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Diss Factsheets

Administrative data

Description of key information

Based on the results of a 90 -day oral gavage study in rats (OECD 408) using the test substance, the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 150 mg/kg/day due to the histopathological changes in the stomach. Test-item related changes observed in the non-glandular region of the stomach (epithelial hyperplasia, ulceration, inflammatory cell infiltration or inflammation) and glandular region of the stomach (erosion) in males and females given 500 mg/kg/day for 13 weeks by oral gavage were considered adverse.

In the combined Repeat-dose/Reproductive study (OECD 422) the 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.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
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
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
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).
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).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle Controls
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
240 mg/kg bw/day (nominal)
Dose / conc.:
720 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Satellite Group - Vehicle Controls
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
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.
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
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
Critical effects observed:
not specified
System:
gastrointestinal tract

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

1stweek

199.87 ± 10.13

204.30 ± 16.08

218.57 ± 19.07

212.42 ± 10.99

2ndweek

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)

7thday

16.77

15.91

17.91

19.35

14thday

19.77

20.43

21.90

23.80

20thday

26.04

22.12

24.99

22.96

3rdday 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:

0without 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:

0without 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)

106/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)

103/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

 103/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)

106/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)

103/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

 103/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.

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.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2018 - March 2019
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)
Deviations:
yes
Remarks:
See details below
Principles of method if other than guideline:
The following deviation from study plan occurred:
Dose observations: On the 28 November 2018, the 1-2 hour post-dose observation was performed ten minutes late. All animals were observed as within normal limits.
This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Test item: N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine
Appearance: Clear pale yellow liquid
Storage conditions: Room temperature (15 to 25ºC) and stored in the dark
Supplier: Sponsor
Batch number: 2305905
Expiry date: 15 June 2020
Purity 98.6%
Species:
rat
Strain:
Wistar
Details on species / strain selection:
RccHan™:WIST rat
Sex:
male/female
Details on test animals or test system and environmental conditions:
Strain/Species: RccHan™:WIST rat.
Supplier: Envigo RMS Limited (UK).
Number of animals: 45 males and 45 females.
Duration of acclimatization: Eight days before commencement of treatment.
Age of the animals at start of treatment: 37 to 43 days.
Weight range of the animals at the start of treatment Males:106 to 145 g, Females: 107 to 135 g

Animal facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%. There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Cages: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Number of animals per cage: Up to four of the same sex.
Bedding: Wood based bedding which was changed at appropriate intervals each week.
Diet: Teklad 2014C pelleted diet.
Availability: Non-restricted (except overnight before blood sampling for hematology and blood chemistry).


Route of administration:
oral: gavage
Details on route of administration:
Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Vehicle:
water
Details on oral exposure:
Animals were teated daily at constant doses in mg/kg/day (10 mL/kg body weight, Individual dose volume calculated from the most recently recorded scheduled body weight)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability of N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine in purified water formulations was assessed at nominal concentrations of 1 mg/mL and 100 mg/mL.
The mean concentrations of N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine in test formulations analysed during the study were within ±7% of nominal which were within the applied limits +10/-15%, confirming the accuracy of formulation.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
The doses used in this study (0, 50, 150 and 500 mg/kg/day) were selected ionthe basis of a previous OECD 422 study (VUOS Study No. 171/12/19) where rats received the test item by gavage for at least 42 days at dose levels of 80, 240 and 720 mg/kg/day. Inflammation and/or erosion and/or oedema was observed in the forestomach at 720 mg/kg/day. No adverse effect was found on reproduction/developmental parameters at 720 mg/kg/day. No adverse effects or test item related histopathological findings were observed at 80 or 240 mg/kg/day. The NOAEL was considered to be 240 mg/kg/day, while for the reproduction/developmental toxicity the NOAEL was considered to be higher than 720 mg/kg day.

The stomach findings at 720 mg/kg/day suggested a response to the irritancy of the test item, rather than a systemic response to treatment but there was the possibility that more severe stomach damage may develop in a study of longer duration and, consequently, the highest dose for this 13-week study was selected at 500 mg/kg/day. The low and intermediate dose levels of 50 and 150 mg/kg/day were selected to allow evaluation of a dose response.

Positive control:
No
Observations and examinations performed and frequency:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.

Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 12 of treatment.
Sacrifice and pathology:
Method of kill: Carbon dioxide asphyxiation with subsequent exsanguination.
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Other examinations:
Clinical observations were presented for each animal, providing detail of type of sign, week of occurrence and information on the duration of the sign applicable.
The following data types were collected:
- Grip strength and motor activity
- Body weight, using gains over appropriate study periods
- Hematology (Hematocrit (Hct)*, Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH)*, Mean cell hemoglobin concentration (MCHC)*, Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count:, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt))
- Blood chemistry (Alkaline phosphatase (ALP), Alanine amino-transferase (ALT), Aspartate amino-transferase (AST), Total bilirubin (Bili), Urea*, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb) by chemical assay)
- Organ weights, absolute and adjusted for terminal body weight

The eyes of the animals were examined by means of a binocular indirect ophthalmoscope during pretreatment and at Week 12 (all animals of Groups 1 and 4)
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.

The following data types were analyzed at each timepoint separately:
Grip strength and motor activity
Body weight, using gains over appropriate study periods
Hematology
Blood chemistry
Organ weights, absolute and adjusted for terminal body weight

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972).
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.

For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The general appearance and behaviour of the animals receiving 50 or 150 mg/kg/day during the detailed physical examination and the arena observations were not affected by treatment, there were no signs associated with dosing observed during the treatment period for these dose levels.

At 500 mg/kg/day, during the detailed physical examination signs of decreased activity, salivation and breathing difficulties (gasping, rapid breathing and wet rales) were observed for some males and a small number of females were also observed having breathing difficulties with a single female having rapid breathing, one female was observed sneezing and two females observed with wet rales.

Signs relating to breathing difficulties were also observed during dosing observations where two males receiving 500 mg/kg/day had wet rales, these signs were recorded both before and after dosing during Weeks 1 and 3 of treatment for one male and during Week 4 of treatment for the second male. Two females receiving 500 mg/kg/day also showed breathing difficulties, one female was observed with labored breathing during Week 7 of treatment and wet rales was observed for the other female during Week 1 of treatment, both signs were observed after dose administration. As a number of incidences of breathing difficulties (rapid, labored and wet rales) were observed during the treatment period for males and females receiving 500 mg/kg/day these signs were considered related to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
A single male (4M No. 39) that received 500 mg/kg/day was euthanized for welfare reasons on Day 60 of dosing. Clinical signs including decreased activity, gasping, sneezing, partially closed eyes and a red aqueous discharge from the nose were observed for this animal prior to the animal’s dispatch to necropsy. Macroscopic examination revealed dark areas seen on the glandular mucosa of the stomach (correlated microscopically with erosion of the glandular mucosa) and depressions on the non-glandular mucosa (correlated microscopically with ulceration). The major factor contributing to the poor clinical condition of this animal was considered to be the ulcerative lesion in the non-glandular mucosa of the stomach.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Group mean body weights and body weight gain for males and females receiving 50, 150 and 500 mg/kg/day were similar to Controls and considered to be unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related ophthalmic findings.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematological investigations during Week 13 of treatment revealed slightly high reticulocyte counts, compared with Controls, for males (1.17X control) and females (1.07X Control) receiving 500 mg/kg/day with statistical significance attained for males only. Mean eosinophil counts were decreased amongst males and females receiving 500 mg/kg/day (0.22 and 0.00X control for males and females, respectively), a marginal decrease in monocyte counts were also evident for females receiving 500 mg/kg/day (0.73X control). Mean prothrombin times were slightly shorter in females receiving 150 and 500 mg/kg/day (0.95 and 0.90X control, respectively).

All other inter-group differences from Controls, including those which attained statistical significance, were generally minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included, but were not limited to, the slight decrease in mean cell hemoglobin concentration in males given 50 mg/kg/day (0.97X control) and the slight decrease in hematocrit concentration in females given 500 mg/kg/day (0.95X control), but these changes lacked a dose-relationship and were confined to one sex. They also included the slightly high red cell distribution width in females given 500 mg/kg/day (1.04X control), this difference from controls was minimal and was restricted to females only.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry investigations during Week 13 of treatment revealed slightly low glucose concentrations for females at all dose levels with evidence of a dose-dependent decrease, statistical significance was attained for the intermediate and high dose groups only (0.79 and 0.76X control, respectively). Total protein concentrations were decreased (0.94 and 0.95X control for males and females, respectively) in both sexes given 500 mg/kg/day and were associated with slightly decreased (0.95X control) albumin concentrations in males and females.
Statistical significance was attained for low alkaline phosphatase and alanine amino-transferase activities in males at all dose levels (maximum reduction of 0.72 and 0.80X control for alkaline phosphatase and alanine amino-transferase activities, respectively). Dose responses were not apparent, and changes of this degree were considered not to be adverse. Alanine amino-transferase activity was also marginally low amongst females given 500 mg/kg/day (0.78X control).

A small number of other differences from Controls attained statistical significance, but these were minor, confined to one sex and lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included, but were not limited to, the slightly low cholesterol and calcium concentrations in males given 500 mg/kg/day, which attained statistical significance, but there was no dose response and these differences were not of a toxicologically significant magnitude. They also included a slight increase in phosphorus concentration in males at 500 mg/kg/day and a slight reduction in sodium concentration amongst females given 500 mg/kg/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There was no effect of treatment on motor activity.

The total mean low beam break scores (cage floor activity) and high beam scores (rearing activity) were slightly low, when compared with the Controls, for all treated groups of males; however, there were no statistically significant differences in the total one hour high and low beam scores. In addition, for total low beam break scores a dose-dependent relationship was not observed.
There were some occasional statistically significant differences from Controls (comprising: reduced low beam (cage floor activity) scores at 60 minutes at all doses in males and at 54 minutes in males given 500 mg/kg/day; increased high beam (rearing activity) scores at 60 minutes at all doses in males and at 36 minutes in males given 500 mg/kg/day) but these were transient and inconsistent differences. Consequently, they were all attributed to normal biological variation.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Group mean body weights and body weight gain for males and females receiving 50, 150 and 500 mg/kg/day were similar to Controls and considered to be unaffected by treatment.

Analysis of organ weights for animals killed after 13-weeks of treatment revealed a statistically significant decrease in body weight-adjusted spleen weights (down to 0.90X control for adjusted values for both sexes) in males and females given 500 mg/kg/day. Absolute and body weight-adjusted kidney weights for males given 500 mg/kg/day were slightly higher than those of the Controls (1.07X Control). Absolute and body weight-adjusted spleen weights for males given 150 and 500 mg/kg/day were also slightly higher than those of the Controls (1.16X Control). The differences from Controls were, however, minor and, consequently, these differences from Controls were considered of no toxicological significance.

All other inter-group differences from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopic examination performed after 13 weeks of treatment revealed the following changes in the stomach:
Depressions of the non-glandular stomach were seen in ten males and eight females receiving 500 mg/kg/day. Dark areas in the glandular mucosa were seen in two females receiving 500 mg/kg/day.

The incidence and distribution of all other findings were considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with N, N, N’, N’, N”, N”-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine were seen in the stomach.
Findings were observed in both sexes given 500 mg/kg/day. Ulceration of the non-glandular epithelium and concurrent inflammation was observed in one male and three females. Hyperplasia and inflammatory cell infiltration of the non-glandular epithelium and erosion of the glandular epithelium were observed in most male and female animals given 500 mg/kg/day.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
The oral administration, by gavage, of N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine to Han Wistar rats for 13 weeks at dose levels of 50 and 150 mg/kg/day was well tolerated, with no test item-related premature deaths or changes in general clinical condition, and no test item-related macroscopic or microscopic findings.

In both sexes receiving 500 mg/kg/day, the test item was considered to have a direct and local effect on the mucosa of the glandular and non-glandular regions of the stomach. Histopathological findings in the non-glandular region of the stomach included ulceration, hyperplasia and inflammatory cell infiltration of the non-glandular epithelium, with erosions observed in the epithelium of the glandular region only. The spectrum of findings in the non-glandular region of the stomach when compared to the glandular region of the stomach reflected a more severe response in the non-glandular mucosa to irritation and subsequent injury. This response was considered to be related to the prolonged contact of the test item with the non-glandular mucosa of the forestomach where digesta is held for longer in comparison to the glandular portion of the stomach.

Key result
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: Dose Groups (Male/Female)

 

 

Control

N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine

Dose Group

1

2

3

4

Dose (mg/kg/day)

0

50

150

500

 

Tables Legend:

MMale

FFemale

SDStandard deviation

NNumber of animals/cages examined

Weeks of pretreatment relate to study weeks, as follows: Phase week P1 Week of study -1

 

*All tables are provided as an attachment. Table numbers are aligned.

 

 

Table 4: Body Weights –group mean values (g) - Males

 

Group/Sex

 

Week

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Change

 

 

P1

0

1

2

3

4

5

6

7

8

9

10

11

12

13

0-13

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1M

Mean

94

130

167

203

238

268

288

312

328

345

357

368

376

385

396

265

 

SD

8.6

10.1

12.1

12.3

15.2

17.3

16.8

18.5

22.2

24.9

26.3

28.5

30.1

31.5

30.9

25.5

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2M

Mean

90

123

157

191

226

255

276

294

310

326

338

351

359

366

375

252

 

SD

9.1

12.0

15.3

17.8

18.9

19.7

20.4

20.1

20.3

22.1

21.9

22.8

24.4

24.5

25.2

17.1

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

% of 1M

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

95

3M

Mean

91

125

160

198

233

266

290

307

324

341

353

363

374

381

390

265

 

SD

9.0

10.0

13.2

15.7

17.2

18.8

21.0

23.6

23.2

24.4

25.5

27.0

26.2

27.0

28.3

21.6

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

% of 1M

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100

4M

Mean

94

127

162

200

237

266

289

304

322

340

354

361

374

383

392

264

 

SD

7.6

9.6

13.2

17.0

21.3

23.1

24.4

28.7

33.2

34.4

33.5

31.8

35.5

37.8

38.2

32.4

 

N

10

10

10

10

10

10

10

10

10

10

9

9

9

9

9

9

 

% of 1M

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4a: Body Weights –group mean values (g) - Females

 

Group

 

Week

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Change

/Sex

 

P1

0

1

2

3

4

5

6

7

8

9

10

11

12

13

0-13

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1F

Mean

99

122

140

158

171

184

193

200

206

214

219

221

221

226

230

108

 

SD

7.1

5.7

5.5

5.3

7.8

8.9

8.2

8.9

10.7

11.1

10.6

9.3

11.8

11.6

9.4

10.0

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2F

Mean

98

121

138

155

169

180

190

198

205

209

214

220

221

223

226

105

 

SD

8.3

7.0

6.6

6.1

8.6

9.0

10.0

11.2

11.0

10.7

10.9

11.9

10.2

12.2

14.0

14.5

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

% of 1F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

97

3F

Mean

95

120

140

160

176

190

200

206

215

219

225

229

232

234

237

117

 

SD

8.9

7.8

11.2

13.2

15.2

15.3

16.4

16.2

16.4

16.3

16.7

16.9

16.7

15.0

15.1

12.1

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

% of 1F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

108

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4F

Mean

99

124

142

161

177

191

199

207

214

221

227

234

234

239

243

118

 

SD

4.6

5.2

6.2

8.4

10.3

7.8

10.0

11.5

13.0

10.5

13.2

14.3

14.5

14.7

15.0

14.2

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

% of 1F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

110

 

 

Table 5: Food Consumption -group mean values (g/animal/week) -Males

 

Group

 

Week

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean

/Sex

 

P1

1

2

3

4

5

6

7

8

9

10

11

12

13

1-13

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1M

Mean

125

147

149

154

159

166

168

163

162

162

161

162

154

153

158

 

SD

11.6

12.5

8.6

12.0

13.8

10.9

7.7

8.2

14.8

16.7

17.5

19.5

8.1

15.5

12.5

 

N

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2M

Mean

117

135

142

149

153

163

157

155

154

154

159

158

152

138

151

 

SD

1.1

3.4

5.9

5.6

8.2

7.6

3.8

5.7

8.5

8.5

4.0

7.2

1.5

6.7

4.4

 

N

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

 

% of 1M

 

 

 

 

 

 

 

 

 

 

 

 

 

 

96

3M

Mean

120

141

153

154

161

171

170

159

158

158

162

161

158

146

158

 

SD

3.0

6.5

12.9

9.8

12.3

11.8

12.8

6.7

7.3

3.4

9.0

6.7

4.7

10.9

8.0

 

N

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

 

% of 1M

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100

4M

Mean

119

137

157

154

161

164

162

157

159

152

155

163

163

140

156

 

SD

3.1

5.9

2.8

15.1

11.6

8.4

13.1

16.0

13.0

10.5

2.1

4.8

6.4

13.4

7.5

 

N

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

 

% of 1M

 

 

 

 

 

 

 

 

 

 

 

 

 

 

98

 

 

 

Table 5a: Food Consumption -group mean values (g/animal/week) –Females

 

Group

 

Week

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean

/Sex

 

P1

1

2

3

4

5

6

7

8

9

10

11

12

13

1-13

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1F

Mean

99

99

109

113

115

123

121

121

119

117

116

118

113

106

115

 

SD

4.6

3.5

3.8

4.6

2.1

1.9

5.8

6.5

5.5

2.6

5.6

6.5

7.3

4.1

3.6

 

N

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2F

Mean

99

99

107

109

111

125

118

116

114

115

115

116

113

104

112

 

SD

6.0

5.1

5.4

6.5

6.3

9.5

8.8

13.0

8.6

11.5

7.0

6.6

9.8

8.4

7.9

 

N

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

 

% of 1F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

98

3F

Mean

104

104

115

115

119

128

126

123

118

118

125

123

116

113

119

 

SD

6.1

8.9

6.9

5.0

6.8

5.3

6.1

5.5

5.0

9.6

8.9

3.1

2.9

18.1

6.4

 

N

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

 

% of 1F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

104

4F

Mean

109

99

112

112

113

121

120

118

117

110

122

119

112

101

114

 

SD

1.0

5.6

5.2

5.8

4.0

9.7

8.5

11.8

11.8

7.3

13.5

10.4

4.4

13.4

8.5

 

N

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

 

% of 1F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

99

 

 

Table 6: Hematology - group mean values during Week 13 of treatment - Males

 

Group

/Sex

 

Hct

L/L

Hb

g/dL

RBC

x1012/L

Retic

x1012/L

MCH

pg

MCHC

g/dL

MCV

fL

RDW

%

WBC

x109/L

N

x109/L

L

x109/L

E

x109/L

B

x109/L

M

x109/L

LUC

x109/L

Plt

x109/L

PT

sec

APTT

Sec

1M

Mean

0.472

16.0

8.71

0.141

18.4

33.9

54.2

13.1

7.63

1.24

6.04

0.09

0.05

0.15

0.04

557

25.5

19.3

 

SD

0.0200

0.46

0.360

0.0217

0.56

0.84

1.48

0.37

1.789

0.483

1.418

0.031

0.015

0.042

0.019

35.6

7.41

5.47

 

N

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

3

10

8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2M

Mean

0.483

15.9

8.86

0.143

17.9

32.9*

54.5

13.3

7.90

1.28

6.23

0.11

0.06

0.17

0.05

566

21.6

18.0

 

SD

0.0178

0.41

0.396

0.0227

0.85

0.90

1.92

0.49

0.832

0.314

0.842

0.060

0.018

0.045

0.020

54.4

1.82

1.87

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

7

7

7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3M

Mean

0.473

16.2

8.53

0.143

19.0

34.2

55.6

13.2

7.41

0.97

6.11

0.07

0.06

0.15

0.05

533

23.2

17.6

 

SD

0.0159

0.47

0.390

0.0236

0.79

0.60

1.62

0.50

1.464

0.294

1.279

0.011

0.028

0.043

0.025

75.9

4.84

2.17

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

6

10

9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4M

Mean

0.475

15.7

8.61

0.165*

18.3

33.1

55.2

13.3

8.48

1.45

6.72

0.02**

0.07

0.18

0.05

649

22.3

17.0

 

SD

0.0143

0.45

0.465

0.0148

0.77

0.84

1.54

0.66

1.358

0.209

1.226

0.009

0.034

0.033

0.021

134.7

1.79

1.39

 

N

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

8

8

8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 6a: Hematology - group mean values during Week 13 of treatment -Females

 

Group

/Sex

 

Hct

L/L

Hb

g/dL

RBC

x1012/L

Retic

x1012/L

MCH

pg

MCHC

g/dL

MCV

fL

RDW

%

WBC

x109/L

N

x109/L

L

x109/L

E

x109/L

B

x109/L

M

x109/L

LUC

x109/L

Plt

x109/L

PT

sec

APTT

sec

1F

Mean

0.430

14.7

7.55

0.164

19.4

34.1

57.0

11.4

4.43

0.73

3.44

0.04

0.01

0.11

0.09

833

21.6

19.5

 

SD

0.0118

0.51

0.194

0.0287

0.75

0.80

1.70

0.33

0.851

0.174

0.687

0.016

0.008

0.046

0.045

107.3

1.40

1.82

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2F

Mean

0.425

14.6

7.43

0.163

19.7

34.4

57.3

11.5

3.91

0.76

2.94

0.04

0.01

0.09

0.08

836

21.3

19.7

 

SD

0.0156

0.24

0.241

0.0172

0.54

1.01

1.33

0.32

0.849

0.313

0.713

0.021

0.006

0.027

0.028

70.6

1.20

1.83

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3F

Mean

0.436

14.8

7.82

0.156

19.0

34.0

55.8

11.7

3.37

0.62

2.56

0.03

0.01

0.09

0.07

844

20.6*

20.8

 

SD

0.0306

0.51

0.541

0.0234

0.84

1.58

1.05

0.44

0.936

0.148

0.828

0.012

0.003

0.031

0.022

100.1

0.90

2.83

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4F

Mean

0.409*

14.3

7.30

0.176

19.6

34.9

56.1

11.8*

4.08

0.74

3.19

0.00**

0.01

0.08*

0.06

838

19.5**

20.9

 

SD

0.0181

0.58

0.311

0.0255

0.58

0.73

1.67

0.48

0.995

0.174

0.971

0.005

0.005

0.016

0.015

85.4

0.96

1.84

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

9

9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                                                                                                                            

 

 

Table 7: Blood chemistry - group mean values during Week 13 of treatment -Males

 

Group

/Sex

 

ALP

U/L

ALT

U/L

AST

U/L

Bili

µmol/L

Urea

mmol/L

Creat

µmol/L

Gluc

mmol/L

Chol

mmol/L

Trig

mmol/L

Na

mmol/L

K

mmol/L

Cl

mmol/L

Ca

mmol/L

Phos

mmol/L

Total Prot

g/L

Alb

g/L

A/G

Ratio

1M

Mean

111

56

67

2

7.55

33

8.56

2.79

1.05

142

4.5

101

2.73

1.85

70

38

1.23

 

SD

13.5

12.1

20.1

0.5

1.003

3.6

2.076

0.261

0.339

1.4

0.52

1.3

0.065

0.115

1.5

1.2

0.059

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2M

Mean

93*

47*

86

2

7.62

32

8.69

2.43

1.02

142

4.5

103

2.66

1.91

69

37

1.19

 

SD

13.2

8.6

14.9

0.5

1.248

3.8

1.000

0.458

0.348

1.3

0.37

1.9

0.058

0.130

2.5

1.1

0.078

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3M

Mean

80**

45*

67

2

6.86

29

9.63

2.58

1.15

142

4.5

102

2.67

1.77

69

37

1.20

 

SD

10.8

11.4

16.9

0.5

1.164

2.0

1.864

0.645

0.470

1.3

0.19

1.5

0.079

0.108

2.6

1.1

0.068

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4M

Mean

88**

47*

81

2

7.18

30

8.84

2.28*

1.40

141

4.4

101

2.65*

2.07**

66**

36**

1.23

 

SD

22.6

6.9

22.2

0.7

0.786

2.4

1.496

0.360

0.699

1.1

0.65

1.0

0.065

0.140

1.4

0.7

0.057

 

N

9

9

9

9

8

8

9

9

9

8

9

9

9

8

9

8

8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 7a: Blood chemistry - group mean values during Week 13 of treatment -Females

 

Group

/Sex

 

ALP

U/L

ALT

U/L

AST

U/L

Bili

µmol/L

Urea

mmol/L

Creat

µmol/L

Gluc

mmol/L

Chol

mmol/L

Trig

mmol/L

Na

mmol/L

K

mmol/L

Cl

mmol/L

Ca

mmol/L

Phos

mmol/L

Total Prot

g/L

Alb

g/L

A/G

Ratio

1F

Mean

46

59

98

2

7.53

32

9.17

2.21

0.75

143

3.9

102

2.78

1.54

75

44

1.40

 

SD

7.1

7.6

29.7

0.6

1.616

4.4

1.430

0.430

0.253

1.3

0.09

1.5

0.099

0.167

3.7

2.6

0.077

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2F

Mean

52

54

94

2

8.40

37

8.33

2.22

0.82

143

3.8

103

2.76

1.51

74

43

1.39

 

SD

15.6

13.6

23.3

0.5

1.438

3.0

1.446

0.261

0.298

1.4

0.27

1.1

0.058

0.202

2.4

2.3

0.095

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3F

Mean

47

52

83

2

7.40

34

7.26**

2.26

0.79

143

3.8

104

2.74

1.42

73

43

1.44

 

SD

7.2

12.2

16.3

0.5

0.915

3.5

0.792

0.411

0.303

0.9

0.21

0.9

0.099

0.171

3.6

2.5

0.070

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4F

Mean

49

46*

97

2

8.36

38**

6.93**

2.02

0.78

142*

3.8

102

2.72

1.66

71*

42

1.44

 

SD

7.2

10.7

28.9

0.7

0.808

5.4

0.895

0.284

0.173

1.0

0.33

1.4

0.052

0.226

5.1

2.5

0.113

 

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 8: Organ weights - group mean absolute and adjusted values (g) for animals killed after 13 weeks of treatment - Males

 

Group /Sex

 

Terminal Body weight

Adrenals

Brain

Epididymides

Heart

Kidneys

Liver

Spleen

Testes

Thymus

1M

Mean

396

0.056

2.082

1.397

1.054

2.073

13.781

0.749

4.054

0.317

 

SD

32

0.007

0.086

0.140

0.088

0.174

1.273

0.070

0.369

0.056

 

N

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

2M

Mean

373

0.060

2.072

1.374

0.971

2.007

12.499

0.670

3.861

0.309

 

SD

25

0.006

0.095

0.170

0.065

0.148

1.332

0.064

0.264

0.064

 

N

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

3M

Mean

389

0.065

2.096

1.414

1.018

2.114

13.275

0.731

3.999

0.325

 

SD

29

0.009

0.079

0.133

0.087

0.146

1.553

0.057

0.250

0.073

 

N

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

4M

Mean

390

0.065

2.080

1.323

1.036

2.198

13.421

0.673

3.875

0.306

 

SD

37

0.008

0.109

0.115

0.084

0.149

1.297

0.072

0.291

0.067

 

N

9

9

9

9

9

9

9

9

9

9

 

Table 8a: Organ weights - group mean absolute and adjusted values (g) for animals killed after 13 weeks of treatment -Females

 

Group

/Sex

 

Terminal Body weight

Adrenals

Brain

Heart

Kidneys

Liver

Ovaries

Spleen

Thymus

Uterus and Cervix

1F

Mean

230

0.066

1.847

0.774

1.405

8.376

0.085

0.595

0.327

0.718

 

SD

13

0.009

0.086

0.072

0.089

0.987

0.017

0.088

0.189

0.272

 

N

10

10

10

10

10

10

10

10

10

9

 

 

 

 

 

 

 

 

 

 

 

 

2F

Mean

226

0.070

1.882

0.738

1.314

8.226

0.086

0.563

0.279

0.803

 

SD

14

0.014

0.108

0.052

0.100

0.645

0.011

0.070

0.041

0.380

 

N

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

3F

Mean

238

0.077

1.947

0.798

1.440

8.587

0.094

0.609

0.286

0.721

 

SD

14

0.012

0.081

0.052

0.142

0.794

0.015

0.069

0.072

0.345

 

N

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

4F

Mean

241

0.072

1.928

0.759

1.500

8.617

0.095

0.569

0.273

0.898

 

SD

14

0.013

0.084

0.041

0.089

0.949

0.018

0.063

0.048

0.373

 

N

10

10

10

10

10

10

10

10

10

10

 

 

Table 9: Macropathology - group distribution of findings for animals killed after 13 weeks of treatment – Males and Females

 

 

Number of animals affected

 

 

Group/Sex

1M

2M

3M

4M

1F

2F

3F

4F

Tissue/Organ and Findings

No. of animals

10

10

10

9

10

10

10

10

 

 

 

 

 

 

 

 

 

 

Number of animals within normal limits

 

10

8

9

0

9

9

9

2

 

 

 

 

 

 

 

 

 

 

Heart

 

 

 

 

 

 

 

 

 

    Dark area(s)

 

0

0

1

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

Kidneys

 

 

 

 

 

 

 

 

 

    Dilated Pelvis

 

0

1

0

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

Liver

 

 

 

 

 

 

 

 

 

    Strangulated

 

0

1

0

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

Lungs and Bronchi

 

 

 

 

 

 

 

 

 

    Pale area(s)

 

0

0

0

0

0

1

1

1

 

 

 

 

 

 

 

 

 

 

Stomach

 

 

 

 

 

 

 

 

 

    Dark area(s)

 

0

0

0

0

0

0

0

2

    Depression(s)

 

0

0

0

9

0

0

0

8

 

 

 

 

 

 

 

 

 

 

Vagina

 

 

 

 

 

 

 

 

 

    Abnormal contents

 

-

-

-

-

1

0

0

0

    Absent

 

-

-

-

-

1

0

0

0

    Imperforate

 

-

-

-

-

1

0

0

0

 

 

 

 

 

 

 

 

 

 

 

Table: Summary of treatment related findings in the stomach for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

50

150

500

0

50

150

500

Ulceration, Non-glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

0

0

2

Slight

0

0

0

0

0

0

0

1

Moderate

0

0

0

1

0

0

0

1

Total

0

0

0

1

0

0

0

4

Hyperplasia, Epithelial, Non-glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

1

0

0

1

Slight

0

0

0

1

0

0

0

1

Moderate

0

0

0

8

0

0

0

5

Marked

0

0

0

0

0

0

0

1

Total

0

0

0

9

1

0

0

8

Inflammation, Non-glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

0

0

3

Moderate

0

0

0

1

0

0

0

0

Total

0

0

0

1

0

0

0

3

Erosion, Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

6

0

0

0

7

Moderate

0

0

0

0

0

0

0

1

Total

0

0

0

6

0

0

0

8

Infiltration, Inflammatory Cell, Mucosal/Submucosal, Non-glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

8

0

0

0

4

Slight

0

0

0

0

0

0

0

1

Total

0

0

0

8

0

0

0

5

Number of tissues examined

10

10

10

9

10

10

10

10

 

Conclusions:
Based on the results of a 90-day repeat-dose study in rats (OECD TG 408), it was concluded that the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity of N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine was 150 mg/kg/day due to the histopathological changes in the stomach. Test-item related changes observed in the non-glandular region of the stomach (epithelial hyperplasia, ulceration, inflammatory cell infiltration or inflammation) and glandular region of the stomach (erosion) in males and females given 500 mg/kg/day for 13 weeks by oral gavage were considered adverse.
Executive summary:

In a 90-day repeat-dose study in rats (OECD TG 408), the test substance  N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine was administered by oral gavage to Han Wistar rats for 13 weeks at dose levels of 50, 150 or 500 mg/kg/day. A control group received the vehicle, purified water, at the same volume dose as the treated groups.

During the treatment period four males and four females receiving 500 mg/kg/day were observed with breathing difficulties, including rapid and laboured breath, gasping and the presence of wet rales.  These signs were observed during the weekly physical examinations and during observations related to dose administration. There were no signs relating to breathing difficulties observed for Control animals or animals receiving 50 or 150 mg/kg/day and therefore due to the number of animals observed with breathing difficulties at 500 mg/kg/day, these signs were considered to be related to treatment.  As there were no associated histopathological findings for these observations, these signs associated with breathing difficulties were considered non-adverse.

Group mean body weights and body weight gain for males and females receiving 50, 150 and 500 mg/kg/day were similar to Controls and considered to be unaffected by treatment (Table 4).

Food consumption was unaffected by treatment (Table 5)

The low body weight-adjusted spleen weights in males and females given 500 mg/kg/day, the high absolute and body weight-adjusted kidney weights in males given 500 mg/kg/day and the high absolute and body weight-adjusted adrenal weights in males given 150 and 500 mg/kg/day were not associated with any macroscopic or microscopic changes.  As these organ weight changes were minimal and were not associated with any other findings, these changes were considered non-adverse.

Analysis of organ weights for animals killed after 13-weeks of treatment revealed a statistically significant decrease in body weight-adjusted spleen weights (down to 0.90X control for adjusted values for both sexes) in males and females given 500 mg/kg/day (Table 8).

Absolute and body weight-adjusted kidney weights for males given 500 mg/kg/day were slightly higher than those of the Controls (1.07X Control). Absolute and body weight adjusted spleen weights for males given 150 and 500 mg/kg/day were also slightly higher than those of the Controls (1.16X Control). The differences from Controls were, however, minor and, consequently, these differences from Controls were considered of no toxicological significance. There were no histopathological correlates for these changes.  

All other inter-group differences from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.

Haematology investigations during Week 13 of treatment (Table 6) showed a slight increase in reticulocyte counts for males and females receiving 500 mg/kg/day, however clear dose-dependent relationships were not apparent, and this was not associated with any changes in other red blood cell parameters.  Eosinophil counts were statistically low for males and females at 500 mg/kg/day and a marginal decrease in monocyte counts was evident for females receiving 500 mg/kg/day.  These changes were not corroborated with any histopathological findings and therefore, these changes were considered non-adverse.  For prothrombin times, only prolonged times are considered adverse and, consequently, the slightly reduced times in females given 150 or 500 mg/kg/day were of no toxicological significance.

Blood chemistry investigations during Week 13 of treatment (Table 7) revealed low glucose in the plasma for females at all dose levels, and the low protein and albumin concentrations in both sexes given 500 mg/kg/day were not associated with any test item related histopathological changes and therefore considered non-adverse. Statistical significance was attained for low alkaline phosphatase and alanine aminotransferase activities in males at all dose levels (maximum reduction of 0.72 and 0.80X control for alkaline phosphatase and alanine amino-transferase activities, respectively). Dose responses were not apparent, and changes of this degree were considered not to be adverse. Alanine amino-transferase activities were also marginally low amongst females given 150 and 500 mg/kg/day (0.88 and 0.78X control, respectively). A small number of other differences from Controls attained statistical significance, but these were minor, confined to one sex and lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included, but were not limited to, the slightly low cholesterol and calcium concentrations in males given 500 mg/kg/day, but there was no dose response and these differences were not of a toxicologically significant magnitude. They also included a slight increase in phosphorus concentration in males at 500 mg/kg/day, a slight reduction in sodium concentration and a slight increase in creatinine concentration amongst females given 500 mg/kg/day.

The downward trend observed for some liver enzymes in males at all dose levels and the low alanine amino-transferase activity in females given 500 mg/kg/day did not impact liver weights or was not associated with any histopathological changes and therefore these differences were considered non adverse.

Body weight (Table 4) and food consumption (Table 5) were unaffected by treatment at all dose levels and there were no treatment-related changes in sensory reactivity, grip strength or motor activity.  In addition, there were no treatment-related ophthalmic findings.

The macroscopic examination performed after 13 weeks of treatment (Table 9) revealed changes in the stomach, characterised by depressions of the non-glandular stomach in ten males and eight females receiving 500 mg/kg/day.

Dark areas in the glandular mucosa were seen in two females receiving 500 mg/kg/day. The incidence and distribution of all other gross pathology findings were considered to be unrelated to treatment.

In both sexes receiving 500 mg/kg/day, the test item was considered to have a direct and local effect on the mucosa of the glandular and non-glandular regions of the stomach.  Histopathological findings in the non-glandular region of the stomach included ulceration, hyperplasia and inflammatory cell infiltration of the non-glandular epithelium, with erosions observed in the epithelium of the glandular region only.  The spectrum of findings in the non-glandular region of the stomach when compared to the glandular region of the stomach reflected a more severe response in the non-glandular mucosa to irritation and subsequent injury. This response was considered to be related to the prolonged contact of the test item with the non-glandular mucosa of the forestomach where digesta is held for longer in comparison to the glandular portion of the stomach.

Based on the results of this study it was concluded that the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 150 mg/kg/day due to the histopathological changes in the stomach.  Test-item related changes observed in the non-glandular region of the stomach (epithelial hyperplasia, ulceration, inflammatory cell infiltration or inflammation) and glandular region of the stomach (erosion) in males and females given 500 mg/kg/day for 13 weeks by oral gavage were considered adverse.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data derived from two valid GLP-compliant OECD guideline studies performed in 2013 and 2019.
System:
gastrointestinal tract
Organ:
stomach

Additional information

In a 90-day repeat-dose study in rats (OECD TG 408), the test substance  N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine was administered by oral gavage to Han Wistar rats for 13 weeks at dose levels of 50 and 150 mg/kg/day.

During the treatment period four males and four females receiving 500 mg/kg/day were observed with breathing difficulties, including rapid and labored breath, gasping and the presence of wet rales.  These signs were observed during the weekly physical examinations and during observations related to dose administration.  There were no signs relating to breathing difficulties observed for Control animals or animals receiving 50 or 150 mg/kg/day and therefore due to the number of animals observed with breathing difficulties at 500 mg/kg/day, these signs were considered to be related to treatment.  As there were no associated histopathological findings for these observations, these signs associated with breathing difficulties were considered non-adverse.

In both sexes receiving 500 mg/kg/day, the test item was considered to have a direct and local effect on the mucosa of the glandular and non-glandular regions of the stomach.  Histopathological findings in the non-glandular region of the stomach included ulceration, hyperplasia and inflammatory cell infiltration of the non-glandular epithelium, with erosions observed in the epithelium of the glandular region only.  The spectrum of findings in the non-glandular region of the stomach when compared to the glandular region of the stomach reflected a more severe response in the non-glandular mucosa to irritation and subsequent injury.  This response was considered to be related to the prolonged contact of the test item with the non-glandular mucosa of the forestomach where digesta is held for longer in comparison to the glandular portion of the stomach.

The low body weight-adjusted spleen weights in males and females given 500 mg/kg/day, the high absolute and body weight-adjusted kidney weights in males given 500 mg/kg/day and the high absolute and body weight-adjusted adrenal weights in males given 150 and 500 mg/kg/day were not associated with any macroscopic or microscopic changes.  As these organ weight changes were minimal and were not associated with any other findings, these changes were considered non-adverse.

There was a slight increase in reticulocyte counts for males and females receiving 500 mg/kg/day, however clear dose-dependent relationships were not apparent, and this was not associated with any changes in other red blood cell parameters.  Eosinophil counts were statistically low for males and females at 500 mg/kg/day and a marginal decrease in monocyte counts was evident for females receiving 500 mg/kg/day.  These changes were not corroborated with any histopathological findings and therefore, these changes were considered non-adverse.  For prothrombin times, only prolonged times are considered adverse and, consequently, the slightly reduced times in females given 150 or 500 mg/kg/day were of no toxicological significance.

The low glucose in the plasma for females at all dose levels, and the low protein and albumin concentrations in both sexes given 500 mg/kg/day were not associated with any test item related histopathological changes and therefore considered non-adverse.  

The downward trend observed for some liver enzymes in males at all dose levels and the low alanine amino-transferase activity in females given 500 mg/kg/day did not impact liver weights or was not associated with any histopathological changes and therefore these differences were considered non adverse.

Body weight and food consumption was unaffected by treatment at all dose levels and there were no treatment-related changes in sensory reactivity, grip strength or motor activity.  In addition, there were no treatment-related ophthalmic findings.

Based on the results of this study it was concluded that the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 150 mg/kg/day due to the histopathological changes in the stomach.  Test-item related changes observed in the non-glandular region of the stomach (epithelial hyperplasia, ulceration, inflammatory cell infiltration or inflammation) and glandular region of the stomach (erosion) in males and females given 500 mg/kg/day for 13 weeks by oral gavage were considered adverse.

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.

Justification for classification or non-classification

Based on the results of a 90 -day oral gavage study in rats (OECD 408) using the test substance, the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 150 mg/kg/day due to the histopathological changes in the stomach. Test-item related changes observed in the non-glandular region of the stomach (epithelial hyperplasia, ulceration, inflammatory cell infiltration or inflammation) and glandular region of the stomach (erosion) in males and females given 500 mg/kg/day for 13 weeks by oral gavage were considered adverse.

In the combined Repeat-dose/Reproductive study (OECD 422) the 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.

In a GLP-compliant OECD 414 guideline study on Pre-Natal Developmental Toxicity (PNDT), three groups of 20 female Han-Wister rats received N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine1,3,5(2H,4H,6H)-Tripropanamineat doses of 80, 240 or 720mg/kg/day by oral gavage administration at a volume dose of 10 mL/kg body weight, from Day 6 to19after mating, inclusive. At the highest dose of 720 mg/kg/day some evidence of maternal toxicity was observed, manifest as a 50% reduction in mean body weight gain during Days 6-9 of gestation associated with a 21% reduction in mean food intake during Days 6-10of gestation.  Thereafter, the body weight gain and mean food intake of females in this group was essentially similar to that of the Controls.  Mean body weight gain and food consumption for females given 80 or 240 mg/kg/day was unaffected. It was concluded that the high dose level of 720 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.

No significant systemic toxicity was observed in the repeat-dose studies. A prinicpal finding in all studies was related to local irritation effects on the stomach of high dose animals.      

According to CLP Criteria ( Regulation (EC) No 1272/2008) the substance is not classifiable for repeat dose toxicity or specific target organ toxicity (STOT).