Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-03-04 until 2015-04-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethyl-N,N-bis(2-ethylhexyl)hexylamine
EC Number:
217-461-0
EC Name:
2-ethyl-N,N-bis(2-ethylhexyl)hexylamine
Cas Number:
1860-26-0
Molecular formula:
C24H51N
IUPAC Name:
2-ethyl-N,N-bis(2-ethylhexyl)hexylamine
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Purity: 99.7 %
- 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: ad libitum (Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: 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:
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.

DOSING SOLUTIONS PREPARATION
- Rate of preparation of dosing solution: at the beginning of the administration period and thereafter at intervals
- Storage temperature of dosing solution: room temperature

VEHICLE
- Justification for use and choice of vehicle: not specified
- Amount of vehicle: 4 mL/kg bw/d (substance in vehicle)
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 / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
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
- Organs examined/Tissue fixed:
1. All gross lesions
2. Adrenal glands
3. Kidneys
4. Liver
5. Mesenteric lymph nodes
6. Ovaries
7. Spleen
8. Stomach
9. Small intestine
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

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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.
Mortality:
no mortality observed
Description (incidence):
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).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
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 significant increased body weight gain value in test group 3 on GD 8-10.
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.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
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.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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 biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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). 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 dose-dependent. Therefore, the mentioned alterations were regarded as incidental and not treatment-related.
Details are given table 1.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
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.
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.
Details are given in table 2.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred only individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: non-neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
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 the mean number of implantation sites or in the the number of resorptions.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
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 the mean number of implantation sites and in the values calculated for the pre- and the postimplantation losses.
Dead fetuses:
no effects observed
Description (incidence and severity):
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 viable fetuses.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
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.
Other effects:
no effects observed
Description (incidence and severity):
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 the mean number of corpora lutea.
All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical biochemistry
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: No adverse effects on fetuses.

Maternal abnormalities

Abnormalities:
effects observed, treatment-related

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
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]). Therefore it was regarded as incidental and not treatment-related.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
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.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
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 table 3. 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.
No external variations were recorded.
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.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
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 table 5. 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.
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.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue malformations occurred in one fetus, each, of the control and the high-dose group (0 and 500 mg/kg bw/d). One male high-dose fetus 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. Details on soft tissue effects are given in table 4.
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.
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.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effects in fetuses.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Clinical chemistry (enzymes and substrates + minerals) results of the female dams

Enzymes

 

 

 

 

 

G 0 / F

G 1 /F

50 mg/kgbw/d

G 2 /F

150 mg/kgbw/d

G 3 /F

500 mg/kgbw/d

ALT

Mean

0.98 v

1.10 *

1.33 **

1.83 **

[µkat/L]

S.d.

0.12

0.21

0.17

0.29

day 20

N

25

23

25

25

 

Median

1.01

1.10

1.35

1.79

AST

Mean

1.24 k

1.28

1.22

1.34

[µkat/L]

S.d.

0.32

0.33

0.30

0.46

day 20

N

25

23

25

25

 

Median

1.17

1.23

1.19

1.23

ALP

Mean

1.16 k

1.03

1.02

0.92

[µkat/L]

S.d.

0.31

0.25

0.30

0.26

day 20

N

25

23

25

25

 

Median

1.03

1.03

0.96

0.96

GGT_C

Mean

0

0

0

0

[nkat/L]

S.d.

0

0

0

0

day 20

N

25

23

25

25

 

Median

0

0

0

0

Substrates + minerals

 

 

 

 

 

 

G 0 / F

G 1 /F

50 mg/kgbw/d

G 2 /F

150 mg/kgbw/d

G 3 /F

500 mg/kgbw/d

 

UREA

Mean

4.52 k

4.72

4.58

4.69

 

[mmol/L]

S.d.

0.51

0.86

0.81

0.66

 

day 20

N

25

23

25

25

 

 

Median

4.48

4.53

4.36

4.62

 

CREA

Mean

27.3 k

26.2

27.1

26.8

 

[µmol/L]

S.d.

2.0

2.2

2.2

3.1

 

day 20

N

25

23

25

25

 

 

Median

27.6

26.8

26.8

26.8

 

GLUC

Mean

5.32 k

5.43

5.39

5.45

 

[mmol/L]

S.d.

0.49

0.51

0.41

0.37

 

day 20

N

25

23

25

25

 

 

Median

5.22

5.40

5.32

5.27

 

TBIL_C

Mean

1.26 v

0.98 **

0.94 **

0.81 **

 

[µmol/L]

S.d.

0.25

0.13

0.15

0.22

 

day 20

N

25

23

25

25

 

 

Median

1.23

1.00

0.92

0.75

 

TPROT

Mean

59.59 v

61.63 *

60.90

59.05

 

[g/L]

S.d.

2.06

3.37

3.51

3.88

 

day 20

N

25

23

25

25

 

 

Median

59.76

61.49

60.09

59.04

 

ALB

Mean

35.10 v

36.50 *

36.03

34.73

 

[g/L]

S.d.

1.19

2.34

2.17

2.34

 

day 20

N

25

23

25

25

 

 

Median

34.86

36.21

35.66

35.04

 

GLOB

Mean

24.49 k

25.13

24.88

24.33

 

[g/L]

S.d.

1.11

1.49

1.51

1.73

 

day 20

N

Median

25

24.54

23

24.94

25

24.85

25

24.62

 

CHOL

Mean

2.08 v

2.24

2.41 **

2.53 **

 

[mmol/L]

S.d.

0.32

0.32

0.32

0.41

 

day 20

N

25

23

25

25

 

 

Median

2.03

2.18

2.38

2.50

 

TRIG

Mean

5.28 v

5.31

5.72

6.67 **

 

[mmol/L]

S.d.

1.68

2.23

1.81

1.78

 

day 20

N

25

23

25

25

 

 

Median

5.01

5.45

5.89

6.66

 

INP

Mean

1.62 v

1.71

1.68

1.54

 

[mmol/L]

S.d.

0.22

0.19

0.17

0.20

 

day 20

N

25

23

25

25

 

 

Median

1.55

1.66

1.66

1.55

 

CA

Mean

2.55 v

2.61 **

2.63 **

2.65 **

 

[mmol/L]

S.d.

0.05

0.06

0.09

0.07

 

day 20

N

25

23

25

25

 

 

Median

2.56

2.62

2.63

2.65

 

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

v=KRUSKAL-WALLIS-WILCOX; k=KRUSKAL-WALLIS

Table 2: Absolute and relative organ weights of the female dams

Absolute weights

Females

Test group mg/kg bw/d

1

(50 )

2

( 150)

3

( 500)

Adrenal glands

102%

108%

114%**

Liver

104%

113%**

113%**

Ovaries

101%

108%

112%**

Spleen

103%

108%*

111%*

Relative weights

Females

Test group mg/kg bw/d

1

(50)

2

(150)

3

(500)

Adrenal glands

102%

106%

115%**

Liver

104%

110%**

113%**

Ovaries

101%

106%

112%**

Spleen

103%

106%

111%**

*: p <= 0.05, **: p <= 0.01

Table 3: External examination of the fetuses

Total external observations

 

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

257

23

210

25

261

25

262

Fetal

incidence

 

N (%)

 

1 (0.4)

 

0.0

 

0.0

 

1 (0.4)

Litter incidence

 

N (%)

 

1 (4.0)

 

0.0

 

0.0

 

1 (4.0)

Affected

fetuses/litter

 

Mean%

 

0.4

 

0.0

 

0.0

 

0.4

Total external unclassified observations

 

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

257

23

210

25

261

25

262

Fetal incidence

 

N (%)

 

0.0

 

0.0

 

0.0

 

1 (0.4)

Litter incidence

 

N (%)

 

0.0

 

0.0

 

0.0

 

1 (4.0)

Affected fetuses/litter

 

Mean%

 

0.0

 

0.0

 

0.0

 

0.4

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

Table 4: Soft tissue examination of the fetuses

Total soft tissue malformations

 

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

121

22

99

25

125

25

124

Fetal incidence

 

N (%)

 

1 (0.8)

 

0.0

 

0.0

 

1 (0.8)

Litter incidence

 

N (%)

 

1 (4.0)

 

0.0

 

0.0

 

1 (4.0)

Affected fetuses/litter

 

Mean%

 

0.8

 

0.0

 

0.0

 

0.8

Total soft tissue variations

 

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

121

22

99

25

125

25

124

Fetal incidence

 

N (%)

 

6 (5.0)

 

2 (2.0)

 

5 (4.0)

 

7 (5.6)

Litter incidence

 

N (%)

 

5 (20)

 

2 (9.1)

 

4 (16)

 

5 (20)

Affected fetuses/litter

 

Mean%

 

4.7

 

5.5

 

3.9

 

5.7

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

Table 5: Skeletal examination of the fetuses

Total skeletal malformations

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

136

23

111

25

136

25

138

Fetal

incidence

 

N (%)

 

1 (0.7)

 

1 (0.9)

 

0.0

 

1 (0.7)

Litter incidence

 

N (%)

 

1 (4.0)

 

1 (4.3)

 

0.0

 

1 (4.0)

Affected

fetuses/litter

 

Mean%

 

0.6

 

1.1

 

0.0

 

1.0

Total fetal skeletal variations

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

136

23

111

25

136

25

138

Fetal incidence

 

N (%)

 

133 (98)

 

108 (97)

 

132 (97)

 

132 (96)

Litter incidence

 

N (%)

 

25 (100)

 

23 (100)

 

25 (100)

 

25 (100)

Affected fetuses/litter

 

Mean%

 

97.7

 

97.2

 

96.9

 

95.4

Total unclassified cartilage observations

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

136

23

111

25

136

25

138

Fetal incidence

 

N (%)

 

119 (88)

 

94 (85)

 

109 (80)

 

110 (80)

Litter incidence

 

N (%)

 

25 (100)

 

22 (96)

 

23 (92)

 

24 (96)

Affected fetuses/litter

 

Mean%

 

88.2

 

80.0

 

78.3

 

80.1

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

Applicant's summary and conclusion

Conclusions:
The administration of the test substance 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.
Executive summary:

SUMMARY

METHODS

Tris-(2-ethylhexyl)amine was tested for its prenatal developmental toxicity in Wistar rats.

The test substance was administered as an oily solution to groups of 25 time-mated female Wistar rats by gavage at doses of 50, 150 and 500 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group.

At terminal sacrifice on GD 20, 23-25 females per group had implantation sites.

OBSERVATIONS

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the adrenal glands, kidneys, liver, ovaries, spleen, unopened uterus and placentas).

For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

RESULTS

Analytics

The various analyses confirmed

• the stability of the test substance preparations over a period of 7 days at room temperature,

• the correctness of the prepared concentrations.

Effects

The following test substance-related adverse effects/findings were noted:

Test group 3 - 500 mg/kg bw/d:

Dams

• Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values

• Increased alanine aminotransferase (ALT) activities

• Increased cholesterol and triglyceride values

• Increased absolute and relative organ weights of liver and spleen

Fetuses

• No test substance-related adverse effects

Test group 2 - 150 mg/kg bw/d:

• No test substance-related adverse effects

Test group 1 - 50 mg/kg bw/d:

• No test substance-related adverse effects

CONCLUSION

Under the conditions of this prenatal developmental toxicity study, the oral administration 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 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.