Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
In the study the effect of apiperidine was investigated on pregnancy and on in utero development of the rat. The test item was administered by whole-body inhalation exposure for six hours daily from Day 6 to Day 15 of pregnancy inclusive. On Day 20, animals were sacrificed and subjected to post mortem examination, litter values were determined and foetuses were subsequently examined for visceral or skeletal anomalies.
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
other: vapour
Details on test material:
- Name of test material (as cited in study report): piperidine
- Analytical purity: 99.3%

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River, Portage, Michigan, U.S.A.
- Age at study initiation: 8 - 10 weeks old, sexually mature
- Weight at study initiation: Batch A (weight range 164 - 205 g); Batch B (weight range 186 - 221 g)
- Housing: five per cage in suspended polypropylene cages
- Diet: Labsure Laboratory Animal Diet No. 1, ad libitum
- Water: tap water, ad libitum

- Temperature (°C): 18.5 - 21.5
- Humidity (%): 33 - 58
- Photoperiod (hrs dark / hrs light): 12 /12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
- The test substance was metered onto the sintered glass disc from a plastic disposable syringe mounted on an infusion pump (Precidor ® type 5003).
- 0il-free dried compressed air was passed through the frit, the test substance evaporated and the vapour laden air was passed into the inlet
of the exposure chamber.

Exposure chamber
- The exposure chambers were constructed from stainless steel and glass and were of approximately 0.5 m³ internal volume.
- Oil-free dried compressed air at 100 liters per minute entered the base of the vapour generator. The vapour-laden air passed through the glass elutriation column and entered the chamber inlet duct.
- The chambers were maintained at 10 mm H2O below ambient pressure by an extract fan connected to the exhaust manifold.
- Each chamber was fitted with parts for withdrawal of chamber air samples for analytical purposes. Routinely a part mid-centre of the chamber side wall was used.
- During exposure the rats were held in individual compartments of stainless steel wire mesh cages.

The animals were put into the exposure cages and placed within the chambers appropriate to each group. The chamber doors were sealed and the air turned on.
The syringes were filled with the test substance in a fume cupboard. The syringes were then connected to the vapour generators with a PTFE tube and mounted onto the infusion pumps. The mechanism was advanced until the test substance just reached the glass frit. The initial volume in each syringe was recorded.
Exposure commenced when the infusion pumps were switched on and working at the setting appropriate to generate the desired chamber-air concentrations.
After six hours the infusion pumps were switched off and the volume of test substance remaining in the syringes was recorded.
The atmosphere within the chambers was allowed to clear before the rats were unladed and returned to their holding cages. Air control rats were sham exposed, under similar conditions to air only.

Chamber temperature and relative humidity
Chamber air temperature and relative humidity were monitored continuously with a wet and dry bulb hygrometer and recorded at hourly intervals throughout each exposure.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The concentration of the test item present in the exposure chambers was determined at least 3 times during each exposure.
Samples of test atmosphere were withdrawn at 2 liters per minute through a fritted glass bubbler containing 2,2,4-trimethylpentane as the trapping agent. The actual volumes withdrawn were measured using a wet-type gas meter (Model DM3B, Alexander Wright and Co. (Westminster) Ltd.).
The sample solutions were made up to volume (25 ml for the Preliminary study or 20 ml for the Main study) in a volumetric flask with further 2,2, 4-trimethylpentane.
Analysis was by flame ionisation gas chromatography using external standards.
Details on mating procedure:
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6 to day 15 of pregnancy
Frequency of treatment:
daily, 6 hours per day
Duration of test:
up to day 20 of pregnancy
No. of animals per sex per dose:
15 of the first batch (A) and 10 of the second batch (B).

Control animals:
yes, sham-exposed
Details on study design:
- Sex: female
- Dose selection rationale: In a preliminary study the signs of evidence of respiratory distress at 160 ppm - during and following exposure -precluded this level from consideration in the present Main study.


Maternal examinations:
- Time schedule: daily, for obvious changes or signs of reaction to treatment.


- Time schedule for examinations: All animals were weighed initially (=Day 2 of pregnancy for Batch A, =Day 1 of pregnancy for Batch B) and on Day 2 (for Batch B), 3, 6, 8, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, from weighday to weighday. As animals were gang-housed, food intake was measured on a cage basis.

- Sacrifice on gestation day 20
- Organs examined: not specified

Group mean values, except for food consumption, were calculated using only the values of animals with live young at termination.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
(a) number of corpora lutea
(b) number and distribution of live young
(c) number and distribution of embryofoetal deaths
(d) individual foetal weight from which the litter weight was calculated
(e) foetal abnormalities

Embryofoetal deaths were classified as:
Early: only placenta visible at termination.
Late: both placental and embryonic remnants visible at termination.

Uteri or individual uterine horns without visible implantations were immersed in a 10 % solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.

Fetal examinations:
- External examinations: Yes: all per litter (Live young were examined externally and weighed.)
- Soft tissue examinations: Yes
- Skeletal examinations: Yes: half per litter for skeletal anomalies
- Head examinations: Yes: half per litter for visceral anomalies

- Half the foetuses in each litter were preserved in Bouin's solution for subsequent free-hand sectioning to discover visceral abnormalities (Wilson technique); the remainder were fixed in 74 OP industrial methylated spirit for subsequent macroscopic examination, evisceration, clearing and alizarin staining (modified Dawson technique) for skeletal examination.
- Young showing suspected abnormalities were processed by the more appropriate technique for clarification of initial observations.
- All foetuses were sexed by gonadal inspection following preservation.

Structural changes were presented as:
- Malformations: rare and/or probably lethal, e.g. exencephialy, anury.
- Anomalies: minor differences from 'normal' that are detected relatively frequently either by free-hand sectioning, e.g. increased renal pelvic dilatation, or at skeletal examination, e.g. bipartite centrum.
- Variants: alternative structures occurring regularly in the control population are classified as variants. These may be permanent structures, e.g. an extra pair of ribs, or they may be transient stages of development, e.g. unossified sternebra(e).

Litter weight and mean foetal weight were calculated from individual foetal weight.

Statistical analysis
Statistical analyses were routinely performed on litter data. Significance tests were normally two-tailed.
- Litter data
The basic sample unit was the litter, and due to the preponderance of non-normal distributions, non-parametric analyses have generally proved the most consistent.
Mean values of litter sizes, pre- and post implantation loss, litter weight, mean pup weight and the incidence of anomalous offspring were generally analysed by the Kruskal-Wallis test.
Intergroup comparisons were made by the non-parametric equivalent of the 't' test together with the Jonckheere test for an ordered series af treatments. Only the test(s) considered to be appropriate were reported.
Where 75 % of the values for a given variable consist of one value, a Fisher's exact test was used and where appropriate, Mantel's test for trend in proportions was also performed.
Individual litter values:
pre-implantation loss was calculated as a percentage from the formula: (No. of corpora lutea - no. of implantations)/No. of corpora lutea * 100
Post implantation loss was similarly calculated from the formula: (No. of implantations - no. of live young)/No. of implantations * 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- Clinical signs:
Signs of reaction to treatment were observed during daily exposure to piperidine at 20 and 80 ppm.
At 80 ppm, during each exposure animals failed to respond to a knock on chamber door and eyes were shut/half-closed. Licking the inside of the mouth, and pilo-erection were observed. Hunched posture was observed on nearly all occasions. Salivation and rubbing af the chin and paws on the cage, and increased respiration were observed on at most two occasions.
At 20 ppm, observations were confined to no response to knock on chamber door. This was observed on every occasion of exposure. There was one single instance of eyes shut/half-closed and hunched posture.
At 5 ppm there were no signs of reaction during exposure observed.
Following the initiation of treatment at 80 ppm, and outside the periods of exposure, some animals were observed to have areas of red/brown staining of the hair, two were observed with "snuffles" and one showed sneezing and salivation.
No similar reactions were observed at the lower exposure concentrations.
There were no mortalities.
- Food consumption:
Following the initiation of treatment food consumption at 80 ppm was reduced and remained lower throughout treatment. Following the withdrawal of treatment, there was some recovery although intake was still slightly reduced compared to controls at termination.
Food consumption at lower levels was generally comparable with controls throughout.
- Body weights:
Following the initiation of treatment, bodyweight gains at 80 ppm were retarded compared to controls and remained so throughout treatment. On withdrawal of treatment, gains showed rapid recovery and were generally comparable to controls through to termination, although mean bodyweight remained lower than the control value at termination.
Bodyweight gains at 5 and 20 ppm were essentially similar to controls throughout.
- Terminal autopsy:
Occasional findings observed at terminal autopsy were not considered to be related to treatment.

Effect levels (maternal animals)

Dose descriptor:
Effect level:
0.071 mg/L air (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
(a) Litter parameters: There were no significant differences in litter parameters: litter size, post implantation loss, litter and mean foetal weight or sex ratios.
(b) Malformations, anomalies and skeletal variants: The incidence and type of malformations and visceral and skeletal anomalies observed did not appear to show a relationship to treatment.
The incidence of skeletal variants was essentially similar among the groups.

Effect levels (fetuses)

Dose descriptor:
Effect level:
0.28 mg/L air (nominal)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Based on the results of this study the NOAEC for teratogenicity was 0.28 mg/L (80 ppm) since no adverse developmental effects were observed up to the highest concentration tested.
Executive summary:

In a GLP conform, non-Guideline developmental toxicity study, 25 pregnant Crl:CD®(SD) GR VAF/Plus strain rats per dose were treated by whole-body to vapour of the test item (99.3%, a.i.) at concentrations of 5, 20, or 80 ppm (0.017, 0.071, and 0.28 mg/mL, respectively) during gestation days (GD) 6-15 for 6 hours/day (BG Chemie, 1990). 25 pregnant rats remained untreated for control.

The dams were observed daily for clinical signs, weighed on GD 2, 3, 6, and at 2-day intervals until GD 20 and had food consumption measured for intervals between weigh days. The dams were killed on GD 20 and the ovaries and uteri were examined.

No maternal mortalities occurred during the study period.

In the highest dose group clinical signs were piloerection, hunched posture, salivation and increased respiration. Food consumption was reduced throughout the treatment period and remained reduced upon withdrawal of the treatment until study termination. The body weight gains were retarded throughout treatment but recovered on withdrawal of the test substance after GD 15. At termination the maternal body weights of the high dose animals remained lower compared to the control. No treatment-related necropsy findings were observed.

In the mid-dose group hunched posture was observed only in one instance. No treatment-related effects on body weights or food consumption and no treatment-related necropsy findings were observed.

No clinical signs were observed in the lowest dose group of 5 ppm. Additionally, no effects on body weights or food consumption and no treatment-related necropsy findings were observed.

Based on the results of the study the NOEC for maternal toxicity was established by the authors as being 0.017 mg/L (5 ppm).

Due to non-toxic effects observed in the dams at this exposure level the NOAEC for maternal toxicity is estimated to 0.071 mg/L (20 ppm).

There were no significant differences in litter parameters (litter size, foetal deaths, number of implants and of corpora lutea, pre- and post-implantation losses, litter and mean foetal weights and sex ratios) and in incidences of malformations, visceral and skeletal anomalies and skeletal variations.