Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1'-[(3-{bis[3-(dimethylamino)propyl]amino}propyl)imino]dipropan-2-ol
EC Number:
816-324-8
Cas Number:
2044770-20-7
Molecular formula:
C19H44N4O2
IUPAC Name:
1,1'-[(3-{bis[3-(dimethylamino)propyl]amino}propyl)imino]dipropan-2-ol
Test material form:
liquid
Details on test material:
Storage conditions: room temperature

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Rat / CD / Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
For this study CD rats bred by Charles River Laboratories Germany GmbH were used. The healthy nulliparous adult animals were randomised and assigned to the treatment groups and cages. The body weight range did not exceed 20% of the mean weight for each sex at the time of selection. The animals were held for 21 days for adaptation.
Health checks were performed on the day of delivery and at first administration.
Test species / Strain / Stock Rat / CD / Crl:CD(SD)

Breeder Charles River Laboratories, Research Models and Services, Germany GmbH
Sandhofer Weg 7
97633 Sulzfeld
Germany

Body weight
(at 1st administration, TD15)
Males:
Females:
381.1 g - 425.3 g
204.4 g - 252.8 g
Age
(at 1st administration, TD15)
Males:
Females:
75 days
75 days

Selection of species The rat is a commonly used rodent species for such studies.

Number and sex of animals Pre-exposure period (TD 1 to TD 14):
60 female animals
A sufficient number of animals in order to grant at least 40 females with a normal oestrus cycle for the main study.
Main study (start on TD 15):
80 animals (40 males and 40 females)
A sufficient number in order to grant at least 8 pregnant females per group for evaluation of the F0 generation.

Adaptation period 21 days

Diet
A certified commercial diet (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany; see Appendix 3 'Composition of the Diet') served as food. This food was offered ad libitum. Food residue was removed and weighed.
Samples of the food are analysed for contaminants based on EPA/USA by LUFA-ITL at least twice a year (see Appendix 3 'Limitation for Contaminants in the Diet'). Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.

Drinking water
Tap water was offered ad libitum.
Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 2001 ] (see Appendix 3 'Limitation for Contaminants in the Drinking Water').
In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the "Deutsche Trinkwasserverordnung 2001, Anlage 1" [German Regulations on Drinking Water 2001, Addendum 1].
No contaminants above the limitations were noted.

Housing
With the exception of the mating period (see section 3.1.10 'Mating procedure'), the males and females (F0-Generation) were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. The room temperature was 22°C ± 3°C (maximum range) and the relative humidity was 55% ± 10% (maximum range).
Deviations from the maximum range caused, for example, during cleaning procedures are dealt with in SOPs. No values exceeding the maximum range were noted during the course of the study.
The animals were placed in the animal room as follows:
Male animals: On one side of the room with an empty row between groups.
Female animals: On the other side of the room with an empty row between groups.
Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week.
Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL (see Appendix 3 'Limitation for Contaminants in the Bedding Material'). Certificates of analysis of the bedding material are included in the raw data.
The rooms were alternately lit (about 150 lux at approximately 1.5 m room height) and darkened in a 12 hours dark/12 hours light cycle. The ventilation rate of the animal room was between fifteen to twenty air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Route of administration: Oral, by gavage.
Frequency of administration: Twice daily at 8.00 am and 3.00 pm
Administration volume: 5 mL/kg b.w./day
The doses were split into 2 administrations of 2.5 mL/kg b.w. each.
Details on mating procedure:
Sexually mature male and female rats were paired monogamously: 1 male and 1 female animal were placed in one cage during the dark period (see table 1 4 'Pairing List'). The female was placed with the same male until pregnancy had occurred. Each morning the females were examined for the presence of sperm or a vaginal plug. The day of conception (day 0 of gestation or GD0) was considered to be the day on which sperm was found.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were analysed using a validated method. The following parameters were determined during validation:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specificty
- Stability
The analysis of the formulation samples is the responsibility of the analytical laboratory and is covered by a GLP program.
The analytical laboratory performed the analysis according its Study Plan and SOPs and in compliance with GLP regulations.
Duration of treatment / exposure:
The study animals were treated during the following periods:

Males:
2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30 until test day 43#1 at maximum) and during the post-mating period until test day 43 (one day before sacrifice on test day 44).

Females:
2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30 until test day 43#1 at maximum) and during the lactation period until test day 64 to 77 (corresponding to lactation day 13#). The last dosing was always one day before sacrifice.

Frequency of treatment:
twice per day in equal doses
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes
Details on study design:
The animals were allocated to the test groups at the last day of the pre-dosing period on test day 14 by using a Provantis -generated randomization based on the body weights of the animals. Only those female animals were used for randomization that passed the estrus cycle test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No changes in behaviour, the external appearance or the appearance of the faeces were noted for the male and female animals of the control group or the dose groups (20, 60 or 200 mg/kg b.w./day).
Mortality:
no mortality observed
Description (incidence):
No prematurely deceased male or female animals were noted in the control group and in the dose groups (20, 60 or 200 mg/kg b.w./day).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related changes in body weight were noted for the male rats between the control group and the dose groups (20, 60 or 200 mg/kg b.w./day).
Accordingly, also no test item-related changes in the body weight gain were noted between the control group and the dose groups (20, 60 or 200 mg/kg b.w./day).
Also for the females, no test item-related influence was noted on the body weight of the treatment groups (20, 60 or 200 mg/kg b.w./day) in comparison to the control groups during the pre-mating period, the gestation period or the lactation period.
Accordingly, no influence on the body weight gain of the female animals was noted after dosing of 20, 60 or 200 mg/kg b.w./day during the pre-mating, gestation or lactation period in comparison to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
During the pre-mating period (TD15 to TD29) no test item-related difference in food consumption was noted between the rats of the control group and the rats of the dose groups (20, 60 or 200 mg/kg b.w./day).
No test item-related differences in food consumption were noted between the female rats of the control group and the female rats of the dose groups (20 or 60 mg/kg b.w./day) during the pre-mating, the gestation and the lactation period.
In the high dose group (200 mg/kg b.w./day), a statistically significantly decreased food consumption in the week between TD22 and TD29 (8.6% below the value of the control group, p ≤ 0.05).
No food intake of female animals was recorded during the mating period as both sexes were housed together.

Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual appraisal revealed no test item-related changes in the consumption of drinking water for the male and female rats treated with 20, 60 or 200 mg/kg b.w./day.
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Males
No test item-related changes were noted for the T4 levels of the male animals of the dose groups (20, 60 or 200 mg/kg b.w./day).
The T4 concentrations in the male animals of the high dose group were slightly decreased (9.8% below the value of the control group in the high dose group, statistically not significant.

Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No changes in behaviour, the external appearance or the appearance of the faeces were noted for the male and female animals of the control group or the dose groups (20, 60 or 200 mg/kg b.w./day).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Males and females
The histopathological examination was conducted on sections of the testes and epididymides of the male animals of the control group and high dose group (200 mg/kg b.w./day) and on ovary sections of the control and the high dose females.
The microscopic examination of the sections revealed no test item-related pathologic changes for the male and female animals of the high dose group.
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
After the allocation of the animals to the test groups and the start of treatment on test day 15, the estrus cycles were further monitored during the pre-mating and mating period until one day before the first sign of mating was noted.
No test item-related differences were noted in the mean number of oestrus cycles per dam during the pre-mating and mating period between the female animals of the control group and the female animals of the dose groups (20, 60 or 200 mg/kg b.w./day).
At 60 mg/kg b.w./day, a statistically significantly decrease was noted for the number of estrus cycles between TD15 and TD28 and between TD15 and one day before mating (1.9 estrus cycles in the intermediate dose group compared to 2.7 estrus cycles in the control group, p ≤ 0.05). However, as this was due to two animals of the intermediate dose group that had only one estrus cycle each, the decreased number of estrus cycles was considered to be spontaneous and not test item-related.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No test item-related influence on the fertility index of the female rats was noted for any of the dose groups (20, 60 or 200 mg/kg b.w./day).
All female rats were successfully mated (confirmed by sperm detection). However, two female animals of the control group and two female animals of the high dose group did not become pregnant, leading to a fertility index of 80% for the female animals of the control group and the high dose group.
The occurrence of two non-pregnant female at the high dose level was considered to be spontaneous, as also two non-pregnant females were noted at the control group.
No influence on the gestation index was noted for the female rats dosed with 20, 60 or 200 mg/kg b.w./day.
All pregnant animals of the control group and the dose groups delivered live pups

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
food consumption and compound intake

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Description (incidence and severity):
No difference between the control group and the dose groups (20, 60 or 200 mg/kg b.w./day) was noted for the viability index between lactation days 1 and 4. The viability index from LD0/1 to LD4 ranged between 97.5% (control and high dose group) and 99.4% (low dose group).

No test item-related influence on the viability index was noted for the low dose group (20 mg/kg b.w./day).
Decreased viability indices (statistically significant for the intermediate dose group at p < 0.01) were noted for the intermediate and high dose groups (60 or 200 mg/kg b.w./day):
In the intermediate dose group (60 mg/kg b.w./day), 6 of 10 dams were noted with dead pups: 5 dams were noted with 3 to up to 8 dead pups. In the high dose group (200 mg/kg b.w./day), 4 of 8 dams were noted with dead pups and of these one dam had three dead pups and one dam had 7 dead pups. In the control group, dead pups were noted for 3 of 8 dams (two dams with one dead pup each and one dam with three dead pups).
As no further influences were noted on the animals of the low dose group the reduced viability index between LD5 and LD 13 was considered to be spontaneous and not test item-related.
The high number of pups that deceased between LD5 and LD13 in the intermediate and high dose group was considered to be test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the dams of the low dose group (20 mg/kg b.w./day) on LD1, LD4 and LD13.
Reductions in pup body weight in comparison to the control group were noted at the intermediate and high dose level (60 or 200 mg/kg b.w./day) on lactation day 13 (at maximum 13.4% below the value of the control group; female pups of the high dose group). These dose-dependently lower pup body weights were considered to be test item-related.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes were noted for the T4 levels of the male and female pups of the low dose group (20 or 60 mg/kg b.w./day).
At 60 mg/kg b.w./day, T4 determination revealed statistically significantly decreased T4 levels for the female pups and the male and female pups combined (at maximum 16.0% below the value of the control group for the female pups, statistically significant at p ≤ 0.05, see figure 5 on the following page). However, these decreased levels were mainly due to only one dam (no. 53) and therefore were considered to be not test item-related.
At the high dose level (200 mg/kg b.w./day), distinctly and statistically significantly decreased T4 levels were noted for the male pups, the female pups and the male and female pups combined (at maximum 22.4% below the value of the control group for the male pups, p ≤ 0.01).
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the absolute and the relative ano-genital distance of the male and the female pups of all treatment groups (20, 60 or 200 mg/kg b.w./day).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (20, 60 or 200 mg/kg b.w./day).
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 20, 60 or 200 mg/kg b.w./day after terminal sacrifice on lactation day 13 or for the pups that died during the lactation period.
Histopathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test item-related differences were noted for the mean number of implantation sites, the mean number of pups born (alive and dead) and the mean number of live born pups between the control group and the dose groups (20, 60 or 200 mg/kg b.w./day).
Accordingly, the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the dose groups.

No test item-related influence on the male to female ratio was noted for all treatment groups (20, 60 or 200 mg/kg b.w./day).
The male to female ratios on LD4 ranged from 0.97 (intermediate dose group) to 1.50 (high dose group).

No test item-related differences for the litter weight was noted between the control group and the low dose group (20 mg/kg b.w./day).
In the intermediate and high dose group (60 or 200 mg/kg b.w./day), reduced values were noted for the litter weights per dam on LD13.
The low litter weight of the low dose group was due to the spontaneously reduced number of live pups on LD13 therefore, was considered to be not test item-related.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Conclusion
The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 421. The test item was administered orally to rats at dose levels of 20, 60 or 200 mg/kg b.w./day.

General toxicity:
Parental male and female animals:
All animals survived until study termination.
No test item-related differences between the control group and the dose groups were noted for the body weight or the body weight gain.
The female animals dosed with 200 mg/kg b.w./day were noted with a reduced food consumption.
No changes in the external appearance, the behaviour or the faeces were noted for the male and female animals of the dose groups.
No pathologic findings were observed during macroscopic post mortem examination the male and female animals.
Reduced thyroid weights were noted for the female high dose animals (200 mg/kg b.w./day).
Determination of the T4 concentration revealed a reduced T4 level in the male animals of the high dose group (200 mg/kg b.w./day).

Reproductive toxicity:
Parental females:
No influence was noted on the fertility of the male and female animals in any of the dose groups.
Pups:
No influence was noted of the pre-natal development (number of implantations, number of resorptions or number of live born pups) for the dose groups.
In the intermediate and high dose group (60 or 200 mg/kg b.w./day), reductions were noted for the pup body weights on LD13, for the litter weights on LD13, for the viability index between LD5 and LD13 and consequently for the number of live pups on LD13.
At 200 mg/kg b.w./day, slightly decreased T4 levels were noted for the male and female pups.

Executive summary:

General Toxicity: NOEAL 60 mg/kg b. w./day p.o.

Reproductive toxicity:

adverse effects  on the reproductive parameters of the parental females: above 200 mg/kg b. w./day p.o.

adverse effects on pre- and postnatal development 20: mg/kg b. w./day p.o.

Categories Display