Fatty acids, tall-oil, compds. with triethanolamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1987

Materials and methods

Test guideline

Version / remarks:

Not stated

Principles of method if other than guideline:

Testing was performed as reported by Galloway et al. (1987). Triethanolamine was sent to the laboratory as a coded aliquot by Radian Corporation. It was tested in cultured Chinese hamster ovary (CHO) cells for induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (Abs), both in the presence and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least three doses of triethanolamine. In the trials conducted without S9, the high dose was limited by toxicity; in the trials with S9, the high dose was limited to 10,100 μg/mL. A single flask per dose was used, and tests yielding equivocal or positive results were
repeated.

In the chromosomal aberrations test without S9, cells were incubated in McCoy’s 5A medium with triethanolamine for 8.5 hours; Colcemid was added, and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the aberrations test with S9 (Arochlor 1254-induced male Sprague-Dawley rat liver S9), cells were treated with triethanolamine and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 8.5 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9.

Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. One hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

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:

Not specified

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cloned Chinese hamster ovary cells (CHO-W-B1)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix

Test concentrations with justification for top dose:

For test with S9, concentrations were 6,040, 8,060 and 10,070 ug/mL. For test without S9, concentrations were 1,510, 2,010 and 4,030 ug/mL. The highest dose tested was 4,030 µg/mL. Initially, dose selection was based on a preliminary test of cell survival 24hr after treatment. The top doses selected for cytogenetics assays were those estimated to reduce growth by 50%.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl Sulfoxide (DMSO)

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Mitomycin-C used as positive control absent S9. Cyclophosphamide used as positive control with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium (McCoy's 5A medium with 10% fetal calf serum, L-glutamine and antibiotics)

DURATION
- Exposure duration: Without S9, 10.5 hours. (8.5 hours + 2 hours colcimid, prior to harvest)
: With S9, 2 hours.(after which the treatment medium was removed, incubated for 8.5 hours + 2 hours Colcemid)
- Expression time (cells in growth medium): 10.5 hours in total
- Fixation time (start of exposure up to fixation or harvest of cells): 10.5 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Not specified

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not specified

NUMBER OF CELLS EVALUATED: 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: Observations on cell growth and cell cycle kinetics from the Sister Chromatid Exchange (SCE) test were used to select the doses and fixation times for the chromosome aberration tests.

OTHER EXAMINATIONS:
- Determination of polyploidy: aberrations in polyploidy not scored
- Determination of endoreplication: recorded but not included in the totals

- OTHER: Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. One hundred first-division metaphase cells were scored at each of the three highest dose groups having sufficient metaphases for analysis. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Rationale for test conditions:

Developed screening protocol for detecting chemically induced cytogenetic changes in vitro [Galloway et al, 1985]
Ref: Galloway SM, Bloom AD, Resnick M, Margolin BH, Nakamura F, Archer P, Zeiger E (1985): Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories. Environ Mutagen 7: 1-51.

Evaluation criteria:

Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. One hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Statistics:

Data Evaluation: The statistical procedures for evaluation of the test data and the assessment of the results have been modified from those described in detail by Galloway et al [1985] Ref: Galloway SM, Bloom AD, Resnick M, Margolin BH, Nakamura F, Archer P, Zeiger E (1985): Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories. Environ Mutagen 7: 1-51 and Margolin et al [1986]. Ref: Margolin BH, Resnick MA, Rimpo JY, Archer P, Galloway SM, Bloom AD, Zeiger E (1986).. Briefly, the analyses examined the evidence for a dose relation and the absolute increase over the solvent control at each dose.

For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, and the test was that described by Margolin et a1 [1983, pp 714-7151. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At highest dose test (4,030 μg/mL), only 56 metaphases analysed

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: No induction of aberrations were observed with or without S9 (Galloway et al., 1987).

Any other information on results incl. tables

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Triethanolamine

Without S9

	Dose (μg/mL)	Total Cells Scored	Number of Aberrations	Aberrations/cells	% cells with abberations
Dimethyl Sulfoxide (solvent control)	-	100	7	0.07	5.0
Mitomycin-C (positive control)	0.5	100	24	0.24	20.0
Triethanolamine	1,510	100	2	0.02	2.0
	2,010	100	1	0.01	1.0
	4,030	56	2	0.00	1.0
P = 0.985 (Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose)

 

With S9

Dose (μg/mL)  Total Cells Scored Number of Aberrations  Aberrations/cell  % Cells with Abberations     Dimethylsulfoxide (solvent control)  - 100  2 0.02  2.0   Cyclophosphamide (positive control) 25  100  32  0.32  26.0   Triethanolamine          6,040  100  3  0.03  3.0  8,060  100  6  0.06  5.0  10,070  100  2  0.02  2.0  P = 0.373

Applicant's summary and conclusion

Conclusions:

An in vitro mammalian chromosome aberration test was perfomormed on cultured Chinese Hamster Ovary (CHO) cells using the test item triethanolamine with and without metabolic activation. Each test consisted of concurrent solvent and positive controls. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 +/- 2 chromosomes). Cytotoxicity was noted at the highest dose tested (4,030 µg/mL) in the aberration test without metabolic activation. No induction of aberrations was observed with or without matabolic activation.

Executive summary:

Triethanolamine (TEA) was tested for Chromosome Aberrations in Chinese Hamster Ovary Cells as an evaluatuion of 108 Chemicals. All chemicals were tested with and without exogenous metabolic activation, using a developed screening protocol for detecting chemically induced cytogenetic changes in vitro [Galloway et al, 1985] .The protocol was designed to allow testing up to toxic doses and concentrations evaluated were from 1,510 - 4,030 µg/mL (with S9) and 6,040 -10,100 µg/mL (without S9) . 100 metaphase cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive and solvent controls. The results were negative even up to doses that induced toxicity (with and without S9). TEA was therefore negative for structural chromosomal aberrations in this in vitro test system. GLP is not specified, however there is clear methodology and reference to evaluation of criteria for a positive response. The study is considered reliable and comparable to a guideline study.

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