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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Combined Repeat Dose Toxicity Study with Reproductive/Developmental Screening (OECD 422)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2012 - April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See details below
Principles of method if other than guideline:
Study Deviation: The decrease of temperature (to 18.6 ºC) in SPF animal room was observed during study. The limit prescribed in OECD Test Guideline No. 422 for temperature in animal room is 19 – 25ºC. The deviations from the limits were short-term and did not influence the wellness of animals and the study results.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine
EC Number:
240-004-1
EC Name:
N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine
Cas Number:
15875-13-5
Molecular formula:
C18H42N6
IUPAC Name:
(3-{3,5-bis[3-(dimethylamino)propyl]-1,3,5-triazinan-1-yl}propyl)dimethylamine
Test material form:
liquid: viscous
Details on test material:
N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine (CAS No. 15875-13-5)
Purity: 98.0% (w/w)
Appearance: Colorless to pale yellow liquid
Batch No.: 1166046
Stability/Expiration:06/2014
Specific details on test material used for the study:
CAS No.:15875-13-5
CAS name: N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine
Purity: 98.0% (w/w)
Impurities: Water (CAS No. 7732-18-5) 0.14 % (w/w)
Appearance: Colorless to pale yellow liquid
Batch No.: 1166046
Stability/Expiration: 06/2014

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal housing
All the study proceeded in SPF (Specified Pathogen Free) animal house of CETA in SPF conditions according to internal SOP No. 46
Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood.

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on 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.
Details on 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.
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).
Details on study schedule:
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.

Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle Controls
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Main Group - Low Dose
Dose / conc.:
240 mg/kg bw/day (nominal)
Remarks:
Main group - Mid dose
Dose / conc.:
720 mg/kg bw/day (nominal)
Remarks:
Main Group - High Dose
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:
Dose levels for study were determined on the basis of results of a dose-range finding study using the same substance.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
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
Oestrous cyclicity (parental animals):
No details provied.
Sperm parameters (parental animals):
In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to internal SOP No. M/45.

Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck ¬– were recorded.
Litter observations:
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.
Postmortem examinations (parental animals):
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffer 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.
After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymis/epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart were recorded (repeated dose toxicity part of study – 6 males and females from each group + satellite groups); testes or ovaries, epididymis or uterus, prostate gland, pituitary gland (reproduction part of study – all animals). Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

Postmortem examinations (offspring):
Macroscopic examination was performed in all pups (except three stillborn pups in the control group – partial cannibalism of mother).
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.
Reproductive indices:
Number and Sex Ratio of Pups
Body Weight of Pups
Offspring viability indices:
Development of Pups
Pathological Examination of Pups

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, 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.
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. Statistical analysis was performed for body weight. Statistically significant differences were not found.
For Females, increased 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.
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 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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination of reproductive system of parental males showed changes of microscopical structure of prostate gland: namely focal mononuclear infiltration and/or oedema of interstitium in all treated groups and control group. In epididymis sporadic changes were recorded: focal mononuclear infiltration and germ cells in lumen of tubules in all groups of males. But frequencies of mentioned effects were not toxicologically relevant according to the Guidance Document for Histologic Evaluation of Endocrine and Reproductive Tests in Rodents, ENV/JM/MONO(2009)11.

Examination of reproductive system of parental females showed changes of uterus in females from all groups. Lobular hyperplasia in mammary gland was detected in all groups. These changes related with previous gravidity or proceeded oestrous cycle.

In the 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:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: slight decreade in highest dose animals (due to unpalatability)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The test substance had no effect on sperm quality of treated males.
Reproductive performance:
no effects observed
Description (incidence and severity):
Abortions were recorded in one female at the lowest dose level and one female at the highest dose level. Increased numbers of corpora lutea were recorded at the middle and highest dose level. Duration of mating and pregnancy of treated groups were similar to the control group. Pre-implantation losses were slightly increased at the middle and highest dose level. Post-implantation losses were similar. Number of pups per litter was slightly increased at the middle dose level. The number of females bearing live pups and females with live pups at day 4 after parturition was increased at the middle and highest dose level. The mating index was increased in all treated groups. The fertility index was slightly increased at the middle and highest dose level. Gestation and survival index of treated groups was well-balanced with the control group. These changes are regarded as spontaneous and biologically insignificant.

Details on results (P0)

All treated and control females were mated. Presence of sperm was not found in two females at the control group, one female at the dose level 80 mg/kg/day and one female at the dose level 240 mg/kg/day. Females with abortion were recorded at the dose level 80 and 720 mg/kg/day (one female at the lowest dose level and one female at the highest dose level).
The number of females achieving pregnancy was higher at the dose level 240 and 720 mg/kg/day. Duration of mating and pregnancy of treated groups were similar to the control group. The number of females bearing live pups and females with live pups at day 4 after parturition was increased at the dose level 240 and 720 mg/kg/day.
The number of corpora lutea was slightly increased at the dose level 240 and 720 mg/kg/day. Number of implantations at the dose level 240 mg/kg/day was higher than at the control group.
Number of pups per litter and mean weight of litters was slightly increased at the dose level 240 mg/kg. The mean weight of pups at all dose level was relative well-balanced. Abnormal pups were not found in all groups.
Biometry of Reproductive Organs
The statistical analysis of the data revealed no significant intergroup difference.
Absolute weight of testes and epididymis were insignificantly decreased at the dose level 80 mg/kg/day. Decreased relative weight of testes and increased absolute weight of pituitary gland at the dose level 240 mg/kg/day was found. At the dose level 720 mg/kg/day increased absolute and relative weight of pituitary gland, increased relative weight of epididymis and decreased absolute weight of prostate gland were detected.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 720 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
720 mg/kg bw/day (nominal)
System:
male reproductive system
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
720 mg/kg bw/day (nominal)
System:
female reproductive system

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean weight of the litters at the dose levels 240 mg/kg/day was increased compared to controls in all intervals of weighting.
The mean weight of pups at all treated groups and control group was relative well-balanced.
No significant changes were recorded.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
A macroscopic examination was performed in all pups (except three stillborn pups in the control group – partial cannibalism of mother).
No macroscopic changes were observed during pathological examination of pups.

Histopathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Number and Sex Ratio of Pups
Statistical evaluation was performed on the number of live pups. No significant changes were recorded.
The total number of live pups (on the day of parturition/1st day after parturition and the 4th day after parturition) at the dose levels 240 and 720 mg/kg/day was higher than at the control.
Mean number of pups per litter at the dose level 240 mg/kg/day was higher than at the control.
Sex ratio of pups was balanced in treated and control groups.
Body Weight of Pups
The mean weight of the litters at the dose levels 240 mg/kg/day was increased compared to control in all interval of weighting.
The mean weight of pups at all treated groups and control group was relative well-balanced.
No significant changes were recorded.
Development of Pups
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.
Pathological Examination of Pups
The macroscopic examination was performed in all pups (except three stillborn pups in the control group – partial cannibalism of mother).
No macroscopic changes were observed during pathological examination of pups.




Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
720 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
720 mg/kg bw/day (nominal)
System:
other: Observation of Pups

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Summary of Effects on Reproductions/Development

Reproduction data

Observed parameters

Dose Group (mg/kg/day)

0

80

240

720

 Pairs started (N)

12

12

12

12

 Females showing evidence of copulation (N)

10***

11***

11***

12

 Females achieving pregnancy (N)

8

8

10

10*

 Females with abortion (N)

0

1

0

1

 Conceiving days (duration of mating) 1 – 5 (N)

8

9

11

11

 Conceiving days (duration of mating) 6 – 10 (N)

1

1

0

1

 Pregnancy ≤ 21 days (N)

0

1

0

0

 Pregnancy = 22 days (N)

4

2

4

5

 Pregnancy ≥ 23 days (N)

3

4

5

4

 Females with live pups born (N)

7

7

9

9

 Females with live pups at day 4 after parturition (N)

7

6

9

9

 Corpora lutea/pregnant females (mean)

14.13

13.25

16.20

15.00

 Implantations/pregnant females (mean)

11.88

10.38

14.00

11.90

 Live pups/mother at birth (mean)

10.00

10.86

13.11

10.67

 Live pups/mother at day 4 after parturition (mean)

10.00

10.57

13.11

10.67

 Sex ratio (M/F) at birth (mean)

5.50/4.88

5.71/5.14

7.11/6.00

5.00/5.89

 Sex ratio (M/F) at day 4 after parturition (mean)

5.25/4.75

5.71/4.86

7.11/6.00

5.00/5.67

 Litter weight at birth (mean)

64.66

65.97

83.16

66.10

 Litter weight at day 4 after parturition (mean)

114.67

120.08

135.78

105.52

 Pup weight at birth (mean)

6.24

6.35

6.36

6.22

 Pup weight at day 4 after parturition (mean)

10.31

10.33

10.63

10.50

ABNORMAL PUPS

 

 Mothers with 0 (N)

7**

6**

9

9**

 Mothers with 1 (N)

0

0

0

0

 Mothers with ≥ 2 (N)

0

0

0

0

Note: (N) –number of animals

*   - females No. 163 died during pregnancy

           ** - stillborn pups were not included

           *** - sperms in vaginal smear in females were not recorded during mating period 

 

 

Table No. 2: Summary of effects on Fertility

Fertility parameters

Calculated parameters

Dose Group (mg/kg/day)

0

80

240

720

 Male mating index

75.00

91.67

91.67

100.00

 Female mating index

75.00

91.67

91.67

100.00

 Males fertility index

66.67

66.67

83.33

83.33

 Females fertility index

88.89

72.72

90.91

83.33

 Gestation index

87.50

87.50

90.00

90.00

 Survival index

100.00

97.37

100.00

100.00

 LOSS OF OFFSPRING

 

 Pre-implantation (corpora lutea minus implants)

 

 Pregnant females with 0 – 5 (N)

8

6

9

7

 Pregnant females with 6 - 10 (N)

0

2

1

3

 Pregnant females with 11 - 15 (N)

0

0

0

0

 Pregnant females with ≥ 16 (N)

0

0

0

0

 Post-implantation (implants minus live births)

 

 Pregnant females with 0 (N)

4

4

3

2

 Pregnant females with 1 (N)

3

2

3

3

 Pregnant females with 2 (N)

0

1

1

2

 Pregnant females with ≥ 3 (N)

1

1

2

3

 Post-natal (live births minus alive at post-natal day 4)

 

 Pregnant females with 0 (N)

7

5

9

9

 Pregnant females with 1 (N)

0

2

0

0

 Pregnant females with 2 (N)

0

0

0

0

 Pregnant females with ≥ 3 (N)

0

0

0

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Histopathological examination of reproductive system of parental males showed changes of microscopical structure of prostate gland: namely focal mononuclear infiltration and/or oedema of interstitium in all treated groups and control group. In epididymis sporadic changes were recorded: focal mononuclear infiltration and germ cells in lumen of tubules in all groups of males.But frequencies of mentioned effects were not toxicologically relevant according to the Guidance Document for Histologic Evaluation of Endocrine and Reproductive Tests in Rodents, ENV/JM/MONO(2009)11.

 

Biometry of organs showed insignificant changes relative and absolute weight of testes, epididymis, prostate gland and pituitary gland in all trated groups.

 

The test substance had no effect on sperm quality of treated males.

Examination of reproductive system of parental females showed changes of uterus in females from all groups. Lobular hyperplasia in mammary gland was detected in all groups. These changes related with previous gravidity or proceeded oestrous cycle.Biometry of organs also proved insignificant decrease of absolute and relative weight of ovary and uterus in treated females. Insignificantly increased relative and absolute weight of pituitary gland was recorded in all treated groups.

Reproduction parameters– females with abortion was recorded at the middle and highest dose level. Increased number of corpora lutea was recorded at the middle and highest dose level.Duration of mating and pregnancy of treated groups were similar to the control group. Pre-implantation losses were slightly increased at the middle and highest dose level. Post-implantation losses were similar. Number of pups per litter was slightly increased at the middle dose level. The number of females bearing live pupsand females with live pups at day 4 after parturition was increased at the middle and highest dose level. The mating index was increased in all treated groups. The fertility index was slightly increased at the middle and highest dose level.Gestation and survival index of treated groups was well-balanced with the control group. These changes are regarded as spontaneous and biologically insignificant.

 

Observation of pups– total number of pups was increased at the middle and highest dose level. The mean weight of litters was increased at the middle dose level. No significant differences in development of all pups were observed. During pathological examination no affected pups were recorded in all groups.

Applicant's summary and conclusion

Conclusions:
The value of NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION and for DEVELOPMENT OF PUPS was established as higher than 720 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 12 Wistar rats (12 males, 12 Females) were exposed to the test substance by oral gavage at dose levels of: 0, 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 considered not releated 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.

Histopathological examination of the reproductive system of parental males showed changes of microscopical structure of prostate gland: namely focal mononuclear infiltration and/or oedema of interstitium in all treated groups and control group. In the epididymis sporadic changes were recorded: focal mononuclear infiltration and germ cells in lumen of tubules in all groups of males. But frequencies of mentioned effects were not toxicologically relevant according to the Guidance Document for Histologic Evaluation of Endocrine and Reproductive Tests in Rodents, ENV/JM/MONO(2009)11.

Biometry of organs showed insignificant changes relative and absolute weight of testes, epididymis, prostate gland and pituitary gland in all trated groups.

The test substance had no effect on sperm quality of treated males.

Examination of the reproductive system of parental females showed changes of uterus in females from all groups. Lobular hyperplasia in mammary gland was detected in all groups. These changes related with previous gravidity or proceeded oestrous cycle. Biometry of organs also proved insignificant decrease of absolute and relative weight of ovary and uterus in treated females. Insignificantly increased relative and absolute weight of pituitary gland was recorded in all treated groups.

Abortion was recorded at the mid- (1 female) and highest (1 female) dose levels. Increased number of corpora lutea was recorded at the middle and highest dose level. Duration of mating and pregnancy of treated groups were similar to the control group. Pre-implantation losses were slightly increased at the middle and highest dose level. Post-implantation losses were similar. The number of pups per litter was slightly increased at the middle dose level. The number of females bearing live pups and females with live pups at day 4 after parturition was increased at the middle and highest dose level. The mating index was increased in all treated groups. The fertility index was slightly increased at the middle and highest dose level. Gestation and survival index of treated groups was well-balanced with the control group. These changes are regarded as spontaneous and biologically insignificant.

The total number of pups was increased at the middle and highest dose level. The mean weight of litters was increased at the middle dose level. No significant differences in development of all pups were observed. During pathological examination no affected pups were recorded in any group.

Administration of the test substance elicited no adverse effect of parameters reproduction parameters - course of mating, pregnancy and lactation, spermiogenesis, number of females bearing live pups, implantations, corpora lutea, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, mating, fertility, gestation and survival index and loss of offspring.

On this basis, the NOAEL (No Observed Adverse Effect Level) for Reproduction and for development of pups was established as higher than 720 mg/kg body weight/day.