Registration Dossier

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:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tris-(2-ethylhexyl)amine
- Physical state: Liquid / colorless, clear
- Analytical purity: 99.7%
- Lot/batch No.: O 3012

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: body weight of the pregnant animals on day 0 varied between 151.5 – 195.7 g
- Housing: individually (Makrolon type M III cages)
- Diet (e.g. ad libitum): ad libitum (Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum):ad libitum
- Acclimation period: yes

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The oily test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.
For the test substance preparations the specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed by shaking until it is dissolved.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in corn oil over a period of 7 days at room temperature were conducted prior to the start of the study.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug / sperm referred to as day 0 of pregnancy
Duration of treatment / exposure:
GD 6-19
Frequency of treatment:
daily
Duration of test:
On GD 20, the females were sacrificed in a randomized order and examined macroscopically. The fetuses were removed from the uterus and investigated.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150, 500 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day on working days or once a day on Saturdays, Sundays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

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 AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption: The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
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: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No
Statistics:
DUNNETT-test (twosided); FISHER'S EXACT test (onesided); WILCOXON-test (onesided); KRUSKAL-WALLIS test
Indices:
The conception rate (in %) was calculated according to the following formula:
number of pregnant animals/number of fertilized animals x100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
number of corpora lutea – number of implantations/number of corpora lutea x 100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
number of implantations – number of live fetuses/number of implantations x100
Historical control data:
Was used to assess whether observed differences were within historical control range.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
One female from test group 1 (50 mg/kg bw/d) was not pregnant and therefore excluded from the analyses.
Mortality:
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 50, 150 or 500 mg/kg bw/d).
Clinical symptoms:
Some females (7 out of 25) of the high-dose group (500 mg/kg bw/d) and some females (5 out of 25) of the mid-dose group (150 mg/kg bw/d) showed transient salivation towards the end of the administration period. Salivation persisted in the respective animals for a few minutes after daily gavage dosing (i.e. up to 10 minutes) and was initially observed on GD 15.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 50, 150 or 500 mg/kg bw/d during the entire study period.
Food consumption:
The mean food consumption of the dams in test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d) was generally comparable to the concurrent control group throughout the study. This includes the slightly, but statistically significantly increased food consumption values in test group 2 (150 mg/kg bw/d) on GD 15-17 and in test group 3 (500 mg/kg bw/d) on GD 3-6 and GD 15-17.
Body weight:
The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (50, 150 and 500 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period. This includes the statistically significantly increased body weight gain value in test group 3 on GD 8-10.
Corrected (net) body weight gain:
The corrected body weight gain of test groups 1-3 (50, 150 and 500 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.
Uterus weight:
The mean gravid uterus weights of the animals of test group 1-3 (50, 150 and 500 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
Reproduction data:
The conception rate was 92% in the low-dose group (50 mg/kg bw/d) and 100% in the control, mid- and high-dose groups (0, 150 and 500 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study. There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 50, 150 and 500 mg/kg bw/d) 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. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Hematology:
In dams of test group 3 (500 mg/kg bw/d) red blood cell (RBC) counts, hemoglobin and hematocrit values were decreased. Hemoglobin values were already lower compared to controls in rats of test group 2 (150 mg/kg bw/d). However, in this test group this was the only altered red blood cell parameter and the mean was within the historical control range (hemoglobin 6.3-7.7 mmol/L). Therefore, in this test group the reduced hemoglobin value was regarded as incidental and not treatment-related. In dams of test groups 2 and 3 (150 and 500 mg/kg bw/d), total white blood cell counts, absolute lymphocyte, monocyte and large unstained cell (LUC) counts were increased. However all values apart from the absolute monocyte counts in test group 3 (500 mg/kg bw/d) were
within historical control ranges (WBC 3.58-6.00 Giga/L; absolute lymphocyte counts 1.89- 3.85 Giga/L; absolute monocyte counts 0.10-0.15 Giga/L; absolute LUC counts 0.01-0.03 Giga/L). Therefore, the changes of the total white and the differential blood cell counts were regarded as incidental and not treatment-related. The mean of the absolute monocyte counts in test group 3 which was marginally above the historical control range was also regarded at least as not adverse.
Clinical chemistry:
In dams of test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d) alanine aminotransferase (ALT) activities were higher compared to controls. In test groups 1 and 2 the increase was marginal (less than 2fold); thus the alteration was regarded as treatment-related, but not adverse. In dams of test group 3 (500 mg/kg bw/d) cholesterol and triglyceride values were increased. Cholesterol levels were already higher in test group 2 (150 mg/kg bw/d), but this was the only relevantly changed clinical chemistry parameter in this test group and therefore it was regarded in this test group as treatment-related but not adverse (ECETOC Technical report No. 85, 2002).
In dams of test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d), total bilirubin values were decreased. A hypoplastic anemia as cause for this decrease is not probable because of the short application time of the compound for 14 days. More probably an increased conjugation rate in the liver cells coupled with an accelerated excretion via bile was the cause for the lower bilirubin values. This effect was regarded as treatment-related but not adverse.
In dams of test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d), calcium levels were higher compared to controls, but the values were within the historical control range (calcium 2.36- 2.68 mmol/L, PART III, Supplement). In dams of test group 1 (50 mg/kg bw/d) total protein and albumin levels were higher compared to controls, but the change was not dosedependent. Therefore, the mentioned alterations were regarded as incidental and not treatment-related.
Gross lesions:
All findings occurred only individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Sex distribution of the fetuses:
The sex distribution of the fetuses in test groups 1-3 (50, 150 and 500 mg/kg bw/d) was comparable to the control fetuses.
Weight of the placentae:
The mean placental weights of the low-, mid- and high-dose groups (50, 150 and 500 mg/kg bw/d) were comparable to the corresponding control group.
Weight of the fetuses:
The mean fetal weights of test groups 1-3 (50, 150 and 500 mg/kg bw/d) were not influenced by the test substance. This includes the statistically significantly higher mean fetal body weight in test group 3, which was well within the historical control range (both sexes combined: mean 3.5 g [2.5 – 5.1 g], see PART III). Therefore it was regarded as incidental and not treatment-related.
Fetal external malformations:
An external malformation was recorded, each, for one control fetus and one fetus of test group 3 (500 mg/kg bw/d), as shown in Tab. 4.3.2.1.1. For the affected fetuses, these external findings were associated either with skeletal or visceral malformations. The total incidence of external malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.
Fetal external variations:
No external variations were recorded.
Fetal external unclassified observations:
One unclassified external observation, i.e. blood coagulum around placenta, was recorded in one fetus of the high-dose group (500 mg/kg bw/d). This finding was not considered biologically relevant.
Fetal soft tissue malformations:
Soft tissue malformations occurred in one fetus, each, of the control and the high-dose group (0 and 500 mg/kg bw/d). Male high-dose fetus No. 99-05 had multiple visceral malformations, i.e. swollen liver lobes (in addition lobular pattern [nutmeg liver]), small kidneys, unexpanded lungs (in addition fused lung lobes), enlarged heart, misshapen and interrupted palatal rugae (in addition to high-arched palate). These visceral findings were associated with an external malformation. The overall incidences of soft tissue malformations were comparable to those found in the historical control data.
Fetal soft tissue variations:
Five soft tissue variations, i.e. misshapen palatal rugae, short innominate, malpositioned carotid branch, dilated renal pelvis and dilated ureter, were detected. In the majority of cases, the incidences were neither statistically significantly nor dose-dependently changed in comparison to the concurrent control group. However, the incidence of ‘short innominate’ was statistically significantly increased in test group 2 (150 mg/kg bw/d). This finding showed no
dose-dependency and was therefore assessed to be without biological relevance. The overall incidences of soft tissue variations were comparable to those
found in the historical control data.
Fetal skeletal malformations:
Some skeletal malformations were detected in all test groups including the control (test groups 0-3; 0, 50, 150 or 500 mg/kg bw/d), as shown in Tab. 4.3.4.1.1. One control fetus had associated external findings. The incidences of these malformations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant.
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. However, the incidence of ‘dumbbell ossification of thoracic centrum (unchanged cartilage)’ was statistically significantly increased in test group 2 (150 mg/kg bw/d). This finding showed no relation to dosing and can be found in the historical control data at a higher frequence. The overall incidences of skeletal variations were also comparable to the historical control data.
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 test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dosing.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

The administartion of Tris-(2-ethylhexyl)amine to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity, such as an anemia and altered liver cell metabolism.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 150 mg/kg bw/d.

There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 500 mg/kg bw/d.

The test substance is not teratogenic in rats.

Applicant's summary and conclusion