Tricyclo[3.3.1.13,7]decan-1-aminium, N,N,N-trimethyl-, hydroxide (1:1)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tricyclo[3.3.1.13,7]decan-1-aminium, N,N,N-trimethyl-, hydroxide (1:1)

EC Number:

610-954-5

Cas Number:

53075-09-5

Molecular formula:

C13 H25 N O

IUPAC Name:

Tricyclo[3.3.1.13,7]decan-1-aminium, N,N,N-trimethyl-, hydroxide (1:1)

Details on test material:

- Name of test substance: Adamantyltrimethylammoniumhydroxide 20%
- Batch identification: ADTAOH 20 %
- CAS-No.: 53075-09-5
- Purity: 20.1g/100g
- Homogeneity: Homogeneous
- Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Date of production: 10 Mar 2008
- Physical state/Appearance: Liquid/colorless, clear
- Storage conditions: Room temperature, under N2

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation: The body weight of the pregnant animals on GD 0 varied between 137.6 - 184.6 g
- Housing: individually from GD 0-20 in type M III Makrolon cages supplied by BECKER & CO., Castrop-Rauxel, Germany (floor area about 800 cm²).
- Diet: ad libitum; ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: GD 0 - GD 6 (6 days)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the period of established stability. To prepare the dose formulations, the specific amount of test substance was weighed, topped up with drinking water in a graduated flask and intensely mixed until it was completely dissolved. A magnetic stirrer was also used to keep the preparations homogeneous during treatment of the animals.

VEHICLE
- Concentration in vehicle: 0, 1000, 3000, 10000 mg/100ml for the 0, 100, 300, 1000 mg/kg bw/d group, respectively
- Amount of vehicle: 10 ml/kg bw/d of the test substance preparations in drinking water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance solutions were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations.
Analytical method: Capillary electrophoresis (CE) with indirect UV detection and evaluation by the method of internal standard.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy (GD0)

Duration of treatment / exposure:

GD 6 - GD 19 (from implantation to one day prior to the expected day of parturition)

Frequency of treatment:

once a day

Duration of test:

until sacrifice on GD 20

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
nominal conc.

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality: A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20). Clinical symptoms: A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-20).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.

FOOD CONSUMPTION: Yes
- The average food consumption of the dams in the test groups was determined.

WATER CONSUMPTION: No

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
- Mortality: There were no substance-related or spontaneous mortalities in any of the groups.
- Clinical symptoms: In total 20 high-dose dams (1000 mg/kg bw/d) showed transient salivation after treatment. Salivation occurred from GD 9 onwards and persisted in the respective females for a few minutes immediately after each administration. After cessation of treatment on GD 19, salivation was no longer observed in these rats. This temporary salivation is considered to be treatment-related. It was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. No clinical symptoms were noted in the low- and the mid dose group (100 and 300 mg/kg bw/d).
- Food consumption: The average food consumption of the dams in test groups 1, 2 and 3 (100; 300 and 1000 mg/kg bw/d) was comparable to the control group and did not show a test substance-related impairment. Differences between control rats and test substance-treated animals did not show a relation to dosing and were considered to be without biological relevance.
- Body weight data: The mean body weights and the average body weight gain of the low-, mid- and high-dose rats (100; 300 and 1000 mg/kg bw/d) were comparable to the concurrent control values. The observable, insignificant differences between test groups and control are not biologically relevant.
- Corrected (net) body weight gain: The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) of all test substance-treated groups (100; 300 and 1000 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights remained also unaffected by the treatment.
- Terminal examinations of the dams:
Uterus weight: The mean gravid uterus weights of the animals of all test groups (100; 300 or 1000 mg/kg bw/d) were not influenced by the test substance. Observed differences between test substance-treated groups and the control group were neither statistically significant nor biologically relevant.
Necropsy findings: At necropsy, no test substance-related findings were observed in the dams. In one mid-dose rat, which was not pregnant at terminal sacrifice, a bilateral hydrometra was found. This isolated gross finding is considered to be spontaneous in nature.
Reproduction data: The conception rate reached 72% in test group 3 (1000 mg/kg bw/d), 80% in test groups 1 and 2 (100 and 300 mg/kg bw/d) and 88% in the control group (0 mg/kg bw/d). A sufficient number of litters was available for the purpose of the study as 18 – 22 rats had implantation sites in the uterus. There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Generally, gestational parameters in the various test groups were within the normal range for animals of this strain and age. One control dam had no live fetuses at all but only early resorptions in the uterus. One dam of test group 3 (1000 mg/kg bw/d) was pregnant by stain and had only one very early resorption in the uterus. If this occurs in isolation, it is not an unusual spontaneous finding in the strain of rats used for this study.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
- Sex distribution of the fetuses: The sex distribution of the fetuses in test groups 1-3 (100; 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.
- Weight of the placenta: The mean placental weights were comparable between the dosed groups (100; 300 and 1000 mg/kg bw/d) and the corresponding control. Differences observed in comparison to the control were neither statistically significant nor biologically relevant.
- Weight of the fetuses: The mean fetal body weights in test groups 1, 2 and 3 (100; 300 and 1000 mg/kg bw/d) were not influenced by the test substance and were comparable to the corresponding control values.
- Fetal external malformations: No external malformations were observed.
- Fetal external variations: One sole external variation (i.e. unilateral hindlimb hyperflexion) occurred in one fetus of the high dose group (1000 mg/kg bw/d). Based on the single observation in the high dose group a relation to treatment could not be excluded. However, the minor degree of this structural deviation from the control is not of biological significance. It is not considered indicative of a potential on the part of the test substance to affect normal development and function of the extremities.
- Fetal external unclassified observations: No external unclassified observations were recorded.
- Fetal soft tissue malformations: Only one soft tissue malformation (i.e. unilateral microphthalmia) was recorded in one fetus of test group 2 (300 mg/kg bw/d). No dose-response relationship was observed. Thus, an association of this finding to the treatment is not assumed.
- Fetal soft tissue variations: Three soft tissue variations (dilated cerebral ventricle, unilateral dilation of renal pelvis and ureter) were detected in different test groups including the control, without any dose-response relationship. The incidences of soft tissue variations were comparable to the historical control data.
- Fetal soft tissue unclassified observations: No unclassified soft tissue observations were recorded.
- Fetal skeletal malformations: Two skeletal malformations (i.e. shortened scapula and humerus) were recorded in one fetus of test group 2 (300 mg/kg bw/d). Although these particular findings are not in the historical control data, they were considered to be spontaneous in nature and not related to treatment, because there is no dose-response relationship.
- Fetal skeletal variations: For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing. Based on the rate of affected fetuses per litter, the incidence of skeletal variations was comparable to the historical control data and was not observed in dose dependency. Therefore, these findings were assessed as not treatment-related.
- Fetal skeletal unclassified cartilage observations: Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings were related to the skull, the sternum and the ribs and did not show any relation to dosing; with the exception of one finding: notched cartilage between basisphenoid and basiocciptal. Regarding the total unclassified cartilage observations, the percentages of affected fetuses per litter are within the overall historical control range (mean value 42.4%; range per study: 22.1 – 64.5%) and do not show any relation to dosing. Thus, a toxicological relevance for these findings is not assumed. A dose-dependent increase of the incidences of notched cartilage between basisphenoid and basioccipital was observed regarding the fetal and litter incidence as well as the affected fetuses per litter. Only in the latter incidence a statistical significance was observed in the high-dose group. This unclassified cartilage observation is present in the historical data of the Wistar rat strain used. However, in this particular study an unusually high incidence of this finding, reaching levels above the historical control range, was noted for all test groups including control. The finding is considered to be a minor morphological change which is not presumed to have a detrimental effect in the stability of the skull in general. Thus, although the increased high-dose incidence might be related to the treatment the finding as such is assessed to have no toxicological relevance. As it was the only dose-dependent effect of the test substance in this study, the singularity of this minor finding and the absence of a malformation/variation pattern support the assumption that the test compound is rather unlikely to cause specific adverse effects on skeletal development.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Abstract of all fetal external, soft tissue and skeletal observations and their assessment

No external malformations were recorded. The one soft tissue and the two skeletal malformations in two fetuses of 2 mid-dose litters were considered to be of spontaneous origin and not related to the test substance. The total incidences of these findings are summarized in the following table:

 

Total fetal malformations:

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	21 192	20 171	20 181	17 143
Fetal incidence	N	0 (0.0%)	0 (0.0%)	2 (1.1%)	0 (0.0%)
Litter incidence	N	0 (0.0%)	0 (0.0%)	2 (10%)	0 (0.0%)
Affected fetuses/litter	Mean%	0.0	0.0	1.3	0.0

 

One external (limb hyperflexion), three soft tissue (dilated cerebral ventricle, dilated renal pelvis and ureter) and/or a broad range of skeletal variations occurred in all test groups including the controls. If all the different types of variations are summarized, none of the incidences showed a relation to dosing (see table below), and comparable incidences of total fetal variations can be found in the historical control data. Therefore, the statistical significantly increased incidence of affected fetuses/litter in the low-dose group was not regarded as toxicologically relevant.

 

Total fetal variations:

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d	HCD Mean% (range)
Litter Fetuses	N N	21 192	20 171	20 181	17 143
Fetal incidence	N	102 (53%)	94 (55%)	98 (54%)	78 (55%)
Litter incidence	N	21 (100%)	20 (100%)	20 (100%)	17 (100%)
Affected fetuses/litter	Mean%	53.0	55.4*	53.5	57.7	55.62 (50.23 – 62.83)

HCD = Historical control data; * = p ≤ 0.05 (Wilcoxon-Test [one-sided])

 

A spontaneous origin is also assumed for several unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3 (0; 100; 300 and 1000 mg/kg bw/d). Distribution and type of these findings do neither suggest a relation to treatment nor that they are regarded to be of toxicological relevance. They reflect the common biological variation inherent in the strain of rats used for this experiment.

Thus, the oral administration of Adamantyltrimethylammoniumhydroxide 20% to pregnant Wistar rats had no adverse effect on fetal morphology at any of the dose levels tested (100; 300 and 1000 mg/kg bw/d).

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL) of Adamantyltrimethylammoniumhydroxide 20% for maternal and prenatal developmental toxicity is 1000 mg/kg bw/day.

Executive summary:

Adamantyltrimethylammoniumhydroxide 20% was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6 - 19). Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance.

There were no test substance-related effects on the dams concerning mortality, food consumption, body weight, body weight gain, gestational parameters, uterine and placental weights, as well as necropsy observations up to and including a dose of 1000 mg/kg bw/day. The high-dose (1000 mg/kg bw/d) caused transient (for a few minutes) salivation directly after treatment, which was likely to be induced by the bad taste of the test substance or by local irritation in the upper digestive tract of dams. It is not considered to be a sign of systemic toxicity. Salivation was not observed after treatment with lower doses.

Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. Adamantyltrimethylammoniumhydroxide 20% shows no direct and specific effect on fetal morphological structures.

In conclusion, the no observed adverse effect level (NOAEL) of Adamantyltrimethylammoniumhydroxide 20% for maternal and prenatal developmental toxicity is 1000 mg/kg bw/day.