N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2012- April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

See details below

Principles of method if other than guideline:

Deviation: The decrease of temperature (to 18.6 ºC) in SPF animal room was observed during study. The limit prescribed in OECD Test Guideline No. 422 for temperature in animal room is 19 – 25ºC. The deviations from the limits were short-term and did not influence the wellness of animals and the study results.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine

EC Number:

240-004-1

EC Name:

N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine

Cas Number:

15875-13-5

Molecular formula:

C18H42N6

IUPAC Name:

(3-{3,5-bis[3-(dimethylamino)propyl]-1,3,5-triazinan-1-yl}propyl)dimethylamine

Test material form:

liquid: viscous

Details on test material:

N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine (CAS No. 15875-13-5)
Purity: 98.0% (w/w)
Appearance: Colorless to pale yellow liquid
Batch No.: 1166046
Stability/Expiration:06/2014

Specific details on test material used for the study:

CAS No.:15875-13-5
CAS name: N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine
Purity: 98.0% (w/w)
Impurities: Water (CAS No. 7732-18-5) 0.14 % (w/w)
Appearance: Colorless to pale yellow liquid
Batch No.: 1166046
Stability/Expiration: 06/2014

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal housing
All the study proceeded in SPF (Specified Pathogen Free) animal house of CETA in SPF conditions according to internal SOP No. 46
Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood.

Housing conditions
Animals were housed in controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 22+3°C, a relative humidity of 30-70% and 12-hour light/12-hour dark cycle (according to internal SOP No. 46).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The application form was prepared by mixing with aqua pro injectione. Two concentrations of application form were prepared (50 mg/10 mL and 1000 mg/10 mL).
The test substance was administered to the stomach by gavage as the solution in aqua pro injectione. Oral way of administration was chosen according to the guideline and it was approved by sponsor. The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The vehicle control group was administered by aqua pro injectione in the same volume. The application form (test substance solution in aqua pro injectione) was prepared daily just before administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity of the substance were determined by evaluation of absorbance measured at maximum of the test substance absorbance spectra by spectrophotometric method.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for
41-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 5 days of lactation.

Frequency of treatment:

The animals were treated 7 days per week at the same time (8.00 – 10.00 am).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle Controls

Doses / concentrations 2

Dose / conc.:

80 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

240 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

720 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Satellite Group - Vehicle Controls

Doses / concentrations 6

Dose / conc.:

720 mg/kg bw/day (nominal)

Remarks:

Satellite Group - Treated

No. of animals per sex per dose:

Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females.

Control animals:

yes, concurrent no treatment

Details on study design:

Preparation of Experimental Animals
During the acclimatisation period the health condition of all animals was controlled daily. Then the animals were randomly divided into the control and test groups and they were marked individually.
Mating Procedure
Animals were mated from the 15th day of study. Mating schedule 1 : 1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Health condition control: daily - during the acclimatization and the experimental part
Body weight: Males - weekly,
Females - weekly in premating and mating period, during pregnancy: 0., 7th, 14th, 20th day, during lactation: 0. or 1st, 3rd and 4th day; pups (litters) – 0. or 1st, 3rd and 4th day;
Satellite males and females - weekly
Food consumption:males - weekly (except the mating period), Females - weekly during premating period during pregnancy and lactation – on the same days as body weight. Satellite males and females – weekly
Water consumption:Satellite males and females – twice a week
Clinical observations: Males and females - daily during the administration period, pups - as soon as possible after delivery and then daily
Mortality control: daily
Detailed clinical observation: before the first application and then weekly (except the mating period)
Functional observation: at the end of administration/observation period
Laboratory examinations:
- vaginal smears: daily in mating period
- urinalysis: last day of administration/observation period – only males
- haematology: at the end of administration/observation period
- biochemistry:at the end of administration/observation period

Sacrifice and pathology:

Males and nonpregnant females – at the end of administration period
Parental females and pups - on the 4th day of lactation
Satellite males and females - at the end of observation period
- weight of organs: during necropsy
- sperm observation: all males after necropsy
- histopathological examination: after necropsy

Statistics:

The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis (the raw data were used for statistical analysis). This statistical analysis was used for the results of body weight, results of haematology, blood biochemistry, urinalysis, biometry of organs and selected reproduction parameters. Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In control males and treated males of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period.
Only sporadic changes of health condition (diarrhoea or dyspnoea or salivation) were observed in treated males at the dose level 80 and 240 mg/kg/day in application period. In males at the dose level 720 mg/kg/day the following changes were observed: salivation and/or chemical odour around animals and/or dyspnoea from the 1st week of study to the end of application period. Difficult application of the test substance was observed at the dose level 720 mg/kg/day from the 3rd week of application to the end of application period.

In control females and treated females of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period.
Only sporadic changes of health condition (dyspnoea or salivation) were observed in treated females at the dose level 80 and 240 mg/kg/day in application period. In females at the dose level 720 mg/kg/day the following changes were observed: salivation and/or chemical odour around animals and/or dyspnoea from the 1st week of study to the end of application period. Difficult application of the test substance was recorded at the dose level 720 mg/kg/day from the 3rd week of application to the end of application period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (No. 163) at the dose level 720 mg/kg/day died on the 20th day of study (pregnancy period).
There were no other unscheduled deaths during the study in all animals.

Two pups at the dose level 80 mg/kg/day died during lactation period. Three stillborn pups at the control group and two stillborn pups at the dose level 720 mg/kg/day were found. No significant differences in development of treated and control pups were observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

For males, decreased body weight was recorded at the dose level 720 mg/kg/day during the whole study and at the dose level 80 mg/kg/day it was slightly decreased. The body weight at the dose level 240 mg/kg/day and control animals was similar during the whole application period. The statistical analysis was performed for body weight. Statistically significant differences were not found.
For Females, oncreased body weight was recorded at the dose levels 240 and 720 mg/kg/day during the whole study (including the week before application). A statistical analysis was performed for body weight. The statistically significant differences were not found.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: The mean food consumption was slightly decreased at the dose level 720 mg/kg/day during the whole study and at the dose level 80 mg/kg/day in the 5th week of application period.
Females: During pre-mating period the food consumption was similar in treated and control females.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

An imbalance of mean food consumption of treated females was recorded in the 20th day of pregnancy period and in the 4th day of lactation period.
Decreased mean food conversion of treated males was recorded during the whole study (except the 2nd week of application period at the dose level 240 mg/kg/day, when it was increased).
Dis-balance of mean food conversion of treated groups was recorded in the pre-mating period. During pregnancy period the mean food conversion was similar in treated and control females. During lactation period the mean food conversion at the dose level 720 mg/kg/day was decreased.

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

delayed changes of haemocoagulation parameters in males and red blood component and haemocoagulation parameters in females

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males: Significantly decreased values of glucose, potassium ions and activity of ALT were recorded at the dose level 720 mg/kg/day. Decreased activity of ALT was also recorded at the dose level 240 mg/kg/day but without statistical significance. Dose-dependent decreased value of urea was recorded in all treated groups (without statistical significance). Insignificantly increased value of bilirubin total was recorded in all treated groups. Value of creatinine at the dose level 720 and 240 mg/kg/day was decreased and on the contrary at the dose level 80 mg/kg/day it was increased. Decreased values of protein total, albumin and activity of AST were detected at the dose level 720 and 240 mg/kg/day without statistical significance (the value of albumin was under historical control limit). Activity of ALP was insignificantly decreased at the dose level 720 mg/kg/day. All other measured parameters were similar to the control group.

Females:
Increased value of albumin was recorded in all treated groups (at the dose level 80 mg/kg/day with statistical significance). Value of protein total was increased at the dose level 80 and 240 mg/kg/day without statistical significance. Activity of ALP at the dose level 80 mg/kg/day was decreased and on the contrary at the dose level 240 mg/kg/day it was increased. Insignificantly decreased value of creatinine at the dose level 240 and 720 mg/kg/day was recorded. Decreased value of glucose was detected at the dose level 240 mg/kg/day. At the dose level 720 mg/kg/day value of urea and activity of AST was insignificantly increased. All other measured parameters were similar to the control group.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males: The statistical analysis was performed for urine volume and pH of urine. Dose-dependent decreased urine volume was recorded in all treated groups with statistical significance. The pH of urine was statistically significantly increased at the dose level 240 and 720 mg/kg/day. The number of males with higher value of specific weight of urine was recorded at the dose level 240 and 720 mg/kg/day. Change of colour of urine (from light yellow to dark yellow) was recorded in five males at the dose level 720 mg/kg/day. At the dose level 720 mg/kg/day presence of protein and leucocytes in all six males and presence of urobilinogen and ketones in two males were recorded. At the dose level 240 mg/kg/day presence of protein in two males, presence of blood in one male and presence of leucocytes in three males were recorded.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The activity (poise, gait, reaction to handling) of all males and females of all treated groups was similar during the study and not different from the activity of males of the control group.
No significant changes were found at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane and secretion. Slight dyspnoea was observed during the study in males at the dose level 720 mg/kg/day.

Reactions to contact, to noise, to pain and pupillary reflex of treated males and satellite treated group were the same as in the control group.
Results of upstanding showed slight decrease in males at the dose level 720 mg/kg/day and in satellite treated males. Results of emiction and defecation in treated males were not the same as in control males but the variation was within the range of the physiological reaction of animals. The values of grip strength of pectoral legs and pelvic legs in males did not show any difference between control and the treated dose levels.

Results of upstanding showed slight decrease in females at the dose level 720 mg/kg/day. In one female at the dose lvel 720 mg/kg/day decreased response to stimuli and gibbous posture was recorded. Results of emiction and defecation in treated females were not the same as in control females but the variation was within the range of the physiological reaction of animals. The values of grip strength of pectoral legs and pelvic legs in females did not show any difference between control and the treated dose levels.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

For males and Females, statistically significant differences in absolute organ weight were not found.

In male animals, iIncreased relative weight of pituitary gland was recorded at the dose level 720 mg/kg/day with statistical significance. Increased relative weight of adrenal glands, epididymis and brain were recorded at the dose level 720 mg/kg/day without statistical significance. At the dose level 240 mg/kg/day relative weight of adrenal glands and testes were decreased without statistical significance and relative weight of thymus was increased. Decreased relative weight of testes was also recorded at the dose level 80 and 240 mg/kg/day.

In female animals, increased relative weight of liver was recorded at the dose level 720 mg/kg/day. At all treated groups relative weight of uterus was decreased and relative weight of pituitary gland was increased. Relative weight of kidneys was increased at the dose level 240 and 720 mg/kg/day. Decreased relative weight of spleen was recorded at the dose level 80 and 240 mg/kg/day and on the contrary at the dose level 720 mg/kg/day it was increased. Increased relative weight of ovaries was detected at the dose level 80 and 240 mg/kg/day. Relative weight of thymus was decreased at the dose level 80 and 720 mg/kg/day. At the dose level 720 mg/kg/day relative weight of adrenal glands, brain and heart were increased.

Relative weight of other organs was well-balanced with the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In 6-5-5-0 males no macroscopic changes were observed. In forestomach focal erosion in 0-0-0-5 males was found out. Other changes were observed only sporadically.

In 6-5-3-2 females no macroscopic changes were observed. In stomach whitish coating on the mucous membrane in 0-0-3-2 females and brown focus in 0-0-0-3 females was found. Focal erosion in forestomach of 0-0-0-2 females was observed.
Other changes were observed only sporadically.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In forestomach focal inflammation in 0-0-0-5 males, focal oedema in 0-0-0-5 males and focal erosion in 0-0-0-3 males were detected. In the prostate gland, the following microscopical changes were found: focal mononuclear infiltration of interstitium in 2-2-0-2 males and focal oedema of interstitium in 1-2-0-1 males. In epididymis focal mononuclear infiltration of interstitium in 1-1-1-1 males and germ cells in lumen of tubules in 0-1-1-0 males were found out. Cysts in pituitary gland in 1-1-1-1 males were detected.

In forestomach focal oedema and subchronic inflammation in 0-0-0-2 females was detected. In reproductive organs the changes related to previous pregnancy were found. In the uterus, the following microscopical changes were found: accumulation of lipophages and siderophages in mesometrium in 5-5-6-6 females, siderophages in mucosa in 0-1-0-2 females and organizing hematoma in 1-2-1-1 females. Lobular hyperplasia in mammary gland in 6-6-5-6 females was recorded.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: histopathological examination results

Target system / organ toxicity

Critical effects observed:

not specified

System:

gastrointestinal tract

Any other information on results incl. tables

Tables for Repeated Dose Toxicity Part of Study

Body weight - groupmean ±standard deviation (g)
MALES
Group code    (rat No.)	0 (rat No.:1 - 6)	80 (rat No.:21 - 26)	240 (rat No.:41 - 46)	720 (rat No.:61 - 66)
Before application	288.03 ± 11.88	288.03 ± 10.98	311.08 ±18.01	291.65 ± 20.14
1st week	328.47 ± 17.62	314.13 ± 12.56	338.10 ± 19.94	310.25 ± 22.05
2nd week	351.40 ± 20.60	334.27 ± 13.03	360.10 ± 21.25	324.25 ± 20.83
3rd week	370.73 ± 25.05	354.50 ± 17.51	373.97 ± 22.91	338.73 ± 17.60
4th week	386.87 ± 25.29	371.78 ± 21.50	393.08 ± 24.41	354.85 ± 20.11
5th week	405.38 ± 26.27	387.25 ± 19.46	407.32 ± 27.25	369.07 ± 20.63
6th week	415.85 ± 28.01	396.45 ± 22.04	415.40 ± 29.77	372.48 ± 19.67

Body weight - groupmean ±standard deviation (g)
FEMALES
Application period		Group code                                                (rat No.)
		0 (rat No. 103, 104, 106, 109, 111, 112)	80 (rat No. 121, 123, 124, 125, 128, 131)	240 (rat No. 144, 145, 148, 149, 150, 152)	720 (rat No. 161, 162, 164, 166, 169, 172)
Befor mating	Before application	193.02 ± 10.17	194.90 ± 12.72	208.75 ± 14.08	208.92 ± 11.38
	1^stweek	199.87 ± 10.13	204.30 ± 16.08	218.57 ± 19.07	212.42 ± 10.99
	2^ndweek	209.18 ± 14.83	211.62 ± 19.58	227.77 ± 17.17	224.93 ± 12.83
		Mating period
Day of pregnancy	0	215.58 ± 13.72	218.10 ± 20.64	234.83 ± 17.42	232.07 ± 14.17
	7	235.28 ± 13.07	237.40 ± 20.93	258.07 ± 16.24	254.92 ± 15.64
	14	262.03 ± 13.63	264.52 ± 28.01	290.00 ± 17.76	282.57 ± 16.08
	20	328.17 ± 21.34	321.48 ± 48.48	352.50 ± 19.33	344.97 ± 26.73
Day of lactation	0/1	255.88 ± 15.39	253.68 ± 24.29	273.37 ± 16.17	268.33 ± 21.62
	3	268.50 ± 17.18	266.22 ± 26.60	291.35 ± 18.24	275.03 ± 19.93
	4	250.22 ± 15.63	245.70 ± 22.99	264.20 ± 18.79	252.58 ± 23.33

Body weight increment – groupmean(grams/animal/day)
MALES					FEMALES
Group code	0	80	240	720	Group code	0	80	240	720
1st week	5.78	3.73	3.86	2.66	1st week	0.98	1.34	1.40	0.50
2nd week	3.28	2.88	3.14	2.00	2nd week	1.33	1.05	1.31	1.79
3rd week	2.76	2.89	1.98	2.07	3rd week	-	-	-	-
4th week	2.31	2.47	2.73	2.30	4th week	-	-	-	-
5th week	2.64	2.21	2.03	2.03	5th week	-	-	-	-
6th week	1.75	1.53	1.35	0.57	6th week	-	-	-	-
Group code	0 S			720 S	Group code	0 S			720 S
1st week	4.15	-	-	2.50	1st week	1.47	-	-	0.99
2nd week	2.45	-	-	2.01	2nd week	0.54	-	-	1.11
3rd week	2.49	-	-	1.92	3rd week	1.11	-	-	1.45
4th week	2.70	-	-	2.07	4th week	0.91	-	-	0.58
5th week	2.56	-	-	2.13	5th week	1.01	-	-	0.73
6th week	1.24	-	-	0.67	6th week	-0.24	-	-	0.47
7th week	2.41	-	-	1.90	7th week	1.57	-	-	0.66
8th week	1.70	-	-	2.22	8th week	0.82	-	-	0.68

Note:for calculation the following femaleswereused:control group – No. 103,104,106,109,111,112

                                                                                        80 mg/kg/day – No. 121,123,124,125,128,131

                                                                                       240 mg/kg/day – No. 144,145,148,149,150,152

                                                                                       720 mg/kg/day – No. 161,162,164,166,169,172

 

Food consumption – group mean (grams/animal/day)
MALES
Group code (rat No.)	0 (rat No.1-6)	80 (rat No.21-26)	240 (rat No.41-46)	720 (rat No.61-66)
1st week	24.66	23.04	24.34	21.55
2nd week	23.85	22.25	23.08	21.85
3rd week	Mating period
4th week
5th week	24.24	22.85	23.42	22.75
6th week	22.61	22.63	23.36	21.78
Group code	0 S			720 S
1st week	25.72	-	-	22.88
2nd week	24.00	-	-	22.60
3rd week	25.35	-	-	21.97
4th week	22.86	-	-	22.74
5th week	23.28	-	-	22.74
6th week	23.80	-	-	21.85
7th week	23.40	-	-	23.57
8th week	23.17	-	-	22.29

Food consumption – group mean (grams/animal/day)
FEMALES
Group code (rat No.)	0 (rat No.101-112)	80 (rat No.121-132)	240 (rat No.141-152)	720 (rat No.161-172)
1st week	16.75	16.34	16.70	16.09
2nd week	15.66	15.35	16.69	16.70
3rd – 4th week	Mating period
PREGNANCY AND LACTATION PERIOD
Group code (rat No.)	0 (rat No.103,104, 106,109,111,112)	80 (rat No.121,123, 124,125,128,131)	240 (rat No.144,145, 148,149,150,152)	720 (rat No.161,162, 164,166,169,172)
7^thday	16.77	15.91	17.91	19.35
14^thday	19.77	20.43	21.90	23.80
20^thday	26.04	22.12	24.99	22.96
3^rdday of lactation	27.75	28.03	42.64	30.08
Group code	0 S			720 S
1st week	17.23	-	-	16.36
2nd week	17.26	-	-	16.25
3rd week	17.71	-	-	16.65
4th week	16.88	-	-	16.30
5th week	17.27	-	-	15.84
6th week	16.82	-	-	15.75
7th week	17.20	-	-	16.38
8th week	17.58	-	-	15.72

 

 

Detailed clinical observation – summary table
MALES
Parameters	0	80	240	720	0S	720S
Skin	1	1	1	1	1	1
Hair (piloerection)	1	1	1	1	1	1
Clonic movements	1	1	1	1	1	1
Eyes (pupil)	1	1	1	1	1	1
Lacrimation	1	1	1	1	1	1
Mucous membranes (visible)	1	1	1	1	1	1
Secretion	1	1	1	1	1	1
Excretion	1	1	1	1	1	1
Respiration	1	1	1	4	1	4
Poise	1	1	1	1	1	1
Gait	1	1	1	1	1	1
Reaction to handling	1	1	1	1	1	1
Clonic-tonic spasm	1	1	1	1	1	1
Stereotypies	0	0	0	0	0	0
Vocalisation	0	0	0	0	0	0
Bizarre behaviour	0	0	0	0	0	0
Other findings	0	0	0	0	0	0

Note:

0–without reaction, 1 – natural reaction, 2 – mild reaction, 3 – increased reaction, 4 – dyspnoea

Detailed clinical observation – summary table
FEMALES
Parameters	0	80	240	720	0S	720S
Skin	1	1	1	1	1	1
Hair (piloerection)	1	1	1	1	1	1
Clonic movements	1	1	1	1	1	1
Eyes (pupil)	1	1	1	1	1	1
Lacrimation	1	1	1	1	1	1
Mucous membrane (visible)	1	1	1	1	1	1
Secretion	1	1	1	1	1	1
Excretion	1	1	1	1	1	1
Respiration	1	1	1	1	1	1
Poise	1	1	1	1	1	1
Gait	1	1	1	1	1	1
Reaction to handling	1	1	1	1	1	1
Clonic-tonic spasm	1	1	1	1	1	1
Stereotypies	0	0	0	0	0	0
Vocalisation	0	0	0	0	0	0
Bizarre behaviour	0	0	0	0	0	0
Other findings	0	0	0	0	0	0

Note:

0–without reaction, 1 – natural reaction, 2 – mild reaction, 3 – increased reaction, 4 – dyspnoea

 

Haematological examination
MALES
Parameter	Units	Group code (rat No.)						Values
		0 (rat No. 1-6)	80 (rat No. 21-26)	240 (rat No. 41-46)	720 (rat No. 61-66)	0 S	720 S
RBC (total erythrocyte count)	10^6/mL	8.44 8.21 ±0.59	7.96 8.03 ±0.63	7.63 7.57 ±0.44	7.52 7.62 ±0.65	7.87 7.86 ±0.46	7.99 8.18 ±0.45	Median Mean +SD
MCV(Mean Corpuscular Volume)	Fl	50.30 50.22 ±1.45	50.40 50.58 ±0.92	51.10 51.70 ±1.75	52.20 51.57 ±1.85	50.35 50.05 ±1.18	50.00 49.33 ±1.68	Median Mean +SD
Haematocrit	%	41.65 41.25 ±3.29	40.45 40.60 ±2.92	40.40 39.13 ±2.54	39.70 39.22 ±2.50	39.55 39.33 ±2.81	40.40 40.28 ±1.02	Median Mean +SD
Haemoglobin	g/100 mL	14.35 14.17 ±0.99	13.45 13.53 ±1.04	13.25 12.97 ±0.85	13.25 13.10 ±0.64	13.30 13.17 ±0.91	13.25 13.22 ±0.32	Median Mean +SD
WBC(total leucocyte count)	10^3/mL	9.30 9.42 ±2.00	7.90 8.15 ±0.84	8.85 9.10 ±1.31	9.65 10.57 ±2.41	8.45 8.83 ±1.94	9.40 9.58 ±2.18	Median Mean +SD
Platelet count	10^3/mL	849.0 842.3 ±98.03	803.0 804.7 ±103.38	764.5 816.5 ±124.75	802.5 812.5 ±85.79	509.50 524.00 ±95.12	805.00 792.00 ±70.37	Median Mean +SD
APTT(Activated Partial Tromboplastin Time)	s	30.55 30.00 ±1.97	25.30 30.17 ±13.33	28.65 29.90 ±12.71	36.30 30.87 ±12.03	56.00 58.82 ±12.39	38.75 37.58 ±15.24	Median Mean +SD
Protrombin Time	s	23.90 23.82 ±2.13	24.15 23.78 ±2.54	23.35 23.12 ±1.47	23.25 21.30 ±4.24	24.55 24.33 ±2.17	24.85 24.72 ±1.96	Median Mean +SD
Granulocytes	%	7.70 8.22 ±3.07	6.60 7.80 ±2.79	6.60 7.25 ±2.53	8.45 8.00 ±2.30	5.25 7.02 ±3.41	5.95 9.37 ±9.70	Median Mean +SD
Lymphocytes	%	87.95 87.08 ±4.01	86.75 84.85 ±6.29	88.65 87.67 ±3.60	87.65 86.87 ±2.03	87.00 85.95 ±4.14	89.85 84.65 ±11.52	Median Mean +SD
Monocytes	%	4.35 4.70 ±0.97	5.85 7.25 ±3.73	4.70 5.08 ±1.48	4.65 5.13 ±1.86	7.05 7.03 ±1.96	5.25 5.98 ±3.44	Median Mean +SD

Note:grey field= values statistically significant on probability level 0.05 (ANOVA test)

All measured parameters were within historical control of the laboratory

 

Haematological examination
FEMALES
Parameters	Units	Group code (rat No.)						Values
		0 (rat No.103,104, 106,109, 111,112)	80 (rat No.121,123, 124,125, 128,131)	240 (rat No.144,145, 148,149, 150,152)	720 (rat No.161,162, 164,166, 169,172)	0 S	720 S
RBC (total erythrocyte count)	10^6/mL	6.46 6.14 ±1.03	6.35 6.53 ±0.54	6.34 6.32 ±0.52	6.08 6.05 ±0.36	7.17 7.13 ±0.16	7.72 7.64 ±0.19	Median Mean +SD
MCV(Mean Corpuscular Volume)	Fl	56.15 57.97 ±5.54	54.80 55.08 ±1.08	55.50 55.00 ±1.90	55.30 55.72 ±3.81	50.85 51.22 ±1.40	52.25 52.32 ±0.97	Median Mean +SD
Haematocrit	%	36.60 35.12 ±3.70	35.45 35.93 ±2.52	35.15 34.70 ±2.49	33.60 33.60 ±1.63	36.50 36.50 ±0.96	39.95 39.98 ±0.68	Median Mean +SD
Haemoglobin	g/100 mL	12.00 11.55 ±1.24	12.10 12.23 ±0.89	11.70 11.62 ±0.87	11.30 11.12 ±0.53	12.35 12.37 ±0.35	13.80 13.63 ±0.39	Median Mean +SD
WBC(total leucocyte count)	10^3/mL	6.95 7.30 ±2.22	5.40 5.48 ±0.50	4.85 5.02 ±0.52	6.15 7.28 ±2.45	6.85 7.02 ±0.79	5.80 5.98 ±1.04	Median Mean +SD
Platelet count	10^3/mL	999.5 1038.3 ±138.89	1095.0 1076.8 ±113.84	1030.5 1001.7 ±136.48	942.5 952.7 ±111.81	535. 0 553.3 ±63.89	785.0 810.2 ±51.16	Median Mean +SD
APTT(Activated Partial Tromboplastin Time)	s	16.20 22.27 ±12.49	15.50 16.05 ±2.14	13.70 13.80 ±1.46	20.45 19.87 ±5.70	40.30 47.45 ±21.16	14.75 21.87 ±15.94	Median Mean +SD
Protrombin Time	s	24.80 24.93 ±2.16	25.05 24.97 ±1.36	21.40 21.38 ±1.20	22.40 22.68 ±1.37	24.45 24.95 ±2.09	24.30 24.68 ±1.62	Median Mean +SD
Granulocytes	%	11.00 10.47 ±3.96	14.60 13.75 ±4.72	11.65 11.33 ±2.49	10.90 11.70 ±4.56	4.10 5.37 ±3.87	3.70 3.63 ±0.54	Median Mean +SD
Lymphocytes	%	82.25 82.08 ±6.98	80.10 80.03 ±5.69	81.40 82.08 ±2.98	79.10 80.15 ±4.89	91.20 89.78 ±3.78	91.50 91.33 ±2.02	Median Mean +SD
Monocytes	%	6.30 7.45 ±4.01	6.05 6.22 ±1.94	6.60 6.58 ±0.77	7.55 8.15 ±4.20	4.85 4.85 ±0.47	4.35 5.03 ±1.87	Median Mean +SD

Note:grey field= values statistically significant on probability level 0.05 (ANOVA test)

        All measured parameters were within historical control of our laboratory.

 

 

 

Biochemical examination
MALES
Parameter	Units	Group code (rat No.)						Values
		0 (rat No. 1-6)	80 (rat No. 21-26)	240 (rat No. 41-46)	720 (rat No. 61-66)	0 S	720 S
Glucose	mmol/L	6.85 6.70 ±0.82	6.20 6.22 ±0.88	6.50 6.60 ±0.28	4.55 4.62 ±0.72	6.55 6.58 ±0.40	5.95 5.80 ±0.80	Median Mean +SD
◙Cholesterol	mmol/L	1.29 - -	1.29 - -	1.29 - -	1.29 - -	1.29 - -	1.29 - -	Median Mean +SD
Urea	mmol/L	6.20 6.60 ±1.35	5.65 5.95 ±0.99	5.10 5.30 ±0.82	4.75 4.98 ±0.72	5.90 5.82 ±0.59	5.05 5.03 ±0.78	Median Mean +SD
Bilirubin total	mmol/L	5.00 5.00 ±0.63	6.00 - -	5.50 - -	7.00 6.80 ±0.41	6.50 6.80 ±1.47	6.00 5.80 ±1.33	Median Mean +SD
AST	mkat/L	1.89 1.91 ±0.18	1.78 1.74 ±0.13	1.55 1.60 ±0.48	1.35 1.45 ±0.32	1.74 1.68 ±0.22	1.67 1.64 ±0.26	Median Mean +SD
∆ALT	mkat/L	0.37 0.36 ±0.08	0.32 0.32 ±0.06	0.19 - -	0.24 - -	0.17 - -	0.20 - -	Median Mean +SD
ALP	mkat/L	4.01 4.24 ±0.69	3.16 3.41 ±0.97	4.12 3.90 ±0.76	2.99 3.27 ±0.94	3.54 3.74 ±1.02	2.86 3.05 ±1.04	Median Mean +SD
Calcium	mmol/L	2.97 2.96 ±0.04	2.95 2.95 ±0.06	2.99 2.98 ±0.05	3.07 3.00 ±0.15	2.95 2.97 ±0.09	3.00 3.03 ±0.11	Median Mean +SD
Phosphorus	mmol/L	1.81 1.80 ±0.07	1.73 1.77 ±0.19	1.90 1.88 ±0.09	1.98 1.93 ±0.22	1.82 1.80 ±0.09	2.00 1.98 ±0.12	Median Mean +SD
Protein total	g/L	70.5 69.8 ±3.49	67.0 68.0 ±3.29	66.5 67.8 ±5.98	63.0 63.0 ±3.10	67.0 67.5 ±4.18	66.0 67.5 ±6.06	Median Mean +SD
Albumin	g/L	39.5 39.0 ±2.19	37.5 37.5 ±1.64	37.0* 37.2 ±1.83	37.0* 36.3 ±1.51	39.5 39.2 ±1.94	38.5 38.7 ±1.86	Median Mean +SD
Creatinine	mmol/L	56.0 57.0 ±7.77	59.0 60.5 ±5.65	53.0 51.8 ±3.87	54.5 55.7 ±4.68	56.0 55.8 ±8.21	57.5 56.5 ±6.28	Median Mean +SD
Sodium	mmol/L	138.5 139.0 ±1.26	140.0 140.3 ±2.25	139.5 138.7 ±2.88	139.5 139.0 ±1.55	140.5 140.7 ±1.75	139.0 138.8 ±1.33	Median Mean +SD
Potassium	mmol/L	4.85 4.78 ±0.15	4.80 4.80 ±0.36	4.80 4.85 ±0.24	4.35 4.22 ±0.38	4.70 4.63 ±0.25	4.45 4.43 ±0.26	Median Mean +SD
Chloride	mmol/L	104.5 103.8 ±1.47	104.5 104.2 ±1.17	103.0 103.2 ±1.33	103.5 103.7 ±1.37	102.0 102.5 ±1.76	102.5 102.8 ±0.98	Median Mean +SD

◙- values of cholesterol in 6 -6 -6 -5 animalsand in 6 -6 satellite animalswere under 1.29mmol/L

 (outside the measure range of analyser)

- values of bilirubin in 0-1-1-0 animalswere under 3mmol/L (outside the measure range of analyser)

_∆- values of ALT in 0-0-2-2 animalsand in 2-1 satellite animalswere under 0.17mmol/L  

 (outside the measure range of analyser)

* the value under historical control

Note: grey field= values statistically significant on probability level 0.05 (ANOVA test)

 

Biochemical examination
FEMALES
Parameter	Units	Group code (rat No.)						Values
		0 (rat No.103,104, 106,109, 111,112)	80 (rat No.121,123, 124,125, 128,131)	240 (rat No.144,145, 148,149, 150,152)	720 (rat No.161,162, 164,166, 169,172)	0 S	720 S
Glucose	mmol/L	5.50 5.73 ±0.80	5.15 5.22 ±0.77	4.65 4.43 ±0.95	5.25 5.47 ±0.56	5.15 5.25 ±0.49	4.65 4.78 ±0.29	Median Mean +SD
◙Cholesterol	mmol/L	1.29 - -	1.29 - -	1.29 - -	1.29 - -	1.29 - -	1.29 - -	Median Mean +SD
Urea	mmol/L	9.20 9.98 ±2.12	10.50 10.17 ±1.39	10.95 11.27 ±1.73	11.15 11.23 ±2.17	7.75 7.33 ±0.85	6.70 6.70 ±0.30	Median Mean +SD
Bilirubin total	mmol/L	3.5 - -	3.5 - -	4.0 - -	4.0 4.5 ±0.84	7.0 6.8 ±0.41	7.0 6.8 ±0.98	Median Mean +SD
AST	mkat/L	1.30 1.25 ±0.25	1.28 1.38 ±0.27	1.49 1.47 ±0.13	1.59 1.65 ±0.29	1.54 1.56 ±0.16	1.17 1.20 ±0.21	Median Mean +SD
∆ALT	mkat/L	0.59 0.56 ±0.21	0.41 - -	0.61 0.64 ±0.20	0.59 0.67 ±0.30	0.23 - -	0.18 - -	Median Mean +SD
ALP	mkat/L	3.05 4.06 ±2.01	1.93 2.39 ±1.36	4.06 4.25 ±1.23	2.54 3.54 ±2.46	2.07 2.27 ±0.86	1.78 1.89 ±0.58	Median Mean +SD
Calcium	mmol/L	2.91 2.87 ±0.11	2.90 2.91 ±0.11	2.88 2.87 ±0.05	2.91 2.90 ±0.14	2.92 2.92 ±0.07	3.05 3.04 ±0.02	Median Mean +SD
Phosphorus	mmol/L	1.51 1.47 ±0.12	1.64 1.65 ±0.25	1.55 1.68 ±0.34	1.75 1.78 ±0.10	1.43 1.41 ±0.13	1.52 1.49 ±0.11	Median Mean +SD
Protein total	g/L	56.5 57.3 ±3.08	60.0 60.0 ±2.83	61.0 61.3 ±2.07	58.5 57.8 ±2.79	57.0 57.0 ±1.41	60.0 60.7 ±2.58	Median Mean +SD
Albumin	g/L	35.0 35.2 ±1.60	40.0 39.3 ±1.51	38.5 38.2 ±1.47	38.5 38.2 ±2.48	40.0 41.3 ±2.16	45.0** 45.0 ±0.89	Median Mean +SD
Creatinine	mmol/L	70.5 68.0 ±7.92	67.0 61.8 ±12.58	56.0 57.7 ±5.32	58.0 63.7 ±16.73	59.5 62.8 ±12.67	59.0 57.7 ±10.95	Median Mean +SD
Sodium	mmol/L	135.0 136.5 ±2.35	139.0 138.5 ±1.38	137.5 137.5 ±3.33	136.0 135.7 ±2.42	141.5 141.2 ±0.98	141.0 140.8 ±0.75	Median Mean +SD
Potassium	mmol/L	4.60 4.48 ±0.37	4.70 4.68 ±0.23	4.45 4.43 ±0.21	4.40 4.38 ±0.29	4.30 4.33 ±0.33	4.15 4.12 ±0.22	Median Mean +SD
Chloride	mmol/L	102.0 101.2 ±2.86	103.5 104.0 ±5.66	101.0 101.3 ±2.42	98.5 100.5 ±4.46	106.0 105.7 ±1.37	104.5 105.3 ±2.50	Median Mean +SD

 

◙- values of cholesterol in 5-6-5-4 animalsand in 5-6 satellite animalswere under 1.29mmol/L

         (outside the measure range of analyser)

- values of bilirubin in 3-3-1-0 animalswere under 3mmol/L (outside the measure range of analyser)

_∆- values of ALT in 0-2-0-0 animalsand in 2-3 satellite animalswere under 0.17mmol/L  

 (outside the measure range of analyser)

** the value above historical control

 

Absolute weight of organs – group mean (g) ± standard deviation
MALES
Organ	Group code (rat No.)
	0 (rat No. 1-6)	80 (rat No. 21-26)	240 (rat No. 41-46)	720 (rat No. 61-66)	0 S	720 S
Liver	10.5562 ± 0.7744	9.8564 ± 0.4675	11.2258 ± 0.8315	9.5842 ± 0.8391	10.8257 ± 0.8869	10.3787 ± 0.8780
Kidneys	2.6955 ± 0.2191	2.4822 ± 0.1493	2.8390 ± 0.2728	2.4200 ± 0.1006	2.6832 ± 0.3159	2.6961 ± 0.2226
Adrenal glands	0.0793 ± 0.0142	0.0783 ± 0.0090	0.0739 ± 0.0026	0.0832 ± 0.0102	0.0695 ± 0.0062	0.0737 ± 0.0110
Testes	3.7811 ± 0.4794	3.3405 ± 0.2025	3.5018 ± 0.6977	3.4016 ± 0.1660	3.7593 ± 0.4723	3.4615 ± 0.2100
Epididymis/Epididymides	0.7354 ± 0.0742	0.6815 ± 0.0192	0.7508 ± 0.1062	0.7074 ± 0.0899	1.5179 ± 0.1273	1.3908 ± 0.1288
Prostate gland	1.1659 ± 0.3180	1.1777 ± 0.2498	1.1545 ± 0.3362	1.0800 ± 0.3797	1.4066 ± 0.3231	1.1707 ± 0.4828
Thymus	0.4685 ± 0.0809	0.4587 ± 0.0612	0.5767 ± 0.0490	0.4880 ± 0.0622	0.4670 ± 0.0624	0.4674 ± 0.0931
Spleen	0.6439 ± 0.0897	0.6044 ± 0.0697	0.7124 ± 0.1429	0.6047 ± 0.0638	0.6242 ± 0.1212	0.6223 ± 0.0682
Brain	2.0968 ± 0.1264	1.9852 ± 0.0581	2.0815 ± 0.1425	1.9704 ± 0.1148	2.0513 ± 0.0859	2.0035 ± 0.0323
Heart	1.1920 ± 0.0938	1.1275 ± 0.1096	1.2294 ± 0.1221	1.0278 ± 0.0466	1.2359 ± 0.1495	1.1621 ± 0.1161
Pituitary gland	0.0113 ± 0.0011	0.0107 ± 0.0014	0.0115 ± 0.0017	0.0124 ± 0.0008	0.0115 ± 0.0013	0.0097 ± 0.0012
Body weight	389.27	372.27	395.45	350.27	395.65	369.57

 Note:grey field= values statistically significant on probability level 0.05 (ANOVA test)

 

Absolute weight of organs – group mean± standard deviation
FEMALES
Organ	Group code (rat No.)
	0 (rat No.103,104, 106,109, 111,112)	80 (rat No.121,123, 124,125, 128,131)	240 (rat No.144,145, 148,149, 150,152)	720 (rat No.161,162, 164,166, 169,172)	0S	720S
Liver	8.9174 ± 0.9628	8.5421 ± 1.8617	9.9625 ± 1.2888	9.8859 ± 1.1394	7.1346 ± 0.7052	6.5246 ± 0.6486
Kidneys	1.7290 ± 0.1734	1.7248 ± 0.3815	1.8933 ± 0.2442	2.0618 ± 0.3800	1.7136 ± 0.1561	1.6210 ± 0.1345
Adrenal glands	0.0964 ± 0.0132	0.0928 ± 0.0177	0.1041 ± 0.0119	0.1097 ± 0.0132	0.0997 ± 0.0206	0.1040 ± 0.0129
Ovaries	0.1158 ± 0.0208	0.1262 ± 0.0199	0.1339 ± 0.0121	0.1135 ± 0.0132	0.1099 ± 0.0213	0.1116 ± 0.0187
Uterus	0.9955 ± 0.1354	0.8377 ± 0.1247	0.9305 ± 0.1818	0.8331 ± 0.1126	1.1091 ± 0.5294	0.8162 ± 0.2047
Thymus	0.3992 ± 0.0617	0.2961 ± 0.0364	0.3800 ± 0.1050	0.3099 ± 0.1173	0.4329 ± 0.0638	0.3492 ± 0.0688
Spleen	0.6399 ± 0.1229	0.5387 ± 0.1091	0.6213 ± 0.1054	0.6860 ± 0.1808	0.4569 ± 0.0511	0.4253 ± 0.0834
Brain	1.8592 ± 0.0824	1.8253 ± 0.0758	1.9323 ± 0.0920	1.8310 ± 0.0649	1.8692 ± 0.0697	1.9199 ± 0.0812
Heart	0.8502 ± 0.0682	0.8112 ± 0.0810	0.8641 ± 0.0694	0.8780 ± 0.0648	0.8462 ± 0.0500	0.8103 ± 0.0948
Pituitary gland	0.0145 ± 0.0024	0.0172 ± 0.0033	0.0168 ± 0.0043	0.0170 ± 0.0019	0.0144 ± 0.0012	0.0148 ± 0.0013
Body weight	250.2	245.7	264.2	235.9	226.4	227.2

Note:grey field= values statistically significant on probability level 0.05 (ANOVA test)

 

Macroscopic findings
Parameter/Organ change	MALES
	0 (rat No. 1-6)	80 (rat No. 21-26)	240 (rat No. 41-46)	720 (rat No. 61-66)	0 S	720 S
Number of examined animals	6	6	6	6	6	6
Number of died animals	0	0	0	0	0	0
Without pathological changes	6	5	5	0	6	1
SPLEEN: pale prominent focus	0	1	0	0	0	0
TESTES: reduced	0	0	1	0	0	0
EPIDIDYMIS: reduced	0	0	1	0	0	0
LIVER: marked structure	0	0	0	1	0	0
STOMACH (margo plicatus): congested mucous membrane	0	0	0	1	0	0
STOMACH: linear focus, area c. 3mm	0	0	0	0	0	1
FORESTOMACH: focal erosion, area c. 3mm	0	0	0	5	0	0
FORESTOMACH: scarred focus, area c. 3mm	0	0	0	0	0	5

 

 

 

Macroscopic findings
Parameter/Organ change	FEMALES
	0 (rat No.103,104, 106,109, 111,112)	80 (rat No.121,123, 124,125, 128,131)	240 (rat No.144,145, 148,149, 150,152)	720 (rat No.161,162, 164,166, 169,172)	0S	720S
Number of examined animals	6	6	6	6	6	6
Number of died animals	0	0	0	0	0	0
Without pathological changes	6	5	3	2	6	3
KIDNEY: enlarged	0	1	0	1	0	0
URETER: dilatation	0	1	0	1	0	0
STOMACH: whitish coating on the mucous membrane	0	0	3	2	0	0
STOMACH : brown focus, area c. 3mm or 4mm	0	0	0	3	0	0
STOMACH: linear focus, area c. 3mm	0	0	0	0	0	2
FORESTOMACH: scarred focus, area c.1mm	0	0	0	0	0	2
FORESTOMACH: focal erosion	0	0	0	2	0	0
LIVER: pale colour	0	0	0	1	0	0
UTERUS: dilatation	0	0	0	0	2	1

 

 

 

Histopathological findings
Parameter/Organ change	MALES
	0 (rat No. 1-6)	80 (rat No. 21-26)	240 (rat No. 41-46)	720 (rat No. 61-66)	0 S	720 S
Number of examined animals	6	6	6	6	6	6
Without pathological changes	0	2	3	0	1	2
PANCREAS: focal vacuolation of acinar cells	1	-	-	1	0	0
PANCRAS: perivascular infiltration	1	-	-	0	0	0
PROSTATE GLAND: focal oedema and/or interstitial infiltration	5	4	0	3	3	1
EPIDIDYMIS: focal mononuclear infiltration of interstitium	1	1	1	1	1	1
EPIDIDYMIS: germ cells in lumen of tubules	0	1	1	0	1	0
TESTES: degeneration of germ epithelium	0	1	1	0	0	0
KIDNEYS: basophile tubules	1	-	-	2	0	1
KIDNEYS: focal glomerulonephrosis	0	-	-	1	0	0
SPLEEN: focal inflammation of spleen capsule (perisplenitis)	0	1	-	0	0	0
PITUITARY GLAND: cysts in adenohypophysis	1	1	1	1	0	0
FORESTOMACH: focal inflammation	0	0	0	5	0	0
FORESTOMACH: focal oedema	0	0	0	5	0	0
FORESTOMACH: focal erosion	0	0	0	3	0	0
FORESTOMACH: focal multiplication of tissue in submucous	0	0	0	0	0	2
RECTUM: focal acute inflammation	0	-	-	1	0	0
HEART: focal necrosis of myocardium	0	-	-	1	0	1
HEART: inflammation of myocardium	0	-	-	1	0	1

Note:Only organs demonstrating treatment-related changes and reproduction organs: stomach, testes, prostate gland, epididymides, seminal vesicle,coagulation glandsand pituitary gland in males and organs with macroscopical changes were examined at the lowest and middle dose level groups.

 

 

 

Histopathological findings
Parameter/Organ change	FEMALES
	0 (rat No.103,104, 106,109, 111,112)	80 (rat No.121,123, 124,125, 128,131)	240 (rat No.144,145, 148,149, 150,152)	720 (rat No.161,162, 164,166, 169,172)	0 S	720 S
Number of examined animals	6	6	6	6	6	6
Without pathological changes	0	0	0	0	1	3
MAMMARY GLAND: lobular hyperplasia	6	6	5	6	0	0
UTERUS: focal accumulation of lipophages and siderophages in mesometrium	5	5	6	6	0	0
UTERUS: siderophages in mucosa	0	1	0	2	0	0
UTERUS:organizing hematoma	1	2	1	1	0	0
UTERUS: hydrometra	0	0	0	0	5	2
VAGINA: squamous cell cyst	0	1	0	0	0	0
LIVER: extramedullary haematopoiesis	2	-	-	1	0	0
SPLEEN: extramedullary haematopoiesis	2	-	-	0	0	0
KIDNEYS: hydronephrosis	0	-	-	1	0	0
FORESTOMACH: focal oedema, subchronic inflammation	0	0	0	2	0	0
URETHER: chronic inflamation	0	-	-	1	0	0
PITUITARY GLAND: cyst	0	0	0	0	0	1

Note:Only organs demonstrating treatment-related changes and reproduction organs: stomach, vagina, ovary, uterus and pituitary gland in females and organs with macroscopical changes were examined at the lowest and middle dose level groups.

Applicant's summary and conclusion

Conclusions:

Based on the results of a Combined Repeat Dose Toxicity Study with Reproductive/Developmental Screening (OECD 422), the NOAEL for repeated dose toxicity was established as 240 mg/kg body weight/day for males and females based on histopathological changes to the forestomach (local irritative effect) at 720 mg/kg bw.
The value of NOEL was established as lower than 80 mg/kg body weight/day. According to Regulation EC No. 1272/2008 and Directive 67/548/EEC, the test substance does not require classification on the basis of the results achieved in this study.

Executive summary:

In a Combined Repeat Dose Toxicity Study with Reproductive/Developmental Screening (OECD 422), three groups of 24 Wistar rats (12 males, 12 Females) were exposed to test substance by oral gavage at dose levels of: 80, 240 and 720 mg/kg b.w.

A vehicle control group was included. Satellite groups of 6 males and 6 females contained one control group (vehicle only) and one treated group (720 mg/kg/day).The dose levels for the study were determined on the basis of results of a dose-range finding study phase.

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 5 days of lactation.Clinical signs, functional observations, body weights, food consumption, reproduction parameters, observations pups, clinical pathology, macroscopy, organ weights, and histopathology were evaluated.

Repeated oral administration of the test substance at the dose level 720 mg/kg/day caused mortality (one female of the highest dose level died during pregnancy period).

Slight changes of body weight and body weight increment in males, clinical status of animals (diarrhoea or dyspnoea), biochemical parameter (significantly increased value of albumin in females), urinalysis in males (significantly decreased urine volume), and microscopical structure of organs (prostate gland in males and uterus and mammary gland in females) were detected at the dose level 80 mg/kg/day. These microscopical changes in reproductive organs did not relate to the test substance treatment.

Slight changes of body weight, body weight increment, food conversion and clinical status (diarrhoea, salivation) of animals were detected at the middle dose level. Haematological parameters (significantly decreased value of protrombin time in females), biochemical parameters (insignificantly decreased albumin in males but under historical control limits) and urinalysis in males (significant decrease of urine volume, significant increase pH of urine, presence of protein and leucocytes) showed treatment-related changes.

Occurrence of microscopical changes of epididymis and testes in males and mammary gland and uterus in females were detected at the dose level 240 mg/kg/day, however these changes were consodered not rleated to treatment.

Slight changes of body weight, body weight increment and food conversion of animals were detected at the highest dose level. The clinical status of animals after application was influenced by the test substance treatment (salivation and/or chemical odour around animals and/or dyspnoea).

Changes in haematological parameters (delayed significantly increased value of platelet count and delayed significantly decreased value of APTT in males and delayed significantly decreased value of APTT, delayed significantly increased value of RBC, haematocrit, haemoglobin and platelet count in females) and blood biochemical parameters (significantly decreased value of glucose, potassium ions and activity of ALT, delayed significantly increased value of inorganic phosphorus ans insignificantly decreased value of albumin in males and delayed significantly decreased activity of AST, delayed significantly increased value of calcium ions, protein total and albumin in females, changes of albumin in both sexes were out historical control limits), urinalysis (significant decrease of urine volume, significant increase pH of urine  and change of colour, presence of protein, urobilinogen, ketones and leucocytes), biometry of organs (delayed significantly increased absolute weight of pituitary gland and significantly increased relative weight of pituitary gland in males, delayed significantly decreased relative weight of liver, kidneys and thymus in females) were detected.  

Occurrence of macroscopical changes (mainly in forestomach in males and females) and microscopical changes (mainly in forestomach, prostate gland and epididymis in males and forestomach, mammary gland and uterus in females) were detected at the dose level 720 mg/kg/day. Pathological changes in forestomach ((occurrence of inflammation and/or erosion and/or oedema) can be considered as attributable to the test substance administration. Also, influence of the test substance on growth of animals and clinical status of animals was observed (salivation, dyspnoea, chemical odour around animals, coughing).

Changes of haematological parameters (delayed changes of haemocoagulation parameters in males and red blood component and haemocoagulation parameters in females), biochemical parameters (changes of glucose, protein total, albumin, potassium and calcium ions, inorganic phosphorus, and ALT and AST activity), biometry of organs (changes weight of pituitary gland in males and liver, kidneys, thymus in females) and urine parameters in males (urine volume, pH of urine, colour, specific weight, occurrence of leucocytes, protein, urobilinogen and ketones) were detected at the highest dose level.

The value of NOAEL (No Observed Adverse Effect Level) for repeated dose toxicity was established as 240 mg/kg body weight/day for males and females.The value was established on the basis of histopathological results in the stomach. The test substance elicited local toxicity effect on the forestomach at 720 mg/kg bw.