Dibutylamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Nov 2007 - 26 Nov 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

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)

Version / remarks:

Jan 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dibutylammonium chloride

EC Number:

228-521-0

EC Name:

Dibutylammonium chloride

Cas Number:

6287-40-7

Molecular formula:

C8H19N.ClH

IUPAC Name:

N-butylbutan-1-aminium chloride

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity: 95 w-%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: analytical verifications of the stability of the test substance in demineralized water for a period of 7 d at room tempreature were carried out before study initiation

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 10 - 12 weeks
- Housing: housed singly
- Diet (e.g. ad libitum): ad libitum, Kliba maintenance diet mouse/rat
- Water (e.g. ad libitum): ad libitum
- Weight at study initiation: 145.4 - 188.4 g

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance solutions were prepared at the beginning of the administration period and thereafter at intervals of 7 days, which took into account the analytical results of the stability verification.
For the preparation of the solutions, appropriate amounts of the test substance was weighed in a beaker, topped up with drinking water and subsequently intensely mixed with a magnetic stirrer.

10 mL/kg bw were administered each day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The results of the analyses of the test substance solutions in drinking water confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

The animals were paired by the breeder("time-mated") and supplied on GD 0 (=detection of vaginal plug/sperm).

Duration of treatment / exposure:

Gestation day 6 - 19

Frequency of treatment:

Once daily

Duration of test:

Dams sacrificed on GD20

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

concentration of the aqueous solution 150 mg/100mL

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

concentration of the aqueous solution 500 mg/100mL

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

concentration of the aqueous solution 1500 mg/100mL

No. of animals per sex per dose:

25 female rats per dose

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 83 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- With the exaption of day 0, the consumption o food was determined on the same days as body weight.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20

MORTALITY was checked twice a day on working days and once a day on saturdays, sundays or public holidays

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Liver, kidney, thyroid glands

other: Clinical pathology (hematology, clinical chemistry)

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
- Other: dead fetuses

Fetal examinations:

All fetus was weighed, sexed and external tissues ans all orifices were examined macroscopically.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter

Statistics:

DUNNETT-test
FISHER`S EXACT
WILCOXON-test
KRUSKAL-WALLIS test

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Dose 50 mg/lg bw: salivation
Dose 150 mg/kg bw: salivation; one dam showed distinct clinical signs of maternal toxicity such as abdominal position, convulsion, twitching and hypersensitivity after treatment on just one occasion during the tretment.
Salivation stopped with exposure on GD 20 but was not considered to be a sign of systemic toxicity (result of the unpleasant taste or local irritation of the upper digestive tract)

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain of the high-dose rats was statistically significantly reduced at initiation of treatment (GD 6-8, about 63% below the concurrent control value) but recovered afterwards. If calculated for the enire treatment phase (GD 6-19), the mean body weight gain of these rats was slightly ans non-signicantly below control (about 5%).
Body weight gain of the dams of test groups 1 and 2 (15 and 50 mg/kg bw/d) was comparable to the concurrent controls. All observable differences in these 2 groups in comparison to the controls are without any biological relevance.

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

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In the dams on GD 20 the red bood cell counts as well as the menoglobin and hematocit values were slightly, but statictically significantly increased beginning in the 50 mg/kg bw/d dose group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

On GD 20, the medians of the alanine aminotransferase (ALT) activity were increased in the dams of the 150 mg/kg bw/d dose group. The inorganic phosphate levels were increased in the dams starting in the 50 mg/kg bw/d dose group. Correspondingly, the calcium levels were increased. This increase showed a statistically weakly significance already in the 15 mg/kg bw/d dose group. However, in this dose group the calcium increase was the only increased parameter in clinical pathology. Because of this exclusiveness, the calcium increase in this dose group was regarded as non-adverse. Beginning in the dams of the 50 mg/kg bw/d dose group the urea as well as the cholesterol values were increased.

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute kidney weights were slightly, but significantly increased in the high-dose group (150 mg/kg bw/d). Neither clinical pathological correlates nor any corroborative gross lesions were noted in this dose group, thus this rather marginal increase of kidney weight was not considered as an adverse effect of systemic toxicity.

The mean gravid uterus weights of the animals of test groups 1, 2 and 3 (15; 50 or 150 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance. They reflect the normal degree of variation for rats of the strain used for this study and have to be assessed in association with the fortuitous fluctuations in the mean number of live fetuses/dam in this study

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Details on maternal toxic effects:

The conception rate reached 96% in test groups 0, 2 and 3 (0; 50 and 150 mg/kg bw/d) and 100% in test group 1 (15 mg/kg bw/d). There were no test substance-related and/or biologically relevant differences among the test groups in the conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses as well as the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

15 mg/kg bw/day

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

2

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

150 mg/kg bw/day

Basis for effect level:

other: highest concentration tested

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were recorded for one fetus each in the mid- and high-dose group (50 and 150 mg/kg bw/d). Both fetuses concerned had multiple malformations. The domed head mirrored the hydrocephaly found in this fetus and is therefore no independent finding. Cleft palates are present in the historical control data at a comparable incidence, thus these, findings were considered to be spontaneous in nature and without a relation to dosing. In the treated groups, the total incidences of external malformation were not significantly different from the control group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One skeletal malformation was noted in one fetus each of test groups 0 and 1 (0 and 15 mg/kg bw/d). Neither statistically significant differences between the test groups nor a dose-response relationship were observed. Based on the rate of affected fetuses per litter, the incidence of skeletal malformations was comparable to the historical control data. No association to the treatment is assumed.
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 the dose. Based on the rate of affected fetuses per litter, the incidence of skeletal variations was comparable to the historical control data

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

The examination of the soft tissues revealed soft tissue malformations in one litter each of test groups 2 and 3 (50 and 150 mg/kg bw/d). Some of these soft tissue malformations are present in the historical control data at comparable incidences and on the whole no specific malformation pattern was obvious. Thus, these individual cases were not considered to be related to the treatment. Malformation incidences showed no statistically significant differences between the test groups and the control.
Three soft tissue variations, i.e. short innominate, uni- or bilateral dilation of the renal pelvis and dilated cerebral ventricle, were detected. These findings were observed in 3 to 4 fetuses of 3 litters in each group including the controls and showed no dose-response relationship. No association to the treatment is assumed.

Description (incidence and severity):

No statistically significant or biological relevant change of the mean placental weights

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Basis for effect level:

other: highest concentration tested

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Examinations of the Fetuses

Total external malformations		Test group 0 0 mg/kg bw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 150 mg/kg bw/d
Litter Fetuses	N N	24 229	25 224	24 201	24 206
Fetal incidence	N	0.0	0.0	1(0.5%)	1(0.5%)
Litter incidence	N	0.0	0.0	1(4.2%)	1(4.2%)
Affectd fetuses/litter	Mean %	0.0	0.0	0.5	0.4
Total fetal soft tissue malformation		Test group 0 0 mg/kg bw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 150 mg/kg bw/d
Litter Fetuses	N N	24 109	25 105	24 95	24 98
Fetal incidence	N	0.0	0.0	1(2.1%)	1(1.0%)
Litter incidence	N	0.0	0.0	1(4.2%)	1(4.2%)
Affectd fetuses/litter	Mean %	0.0	0.0	2.1	0.8
Total soft tissue variations		Test group 0 0 mg/kg bw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 150 mg/kg bw/d
Litter Fetuses	N N	24 109	25 105	24 95	24 98
Fetal incidence	N	3 (2.8%)	4 (3.8%)	4(4.2%)	4 (4.1%)
Litter incidence	N	3 (2.8%)	3 (12%)	3 (13%)	3 (13%)
Affectd fetuses/litter	Mean %	2.7	3.3	2.1	0.8
Total skeletal malformations		Test group 0 0 mg/kg bw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 150 mg/kg bw/d
Litter Fetuses	N N	24 120	25 119	24 106	24 108
Fetal incidence	N	1 (0.8%)	1 (0.8%)	0.0	0.0
Litter incidence	N	1 (4.2%)	1 (4.0%)	0.0	0.0
Affectd fetuses/litter	Mean %	0.8	0.8	0.0	0.0
Total skeletal variations		Test group 0 0 mg/kg bw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 150 mg/kg bw/d
Litter Fetuses	N N	24 120	25 119	24 106	24 108
Fetal incidence	N	120 (100%)	119 (100%)	106 (100%)	108 (100%)
Litter incidence	N	24 (100%)	25 (100%)	24 (100%)	24 (100%)
Affectd fetuses/litter	Mean %	100.0	100.0	100.0	100.0

Total fetal malfromations		Test group 0 0 mg/kg bw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 150 mg/kg bw/d
Litter Fetuses	N N	24 229	25 224	24 201	24 206
Fetal incidence	N	1 (0.4%)	1 (0.4%)	2 (1.0%)	1 (0.5%)
Litter incidence	N	1 (4.2%)	1 (4.0%)	1 (4.2%)	1 (4.2%)
Affectd fetuses/litter	Mean %	0.5	0.4	1.0	0.4

 

Total fetal variations		Test group 0 0 mg/kg bw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 150 mg/kg bw/d
Litter Fetuses	N N	24 229	25 224	24 201	24 206
Fetal incidence	N	123 (54%)	123 (55%)	110 (55%)	112 (54%)
Litter incidence	N	24 (100%)	25 (100%)	24 (100%)	24 (100%)
Affectd fetuses/litter	Mean %	53.7	55.1	54.7	53.7

mg/kg bw/d = milligram per kilogram body weight per day; N = mumber; % = per cent

 

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL) for maternal toxicity is 15 mg/kg bw/d based on clinical pathological effects at dose levels of 50 mg/kg bw/d and above, as well as additional clinical findings and a temporarily reduced body weight gain at a dose level of 150 mg/kg bw/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 150 mg/kg bw/d, the highest tested dose.

Executive summary:

In this GLP-compliant study performed according to OECD TG 414, the test substance was administered to pregnant Wistar rats daily by stomach tube from implantation to one day prior to expected day of parturition (GD 6 -19). There were no test substance-related effects on the dams concerning mortality, food consumption, gross and corrected (net) body weights, gestational parameters, uterine and placental weights, as well as necropsy observations up to and including a dose of 150 mg/kg bw/day. One individual high-dose dam (No. 88) showed distinct clinical signs of maternal toxicity such as abdominal position; convulsion, twitching and hypersensitivity after treatment on just one occasion during the treatment (GD 7). Although this is only one case, a relation of these findings to the treatment cannot be excluded. Some mid- and high-dose dams showed transient salivation for a few minutes immediately after each treatment, which 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. Salivation was not observed after treatment with the low doses. Body weight gain of the high-dose rats was significantly reduced (by 63%) at initiation of treatment (GD 6-8) but recovered afterwards. This temporary, significant reduction is considered to be related to the test compound although it hat no dramatic impact on the average body weight gain throughout the entire treatment phase (GD 6-19).  The increase of the red blood cell counts, the hemoglobin as well as the hematocrit values in the dams on GD 20 beginning in the 50 mg/kg bw/d dose group was an indication of hemo-concentration, most probably because of stress. This assumption was confirmed by the increase of the total white blood cell counts, which was due to higher lymphocyte counts, in the rats of the 150 mg/kg bw/d dose group. Correspondingly, the increase of the urea as well as of the cholesterol values in the dams beginning in the 50 mg/kg bw/d dose group can be interpreted as a consequence of stress accompanied by an increased protein metabolism. Additionally, the latter effect can be partly explained by an increased bone metabolism, which was also expressed by increased inorganic phosphate and calcium blood levels in the rats beginning in the 50 mg/kg bw/d dose group. A slight affection of the liver cells in the dams of the 150 mg/kg bw/d dose group was indicated by the increase of the ALT activity. Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. The test substance had no direct and specific effect on fetal morphological structures. 

 

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 15 mg/kg bw/d based on clinical pathological effects at dose levels of 50 mg/kg bw/d and above, as well as additional clinical findings and a temporarily reduced body weight gain at a dose level of 150 mg/kg bw/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 150 mg/kg bw/d, the highest tested dose.