71.13
[U/L]	S.d.	37.35	30.33	24.57	30.79
day 91	N	9	10	10	9
	Deviation Vs Control [%]		20.77	19.66	16.48
AP	Mean	47.722 a	66.404	69.958	50.442
[U/L]	S.d.	32.024	28.615	42.319	26.699
day 91	N	9	10	10	9
	Deviation Vs Control [%]		39.147	46.594	5.7
Na	Mean	157.9 k	156.4	147.8	153.5
[mmol/L]	S.d.	5.3	3.1	23.4	3.5
day 91	N	9	10	10	10
	Deviation Vs Control [%]		-0.9	-6.4	-2.8
K	Mean	5.180 a	5.46	5.031	5.632
[mmol/L]	S.d.	0.591	0.726	1.336	1.321
day 91	N	9	10	10	10
	Deviation Vs Control [%]		5.405	-2.876	8.726
TP	Mean	48.42 a	53.36	50.52	52.83
[g/L]	S.d.	14.68	10.58	9.45	8.42
day 91	N	9	10	10	9
	Deviation Vs Control [%]		10.2	4.33	9.11
ALB	Mean	32.114 k	27.491	26.543	27.56
[g/L]	S.d.	2.442	6.451	6.91	7.34
day 91	N	9	10	10	9
	Deviation Vs Control [%]		-14.397	-17.349	-14.182
TBIL	Mean	1.69 k	1.98	2.09	2.03
[µmol/L]	S.d.	0.3	0.37	0.66	0.48
day 91	N	9	10	10	9
	Deviation Vs Control [%]		17.24	23.75	20.39
CHOL	Mean	1.823 a	1.761	1.676	1.698
[mmol/L]	S.d.	0.261	0.255	0.275	0.258
day 91	N	6	7	5	5
	Deviation Vs Control [%]		-3.395	-8.08	-6.874
TBA	Mean	29.983 a	27.067	20.185	29.147
[µmol/L]	S.d.	9.863	8.877	9.423	18.154
day 91	N	9	10	10	9
	Deviation Vs Control [%]		-9.727	-32.679	-2.79
TG	Mean	0.73 a	0.79	0.5	0.58
[mmol/L]	S.d.	0.26	0.31	0.22	0.26
day 91	N	9	10	10	8
	Deviation Vs Control [%]		7.59	-32.23	-20.91
Urea	Mean	6.131 k	6.694	6.124	8.121
[mmol/L]	S.d.	1.158	1.276	1.906	3.673
day 91	N	9	10	10	9
	Deviation Vs Control [%]		9.181	-0.116	32.457
HDL	Mean	1.195 a	1.259	1.048	1.226
[mmol/L]	S.d.	0.169	0.123	0.421	0.17
day 91	N	6	7	4	5
	Deviation Vs Control [%]		5.32	-12.343	2.594
LDL (calculated)	Mean	0.489 a	0.34	0.477	0.475
[mmol/L]	S.d.	0.401	0.254	0.317	0.194
day 91	N	6	7	4	4
	Deviation Vs Control [%]		-30.412	-2.454	-2.863

 

		C/F	LD / F	MD / F	HD / F
ALAT	Mean	26.18 a	29.3	29.12	29.32
[U/L]	S.d.	8.53	10.69	7.07	9.16
day 91	N	10	10	10	10
	Deviation Vs Control [%]		11.92	11.23	11.99
Gluc	Mean	6.648 a	7.11	5.648	6.567
[mmol/L]	S.d.	1.25	1.174	1.207	2.565
day 91	N	10	10	10	10
	Deviation Vs Control [%]		6.949	########	-1.218
Crea	Mean	29.5 a	23.9	26.7	23.2
[µmol/L]	S.d.	5.8	6.3	5.4	5.2
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-19	-9.5	-21.4
ASAT	Mean	86.14 a	71	92.37	85.5
[U/L]	S.d.	26.78	20.85	19.4	24.13
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-17.58	7.23	-0.74
AP	Mean	36.005 a	32.742	40.97	44.408
[U/L]	S.d.	21.205	10.974	17.304	23.358
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-9.063	13.79	23.338
Na	Mean	159.9 k	158	156	159.8
[mmol/L]	S.d.	10.4	2.8	10.3	5.9
day 91	N	8	9	10	10
	Deviation Vs Control [%]		-1.2	-2.4	0
K	Mean	4.879 k	4.632	4.777	4.245
[mmol/L]	S.d.	1.526	1.122	1.25	0.522
day 91	N	8	9	10	10
	Deviation Vs Control [%]		-5.053	-2.086	-12.99
TP	Mean	61.15 k	58.66	63.5	59.11
[g/L]	S.d.	9.29	9.11	3.88	5.66
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-4.07	3.84	-3.34
ALB	Mean	33.941 k	35.086	37.175	34.053
[g/L]	S.d.	6.426	4.016	1.813	5.059
day 91	N	10	10	10	10
	Deviation Vs Control [%]		3.374	9.528	0.33
TBIL	Mean	2.80 k	2.55	3.12	2.3
[µmol/L]	S.d.	0.67	0.51	0.93	0.33
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-8.93	11.43	-17.86
CHOL	Mean	1.594 a	1.599	1.562	1.551
[mmol/L]	S.d.	0.278	0.309	0.383	0.39
day 91	N	8	9	10	9
	Deviation Vs Control [%]		0.322	-1.992	-2.675
TBA	Mean	45.823 u	27.922	38.769	16.099 **
[µmol/L]	S.d.	21.993	15.631	40.037	7.575
day 91	N	10	10	10	10
	Deviation Vs Control [%]		########	########	-64.867
TG	Mean	0.40 k	0.36	0.35	0.41
[mmol/L]	S.d.	0.12	0.11	0.1	0.3
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-11.17	-13.15	0.74
Urea	Mean	6.917 a	6.725	6.457	5.891
[mmol/L]	S.d.	0.771	1.313	0.619	0.829
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-2.776	-6.65	-14.833
HDL	Mean	1.236 a	1.227	1.232	1.211
[mmol/L]	S.d.	0.138	0.267	0.259	0.252
day 91	N	8	9	10	9
	Deviation Vs Control [%]		-0.775	-0.344	-2.033
LDL (calculated)	Mean	0.270 a	0.302	0.26	0.258
[mmol/L]	S.d.	0.169	0.129	0.127	0.109
day 91	N	8	9	10	9
	Deviation Vs Control [%]		11.563	-3.882	-4.703

k=KRUSKALL-WALLIS; a=ANOVA; u=KRUSKALL-WALLIS-DUNN; ** = p < 0.01

 

NECROPSY INCIDENCES BY ANIMAL NUMBERS

		M	M	M	M
		C / M	LD / M	MD / M	HD / M
BRAIN	Total affected/examined	0/10	0/10	0/10	0/10
SPINAL CORD, CERVIC	Total affected/examined	0/10	0/10	0/10	0/10
SPINAL CORD, THORAC	Total affected/examined	0/10	0/10	0/10	0/10
SPINAL CORD, LUMBAR	Total affected/examined	0/10	0/10	0/10	0/10
SCIATIC NERVE, LEFT	Total affected/examined	0/10	0/10	0/10	0/10
HEART	Total affected/examined	0/10	0/10	0/10	0/10
AORTA	Total affected/examined	0/10	0/10	0/10	0/10
TRACHEA	Total affected/examined	0/10	0/10	0/10	0/10
LUNG	Abnormal color (dark red, right caudal lobe)	4+
	Total affected/examined	1/10	0/10	0/10	0/10
TONGUE	Total affected/examined	0/10	0/10	0/10	0/10
ESOPHAGUS	Total affected/examined	0/10	0/10	0/10	0/10
STOMACH	Total affected/examined	0/10	0/10	0/10	0/10
DUODENUM	Total affected/examined	0/10	0/10	0/10	0/10
JEJUNUM	Total affected/examined	0/10	0/10	0/10	0/10
ILUEM	Total affected/examined	0/10	0/10	0/10	0/10
PEYER'S PATCHES	Total affected/examined	0/10	0/10	0/10	0/10
CECUM	Total affected/examined	0/10	0/10	0/10	0/10
COLON	Total affected/examined	0/10	0/10	0/10	0/10
RECTUM	Total affected/examined	0/10	0/10	0/10	0/10
LIVER	Total affected/examined	0/10	0/10	0/10	0/10
PANCREAS	Total affected/examined	0/10	0/10	0/10	0/10
KIDNEYS	Total affected/examined	0/10	0/10	0/10	0/10
URETURS	Total affected/examined	0/10	0/10	0/10	0/10
URINARY BLADDER	Total affected/examined	0/10	0/10	0/10	0/10
TESTED	Total affected/examined	0/10	0/10	0/10	0/10
EPIDIDYMES	Total affected/examined	0/10	0/10	0/10	0/10
PROSTATE GLAND	Cyst				31
	Total affected/examined	0/10	0/10	0/10	1/10
CYST	Total affected/examined	0/10	0/10	0/10	0/10
COAGULATING GLANDS	Total affected/examined	0/10	0/10	0/10	0/10
SEMINAL VESICLES	Total affected/examined	0/10	0/10	0/10	0/10
PITUTARY GLAND	Total affected/examined	0/10	0/10	0/10	0/10
THYROID GLAND	Total affected/examined	0/10	0/10	0/10	0/10
PARATHYROID GLANDS	Total affected/examined	0/10	0/10	0/10	0/10
ADRENAL GLANDS	Total affected/examined	0/10	0/10	0/10	0/10
SPLEEN	Total affected/examined	0/10	0/10	0/10	0/10
BONE MARROW	Total affected/examined	0/10	0/10	0/10	0/10
THMUS	Total affected/examined	0/10	0/10	0/10	0/10
AUXILLARY LYMPH NODES	Total affected/examined	0/10	0/10	0/10	0/10
MESENT. LYMPH NODES	Total affected/examined	0/10	0/10	0/10	0/10
SALIVARY GLANDS	Total affected/examined	0/10	0/10	0/10	0/10
HARDERIAN GLANDS	Abnormal color spotted				31
	Total affected/examined	0/10	0/10	0/10	1/10
MAMMARY GLANDS	Total affected/examined	0/10	0/10	0/10	0/10
SKIN/SUBCUTIS	Total affected/examined	0/10	0/10	0/10	0/10
SKELETAL MUSCLE	Total affected/examined	0/10	0/10	0/10	0/10
EYES	Total affected/examined	0/10	0/10	0/10	0/10
OPTIC NERVES	Total affected/examined	0/10	0/10	0/10	0/10
FEMUR WITH KNEE JOINT	Total affected/examined	0/10	0/10	0/10	0/10
STERNUM	Total affected/examined	0/10	0/10	0/10	0/10

 

		M	M	M	M
		C / M	LD / M	MD / M	HD / M
BRAIN	Total affected/examined	0/10	0/10	0/10	0/10
SPINAL CORD, CERVIC	Total affected/examined	0/10	0/10	0/10	0/10
SPINAL CORD, THORAC	Total affected/examined	0/10	0/10	0/10	0/10
SPINAL CORD, LUMBAR	Total affected/examined	0/10	0/10	0/10	0/10
SCIATIC NERVE, LEFT	Total affected/examined	0/10	0/10	0/10	0/10
HEART	Total affected/examined	0/10	0/10	0/10	0/10
AORTA	Total affected/examined	0/10	0/10	0/10	0/10
TRACHEA	Total affected/examined	0/10	0/10	0/10	0/10
LUNG	Abnormal color (dark red, right caudal lobe)
	Total affected/examined	0/10	0/10	0/10	0/10
TONGUE	Total affected/examined	0/10	0/10	0/10	0/10
ESOPHAGUS	Total affected/examined	0/10	0/10	0/10	0/10
STOMACH	Focus (black, few (2-6), 0.1cm				74
	Total affected/examined	0/10	0/10	0/10	1/10
DUODENUM	Total affected/examined	0/10	0/10	0/10	0/10
JEJUNUM	Total affected/examined	0/10	0/10	0/10	0/10
ILUEM	Total affected/examined	0/10	0/10	0/10	0/10
PEYER'S PATCHES	Total affected/examined	0/10	0/10	0/10	0/10
CECUM	Total affected/examined	0/10	0/10	0/10	0/10
COLON	Total affected/examined	0/10	0/10	0/10	0/10
RECTUM	Total affected/examined	0/10	0/10	0/10	0/10
LIVER	Total affected/examined	0/10	0/10	0/10	0/10
PANCREAS	Total affected/examined	0/10	0/10	0/10	0/10
KIDNEYS	Total affected/examined	0/10	0/10	0/10	0/10
URETURS	Total affected/examined	0/10	0/10	0/10	0/10
URINARY BLADDER	Total affected/examined	0/10	0/10	0/10	0/10
OVARIES	Total affected/examined	0/10	0/10	0/10	0/10
OVIDUCTS	Total affected/examined	0/10	0/10	0/10	0/10
UTERUS
both sides					80
complete		50		70	71, 73, 74
	Total affected/examined	1/10	0/10	1/10	4/10
CERVIX	Total affected/examined	0/10	0/10	0/10	0/10
VAGINA	Total affected/examined	0/10	0/10	0/10	0/10
PITUTARY GLAND	Total affected/examined	0/10	0/10	0/10	0/10
THYROID GLAND	Total affected/examined	0/10	0/10	0/10	0/10
PARATHYROID GLANDS	Total affected/examined	0/10	0/10	0/10	0/10
ADRENAL GLANDS	Total affected/examined	0/10	0/10	0/10	0/10
SPLEEN	Total affected/examined	0/10	0/10	0/10	0/10
BONE MARROW	Total affected/examined	0/10	0/10	0/10	0/10
THYMUS	Total affected/examined	0/10	0/10	0/10	0/10
AUXILLARY LYMPH NODES	Abnormal color red				77
	Total affected/examined	0/10	0/10	0/10	1/10
MESENT. LYMPH NODES	Total affected/examined	0/10	0/10	0/10	0/10
MANDIB.LYMPH NODES	Abnormal color red				73
	Total affected/examined	0/10	0/10	0/10	1/10
SALIVARY GLANDS	Total affected/examined	0/10	0/10	0/10	0/10
HARDERIAN GLANDS	Abnormal color spotted
	Total affected/examined	0/10	0/10	0/10	0/10
MAMMARY GLANDS	Total affected/examined	0/10	0/10	0/10	0/10
SKIN/SUBCUTIS	Total affected/examined	0/10	0/10	0/10	0/10
SKELETAL MUSCLE	Total affected/examined	0/10	0/10	0/10	0/10
EYES	Total affected/examined	0/10	0/10	0/10	0/10
OPTIC NERVES	Total affected/examined	0/10	0/10	0/10	0/10
FEMUR WITH KNEE JOINT	Total affected/examined	0/10	0/10	0/10	0/10
STERNUM	Total affected/examined	0/10	0/10	0/10	0/10

 

MICROSCOPIC FINDINGS

	C/M	C/F	LD/M	LD/F	MD/M	MD/F	HD/M	HD/F
HEART :	10	10	—	—	—	—	10	10
— Mononuclear Infiltr.:	1	-	—	—	—	—	2	—
— Cartilag.Metaplasia :	2	1	—	—	—	—	—	1
— Degeneration	-	-	—	—	—	—	1	—
TRACHEA :	10	10	—	—	—	—	10	10
— Glandular Ectasia :	3	6	—	—	—	—	5	7
LIVER	10	10	—	—	—	—	10	10
— Necrosis	—	1	—	—	—	—	—	—
— Mixed Cell Infiltr. :	—	1	—	—	—	—	—	—
— Hematopoiesis	9	6	—	—	—	—	7	4
MESENT. LYMPH NODE	10	10	—	—	—	—	10	10
— Lymphoid Depletion	—	—	—	—	—	—	—	1
— Hemorrhage	—	1	—	—	—	—	2	1
— Erythrophagocytosis :	—	1	—	—	—	—	—	—
KIDNEYS :	10	10	—	—	—	—	10	10
— Mineralization :	—	1	—	—	—	—	—	3
— Hyaline Droplets :	7	—	—	—	—	—	5
— Pelvic Dilation :	1	--	—	—	—	—	1	1
— Tubul.Simple Dilat. :	1	1					2
STOMACH	10	10	—	—	—	—	10	10
— Hyperkeratosis		1
LUNG	10	10	—	—	—	—	10	10
— Alveolar Hemorrhage :	1	—	—	—	—	—	—	—
— Alveol.Histiocytosis:	4	1	—	—	—	—	1	1
— Atelectasis :	5	3	—	—	—	—	3	—
— Mixed Cell Infiltr. :	1	—	—	—	—	—	—	—
— Vascular Mineraliz. :	7	3	—	—	—	—	5	3
THYMUS :	10	10	—	—	—	—	10	10
— Lymphoid Depletion :	—	1	—	—	—	—	1	—
— Hemorrhage :	1	1	—	—	—	—	—	2
— Hemosiderin Deposits:	—	—	—	—	—	—	—	1
AXILLARY LYMPH NODES :	10	9	—	—	—	—	10	10
— Lymphoid Depletion :	2	2	—	—	—	—	3	—
								—
ADRENAL CORTICES :	10	10	—	—	—	—	10	10
— Vacuolation :	3	—	—	—	—	—	1	—
								—
SKELETAL MUSCLE :	10	10	—	—	—	—	10	10
— Mononuclear Infiltr.:	2	1	—	—	—	—	2	—
								—
PROSTATE GLAND	10	—	—	—	—	—	10	—
— Cyst	—	—	—	—	—	—	1	—
— Hemorrhage	—	—	—	—	—	—	1	—
								—
VAGINA	—	10	—	—	—	—	10	—
— Cycle:Proestrus	—	1	—	—	—	—	—	—
— Cycle:Estrus	—	5	—	—	—	—	5	—
— Cycle:Diestrus	—	4	—	—	—	—	5	—
								—
HARDERIAN GLANDS	—	—	—	—	—	—	1	—
— Increased Porphyrin :	—	—	—	—	—	—	1	—
— Glandular Ectasia	—	—	—	—	—	—	1	—

 

 

 

 

 

 

 

 

 

 

 

 

Conclusions:

The no observed adverse effect level (NOAEL) of the test item MPAAU in this study is considered to be 1000 mg/kg body weight/day.

Executive summary:

MPAAU was assessed in a 90-Day Repeated Dose Oral Toxicity study in male and female Wistar rats, with dose levels of 100, 300 and 1000 mg/kg body weight/day. No test-item related mortality was observed and no adverse effects of the test item were found for male and female clinical observations, functional observations, body weight development, food consumption, haematology and coagulation, clinical biochemistry and hormone analysis, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treated dose groups.

Hence, the no observed adverse effect level (NOAEL) of the test item MPAAU in this study is considered to be 1000 mg/kg body weight/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

High quality GLP-guideline study.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

Currently, no repeated dose toxicity studies with MPA are available. However, the source substance, MPAAU was assessed in a 90-day as well as a 28-day repeated dose toxicity study. In the 28-day sub-acute study, repeated dosing of MPAAU was examined in Wistar rats. The test substance was administered as a solution in tap water by oral gavage to groups of 5 rats/sex, once daily during 28 consecutive days. The dose levels were 0 (tap water only), 50, 300 or 1000 mg MPAAU/kg body weight/day. The administration of the test substance was well tolerated at all dose levels and did not induce any relevant changes in general condition, mortality, growth, feed intake, neurobehavioural observations, haematology, clinical chemistry, fertility parameters or in macroscopic- and microscopic findings. Because the administration of the test substance at levels up to 1000 mg/kg bw/day did not induce any changes that were considered to be of toxicological significance, the no-observed-adverse-effect level (NOAEL) was placed at 1000 mg MPAAU/kg bw/day. Similarly, MPAAU was also assessed in a 90-Day Repeated Dose Oral Toxicity study in male and female Wistar rats, with dose levels of 100, 300 and 1000 mg/kg bw/day. No test-item related mortality was observed and no adverse effects of the test item were found for male and female clinical observations, functional observations, body weight development, food consumption, haematology and coagulation, clinical biochemistry and hormone analysis, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treated dose groups. Hence, the no observed adverse effect level (NOAEL) of the test item MPAAU in this study is considered to be 1000 mg/kg body weight/day.

Repeated dose toxicity: inhalation

As per the Regulation (EC) No. 1907/2006, testing by the inhalation route is appropriate if: — exposure of humans via inhalation is likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.  Taking the low vapour pressure (0.00026664477 Pa) into account, vaporization and subsequent inhalation exposure is not likely to occur. MPA is highly soluble in water, hence if inhaled may get cleared by mucociliary mechanism of respiratory tract, thereby further reducing the absorption. Further, irritation or corrosion of the upper airways would be the major effect of toxicity upon significant inhalation of hot fumes of the test substance when taking the melting temperature (103 °C) and the corrosive properties of the substance into account. No adverse effects are reported in 90 day oral gavage repeated dose toxicity with the source substance, MPAAU. Therefore testing for repeated dose toxicity via  inhalation route is considered to be not appropriate and safety can be assessed using the extrapolation methods.

Repeated dose toxicity: dermal

As per the Regulation (EC) No. 1907/2006, testing by the dermal route is appropriate if:

- skin contact in production and/or use is likely; and

- the physicochemical properties suggest a significant rate of absorption through the skin; and

- in vitro tests indicate significant dermal absorption.

(1) MPA has been classified as corrosive to skin and eyes, hence appropriate PPEs are to be used as Risk Management Measures. Hence possibility of skin contact is considered to be minimal.

(2) The physicochemical properties (log Kow -1.5559, water solubility >20,000 mg/l) indicate that substance is too hydrophilic to cross the lipid rich environment of the stratum corneum. Hence, dermal uptake for MPA will be low.

(3) Dermal penetration studies with a substance containing about 50 % of MPA demonstrated no significant uptake through the skin.

Hence taking into account all the above information, testing for repeated dose toxicity via dermal route is considered to be not appropriate.

Justification for classification or non-classification

Based on the above stated assessment of the specific target organ toxicity potential after repeated oral exposure of MPA, this substance does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) or according to CLP (Regulation (EC) No. 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.