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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Embryotoxicity of Sixteen Industrial Amines to the Chicken Embryo
Author:
Korhonen, A., Hemminki, K., and Vainio, H.
Year:
1983
Bibliographic source:
Institute of Occupational Health, Department of Industrial Hygiene and Toxicology, Haartmaninkatu 1, SF-002090 Helsinki 39, Finland.

Materials and methods

Test guideline
Guideline:
other: Not specified
Principles of method if other than guideline:
The embryotoxic effects of TEA were tested in the chicken embryo assay. White Leghorn chicken eggs (3-days old) were used for the study. Acetone (p.a. grade) was used as the solvent for Triethanolamine (TEA, technical grade). The solution was injected into the egg in a total volume of 5 µL. Ten control eggs, injected with 5 µL of acetone, were incubated with each batch of eggs. Results from solvent controls were not subtracted from experimental values. The background level of 1-2% was insignificant against the variation of the method.

Three-day (72-76 h) embryos were selected by candling. The method used involved the injection on the inner shell membrane, focusing the solution drop exactly on the embryo. After two days post injection, the eggs were candled again. Eggs containing dead embryos were counted and discarded, the remaining eggs were then candled every second or third day. Those containing dead embryos were opened and checked for external malformations and the developmental stage according to V. Hamburger and H. L. Hamilton, A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92 (1951 ).

Incubation terminated was 11 days post the injection, after a total incubation of 14 days. The remaining eggs were opened and the embryos inspected for survival and for external malformations. The temperature was kept at 37.7 “C and the humidity between 66% and 71% throughout the incubation period. The eggs were turned two-four times per day.

The affected embryos were classified into the following categories:

(1) Early deaths, embryos that died before day 5 of incubation, within two days of treatment.
(2) Late deaths, non-malformed, externally normal embryos that died between days 5 and 14.
(3) Late deaths, malformed, externally malformed embryos that died between days 5 and 14.
(4) Malformed survivors, externally malformed embryos alive on day 14 of incubation.

LD50 and ED50 values were calculated according to the method described by A. P. Rosiello, J. M. Essigmann and G. N. Wogan, Rapid and accurate determination of the median lethal dose (LD,,) and its error with a small computer. J. Toxicol. Environ. Heaffh 3, 8. K. S. Khera, Ethylenethiourea. Teratogenicity study in rats 797-809 (1977). using a Wang table computer.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2',2''-nitrilotriethanol
EC Number:
203-049-8
EC Name:
2,2',2''-nitrilotriethanol
Cas Number:
102-71-6
Molecular formula:
C6H15NO3
IUPAC Name:
2,2',2''-nitrilotriethanol
Test material form:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Merck, Darmstadt. Lot/batch number not specified
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified (p.a grade)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not specified
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not specified

Test animals

Species:
other: Chicken
Strain:
other: White Leghorn
Remarks:
Test conducted on White Leghorn chicken embryos within the egg
Details on test animals or test system and environmental conditions:
- CHICKEN EMBRYOS
- Source: Hatchery of Siipikarjanhoitajain Liitto ry, Hãmeenlinna, Finland
- Age at study initiation: 3 day embryos

ENVIRONMENTAL CONDITIONS
- Incubator: Commercial type incubator (Manufactured by Hãmeen Insinööritoimisto Oy, Hãmeenlinna.
- Temperature (°C): 37.7 “C
- Humidity (%): 66 - 71 %
- Photoperiod (hrs dark / hrs light): Not specified

IN-LIFE DATES: 3 day old embryos with incubation terminated on day 11 (total incubation period 14 days)

Administration / exposure

Route of administration:
other: Injection into egg
Vehicle:
acetone
Remarks:
p.a. grade
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Treatment concentrations of 1.3, 2.6, 5.2 and 10.5 µmol per egg TEA in acetone injected into the egg in a total volume of 5 µL/per egg.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified, publication details doses soluble or suspendable in acetone in 5 ul of acetone
- Concentration in vehicle: Not specified
- Lot/batch no. (if required): Not specified
- Purity: Not specified (p.a. grade)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
11 days
Frequency of treatment:
Single treatment in 3 day old embryos which were exposed to TEA in acetone for 11 days
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
1.3 other: µmol
Remarks:
1.3 µmol per egg
Dose / conc.:
2.6 other: µmol
Remarks:
2.6 µmol per egg
Dose / conc.:
5.2 other: µmol
Remarks:
5.2 µmol per egg
Dose / conc.:
10.5 other: µmol
Remarks:
10.5 µmol per egg
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not specified
- Rationale for animal assignment (if not random): Not specified.
- Other: Three days embryos were selected by candling to undergo injection of test item. Two days after the injection with TEA in acetone (as solvent), the eggs were candled again. Eggs containing dead embryos were counted and discarded. The remaining eggs were then candled every second or third day. Those containing dead
embryos were opened and the embryos checked for external malformations and the developmental stage according to Hamburger and Hamilton. The incubation was terminated 11 days after the injection, after a total incubation of 14 days. The remaining eggs were opened and the embryos inspected for survival and for external malformations.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:
other:
Remarks:
Not relevant for this type of study
Effect level:
> 0 - < 0 mg/kg bw/day (nominal)
Based on:
other: Not relevant
Remarks:
Not relevant for this type of test
Basis for effect level:
other: Not applicable for this type of test
Remarks on result:
not measured/tested

Results (fetuses)

Details on embryotoxic / teratogenic effects:
No effects observed with the lowest dose of 1.3 µmol per egg.

At 2.6 µmol per egg, 40% of embryos were observed to have had early deaths (before day 5); 3% had a late death/were non-malformed embryos; and 3% were observed to be malformed embryos at days 6-14. Total was 47% of all affected embryos at this dose.

At 5.2 µmol per egg, 80% of embryos were observed to have had early deaths (before day 5); 3% had a late death/were non-malformed embryos; 3% were observed to be malformed embryos at days 6-14; and 3% were observed to be malformed survivors on day 14. Total was 90% of all affected embryos at this dose.

At the maximum dose of 10.5 µmol per egg, 95% of embryos were observed to have had early deaths (before day 5). Total was 95% of all affected embryos at this dose.

Effect levels (fetuses)

Dose descriptor:
other:
Remarks:
Not applicable for this type of toxicity test
Effect level:
> 0 - < 0 mg/kg bw/day (nominal)
Based on:
other: Not relevant for this type of test
Remarks:
Not applicable tot his type of toxicity test
Basis for effect level:
other: Not applicable
Remarks on result:
not measured/tested
Remarks:
Not applicable for this type of toxicity test

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
2.6 other: µmol per egg
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1. Embryotoxic effects of triethanolamine on White Leghorn chicken embryos


Treatment

(µmol per egg)     

 Treated

embryos (n)   

Early deaths

(before day 5)  

Late deaths

non-malformed

embryos    

(Days 6 -14)

malformed

embryos    

Malformed

survivors    

All affected

embryos    

(n) 

(%) 

(n) 

(%) 

(n) 

(%) 

(n) 

(%) 

(n) 

(%) 

1.3 

30 

2.6 

30 

12 

40 

14 

47 

5.2 

30 

24 

80 

27 

90 

10.5 

20 

19 

95 

19  95 
 

Table 2. ED50 and LD50 values on different embryotoxic effects of Triethanolamine on White Leghorn chicken embryos


Total effect   

ED50 

(µmol per egg)

95% confidence limits   

 

Total mortality    

Early deaths

(LD50)    

(µmol per egg)   Tan α   (µmol per egg)   Tan  α (µmol per egg)   Tan α  
2.6  (1.9 - 3.5)  1.6  2.6  1.6  3.0  1.5 
 

Table 3. Malformation types produced in White Leghorn chicken embryos by Triethanolamine


   Small head 

Small eye

cup    

Defects of

lids and

cornea    

Defects of

beak    

Encephalocoele

or skin pimple

in head    

Open

coelom    

Short back

or neck    

Defects

of wings    

Defects

of legs    

 

Oedema

and

lymph

blebs 

 

Number

of

malformed

embryos 

Number   

of

malformations 

 (n)

(%) 

(n) 

(%) 

(n) 

(%) 

(n) 

(%) 

(n) 

(%) 

(n) 

(%) 

(n) 

(%) 

(n) 

(%) 

(n) 

(%) 

(n) 

(%) 

-

100 

33 

33 

 

 3

 

 

Applicant's summary and conclusion

Conclusions:
The embryotoxic effects of TEA were tested in the chicken embryo assay to evaluate embryotoxicity as part of the evaluation of 16 industrial chemicals. Three-day-old white Leghorn chicken eggs were injected with 1.3-10.5 µmol of TEA in acetone. Eleven days after the injection (total incubation period 14 days), eggs were opened and the embryos inspected for survival and external malformations. The ED50 for embryotoxic effects was 2.6 µmol/egg. The embryotoxic effects included early mortality and malformations (open coelom, short back or neck, edema, and lymph blebs). The incidence of malformations (3%-6%) in the TEA-treated groups was not significantly different from that of controls.
Executive summary:

Triethanolamine (TEA, p.a grade) was tested for embryotoxic effects in the chicken embryo assay to evaluate embryotoxicity as part of the evaluation of 16 industrial chemicals. Three-day-old white Leghorn chicken eggs were injected with 1.3-10.5 µmol of TEA in acetone. Eleven days after the injection (total incubation period 14 days), eggs were opened and the embryos inspected for survival and external malformations. The ED50 for embryotoxic effects was 2.6 µmol/egg. The embryotoxic effects included early mortality and malformations (open coelom, short back or neck, edema, and lymph blebs). The incidence of malformations (3%-6%) in the TEA-treated groups was not significantly different from that of controls. GLP and study validity are not specified, however the study is clearly documented with generally accepted scientific principles considered acceptable for assessment.

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