3-[[3-(dimethylamino)propyl]amino]propiononitrile

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The reproductive toxicity of Aradur 1019 was assessed using a method that pre-dates modern published methods such as the OECD guidelines

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 1980 - June 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

The albino rats used in the present study were Sprague Dawleyderived (Tif: RAIf (SPF) and obtained from a closed breeding colony (CIBA-GEIGY, WST).

On arrival the animals were about 6 weeks old and their initial body weight was 140-160 g.

The rats were kept in Macroion cages equipped with a wire mesh top and water bottles, saw dust (granulated forra) serving as bedding material. The cages were placed in movable racks kept in air-conditioned room at a temperature of 21 C + 1 C and humidity of 60% + 5%; the room was illuminated for 10 hours daily (7 a.m. - 5 p.m.).

A Standard cube diet (Nafag No. 890) and tap water were available at all times throughout the experiment.

After a period of acclimatisation of 6 days, the animals were assigned by randomisation to experimental groups and controls and identified by colour code. Throughout the experiment the males were kept one per cage. The females were kept 5 per cage prior to mating, thereafter one per cage.

Each cage was identified by a coloured label according to the group and recording test number and on which the code number of test material, the dose, and data on treatment were recorded

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The F0 males received the test material at least 60 days prior to mating, through the 12 days mating period, and treatment was continued up to weaning of the young. The F0 females were treated from at least 14 days prior to mating, through the mating period, until day 2 0 of pregnancy (Group A) or up to weaning of the young (Group B). The F1 rats remained untreated.

Organisation:
Group mg/kg/day Animal Number wMales Females
Vehicle Control 0 1 – 15 1 – 30
Low dose 25 16 – 30 31 – 60
Intermediate dose 75 21 – 45 61 – 90
High dose 1 50 46 – 60 91 – 120

The test material was dissolved in tap water and administered orally by intubation-at a rate of 1 ml/100 g of body wéight. The dcsage volume remained constant for each female from day 15 of pregnancy until day 20 of pregnancy (Group A) or the day of delivery (Group B) and was then adjusted to the daily body weight. During the periods of treatment, general bodily condition, weight gain, food consumption (not determined throughout the mating periods) and symptoms were checked.

Details on mating procedure:

During the 12 days mating period females were mated overnight with males from the same group in the ratio of 1 male : 2 females (for mating Schedule cf. Appendices 1 and 5). The day on which spermatozoa were found in the vaginal smear or a vaginal plug, was designated as day 0 of pregnancy. The mated emales were transferred to individual cages and the day of either sacrifice (Group A) or delivery (Group B) was determined. Those females which were not mated, were sacrificed about 12 days after the termination of the mating period.

Duration of treatment / exposure:

Dams were treated until day 20 post coitum

Frequency of treatment:

Daily ad libitum

Details on study schedule:

The pups were separated from their mothers after weaning on day 28 after birth and males and females kept separately in groups of between 3 to 9 animals per cage. Part of the young rats were selected at random for mating in a ratio of one male from one litter to two females from other litters to breed the F2 generation (12 days mating period). The remaining young rats were autopsied about 45-50 days after birth. The group B females without litters were autopsied about 5 days after the calculated term. The uterine horns were placed into a solution of ammonium sulphide to detect the possible occurrence of abortion sites or resorption sites.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

75 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

No. of animals per sex per dose:

15 males and 30 females

Control animals:

yes, concurrent vehicle

Details on study design:

The F0 males received the test material at least 60 days prior to mating, through the 12 days mating period, and treatment was continued up to weaning of the young. The F0 females were treated from at least 14 days prior to mating, through the mating period, until day 2 0 of pregnancy (Group A) or up to weaning of the young (Group B).

Parental animals: Observations and examinations:

Treated females:
The approximate duration of pregnancy was determined and observations on possible impairment of parturition were recorded. As far as possible, the number of stillbirths was noted. The pups surviving were counted, sexed, and tattooed individually with Indian ink; at the onset of hair growth identification was made by colour code.

Control Females:
Dams were treated until day 20 post coitum and killed by asphyxiation with C02 (dry ice) on day 21 p.c. and foetuses were removed by caesarean section. They were then weighed individually and submitted to the following procedures:
(1) Examination of the viscera according to the slicing technique of Wilson*. One third of the live foetuses (per litter) were fixed in a mixture of ethyl alcohol and formol to which acetic acid had been added.
(2) Skeletal assessment in two thirds of the foetuses (per litter) following clearing in potassium hydroxide and staining with alizarine red S according to the technique of Dawson**.

Uterine horns showing no evident implantation sites were placed in a solution of ammonium sulphide to visualize possible haemmorhagic alterations in such sites***.

* WILSON, J.G. in Teratology, Principles and Techniques: J.G. Wilson and J. Warkany, eds., The University of Chicago Press, Chicago and London, 1965, pp. 265-277
** DAWSON, A.B., Stain Tech. 1 (1926), 123-124
*** SALEWSKI, E., Arch.exp.Path.Pharmak. 247 (1964), 367-368

Litter observations:

The pups were weighed individually on days 4, 7, 14, 21, 28 and 35 post partum. Developmental and behavioural abnormalities as well as spontaneous deaths were recorded. The main developmental parameters checked were as follows: pinna unfolding (day 4 post partum), incisor eruption (day 8 p.p.), onset of coat development (day 5 p.p.), eye opening (day 14 p.p.), descensus of testes (day 25 p.p.), opening of vagina (day 30 p.p.) .

The following behavioural tests were carried out:
- days 14 post partum:
Righting reflex (placing of the head horizontally after lifting by the root of the tail).
- days 21-23 post partum:
Photophobotaxis (the animal's ability to prefer dark tubes in doublé Y-tube system); 2 trials per animal; animal kept in the dark for at least 10 minutes.
- days 29-32 post partum:
a) cliff avoidance
b) palmar grasp ability
c) negative geotaxis (slope 45°)
d) exploratory locomotion pattern (ELP) in a cylindrical cage (21 cm diameter, wire mesh wall)
- days 35-40 post partum:
Direct pupillary reflex (ophthalmoscope; animals kept in the dark for at least 10 minutes).
- days 40-45 post partum:
Hearing ability by startle response (10*000 Hz, 100 msec, 80 Db.)f 5 trials per animal, each tone delivered 5 times at approximately 2 sec. intervals; the absence of Preyer*s reflex in 3 out of the 5 trials indicates missing hearing ability.

ELP was assessed by observing the behaviour of young rats within the cylinder for 30 seconds:
- Reposing (0 points)
- searching and moving over the floor of the cylinder (1 point)
- standing up at the wire mesh wall (2 points)
- climbing up the wire mesh wall (3 points)

Furthermore an activity index was calculated: total number of points

"activity index" = total number of points / total number of pups examined

The pups were separated from their mothers after weaning on day 28 after birth and males and females kept separately in groups of between 3 to 9 animals per cage. Part of the young rats were selected at random for mating in a ratio of one male from one litter to two females from other litters to breed the F2 generation (12 days mating period). The remaining young rats were autopsied about 45-50 days after birth. The group B females without litters were autopsied about 5 days after the calculated term. The uterine horns were placed into a solution of ammonium sulphide to detect the possible occurrence of abortion sites or resorption sites.

Postmortem examinations (parental animals):

The females which gave birth to a litter were autopsied up from day 28 post partum of the young by asphyxiation with co2. All the females which died perinatally were carefully autopsied and, as far as possible, the contents of the uterus were examined and/or the number of implantation sites determined.

The males were killed with CO2 shortly after weaning of the young. Testes of some males, in particular of those which failed to inseminate either female, were weighed and preserved in Bouin's solution for eventual histological examination.

Postmortem examinations (offspring):

During the period of gestation of the (untreated) F^ mothers their general condition and body weight gain as well as food consumption were checked.

Dams were killed and foetuses removed by caesarean section on day 21 of pregnancy.

The examinations of dams and progeny were carried out according to group A females; however, examination of viscera and skeletal assessment was not performed.

The parent F^ males were autopsied one week after the termination of the mating period.

Statistics:

The mean values and Standard deviations were calculated for corpora lutea, implantations, foetal body weight and pup weight.
Whenever feasible, further statistical evaluation of data was performed

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body-weight gain was slightly depressed in the three experimental groups (treated) for parental males. The average food intake was comparable for all groups.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of toxicity at the maximum tested dose

Remarks on result:

not determinable due to absence of adverse toxic effects

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body-weight gain of the pups (Fig. 3) was slightly depressed in the 75 mg/kg and 150 mg/kg dose groups.
However this is not seen as statistically relevant as there are many factors that can influence body weight.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects see at top dose

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

no

Conclusions:

TK 12 271 when administered continuously by gavage at doses of 0, 25, 75 and 150 mg/kg to male and female rats (F0) through premating, mating, gestation and lactation periods,had no adverse affects on reproductive performance of either the parent (F0) animals or the F1 generation under the conditions of the present experiment.

The postnatal physiological growth of the F1 pups was slightly retarded in the intermediate and high dose groups as indicated by a reduction in body-weight gain. Otherwise, these pups were normally developed.

Executive summary:

TK 12 271 was studied for its effects on fertility and general reproductive performance in albino rats.

The test material was dissolved in tap water and administered orally by intubation at the rate of 1 ml/100 g of body weight.

The doses were 0, 25, 75 and 150 mg/kg/day.

Each group, experimental and control, consisted of 15 males and 30 females (F0 rats).

The males were treated for at least 60 days and females for at least 14 days prior to mating; the treatment was continued through the mating period, gestation and lactation periods.

About half of the females was killed shortly before term on day 21 of pregnancy, the other half of the females was allowed to deliver and raise their offspring.

Part of the pups was selected for breeding the F2 generation; the remaining young were killed between days 45 - 50 post partum.

The F0 males of the three experimental groups reacted to the treatment by a slight reduction in body-weight gain.

The F0 females remained unaffected by the treatment. A slight increase in food consumption was recorded for the females of the experimental groups and associated with great litter size in part of them.

No experimental significance was attached to a slightly reduced fertility rate recorded for the low-dose group of F0 rats; the indices were comparable for the control group, the intermediate and high dose groups.

The overall implantation rates and pre-implantation loss of embryos were comparable for all groups.

The reproduction data recorded for the dams killed on day 21 of pregnancy were not altered.

By applying the slicing technique on about one third per litter of the live foetuses, two instances of pathological change of the brain were observed among the foetuses of the 150 mg/kg dose group. The histopathological examination of the slices revealed cerebral meningeal vascular hamartia (cf. addendum). Visceral abnormalities of a similar type were occasionally found in untreated controls of the breed of rats used for this study at the incidence of 0.08 per cent. The experimental significance of the present finding remains doubtful.

The litter data recorded for the dams raising their offspring were similar in the control group and the low-dose group. In the intermediate and high dose groups the average body-weight gain of the pups was slightly depressed.

The parameters of general behaviour of the pups, as well as "activity indices", pupillary reflex and hearing ability were normal in all the animals of either group.

In the F1 rats selected for breeding the F2 generation, the results concerning fertility, pregnancy and reproduction were comparable for all groups. A significant shift of the sex ratio in favour of the female foetuses was recorded for the high-dose group. This finding, however, is considered to be of a doubtful experimental significance, since the F1 parents remained untreated for at least 70 days before being mated and, otherwise, there was no change of sex ratio in the F1 foetuses and/or pups of the 150 mg/kg dose group.

One malformation (omphalocele) was found among the F2 foetuses derived from the intermediate dose group and considered to be of a spontaneous origin.

To summarize, TK 12 271 when administered continuously by gavage at doses of 0, 25, 75 and 150 mg/kg to male and female rats (F0) through premating, mating, gestation and lactation periods,had no adverse affects on reproductive performance of either the parent (F0) animals or the F1 generation under the conditions of the present experiment.

The postnatal physiological growth of the F1 pups was slightly retarded in the intermediate and high dose groups as indicated by a reduction in body-weight gain. Otherwise, these pups were normally developed.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

chronic

Species:

rat