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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Health effects testing guidelines 40 CFR Part 798.5300
Deviations:
no
Remarks:
none specified
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Remarks:
not specified
Qualifier:
according to
Guideline:
other: EEC In vitro mammalian cell gene mutation test Official Journal of the European Communities, L133, 31, 61-63
Deviations:
no
Remarks:
none specified
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity was 99.21%

Analysis of Dose Solutions by HPLC with Refractive Index Detection

Expt. B1 Expt. B2

Target Observed Target Observed
mg/ml mg/ml mg/ml mg/ml
500 551 500 542
167 189 167 184
50 60.5 50 57.8
17 20.5 16.7 20.0
5 7.22 5 6.97

Method

Target gene:
HGPRT
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
In cultured chinese hamster ovary (CHO) cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver homogenate
Test concentrations with justification for top dose:
50, 167, 500, 1667 and 5000 µg/ml
Vehicle / solvent:
water
Details on test system and experimental conditions:
CHO/HPGRT Mutation Assays

In this study, Triisopropanolamine 99 was tested in two independent assays. Duplicate cultures seeded with 5x10E5 cells/flask were used at each dose level.

For parallel toxicity determination, three plates with 200 cells/plate were used at each dose level. The cells were treated for 5 hours with and without metabolic activation.

In the mutation assay, the cells were treated at test article concentrations of 50, 167, 500, 1667, and 5000 µg/ml both with and without metabolic activation. Appropriate positive, solvent, and untreated controls were also included in the assays. After the exposure time, the cells were washed with HBSS, refed with complete Hx-free medium, and allowed to grow for 18-24 hours. The cells were then subcultured to initiate cultures for the expression of mutant phenotype. The cytotoxicity determination was performed as described in the rage finding test. The cytotoxicity was expressed as RCS.

For the expression of 6-thioguanine-resistant mutants (HGPRT locus mutants), the cells from each of the duplicate culture flasks were subcultured with 5% dialyzed HIFBS, 2mM L-glutamine, 50 Units/ml of penicillin and 50 µg/ml streptomycin (cloning medium) at a density of 1x10E6 cells/75 cm2 flask. The cells were subcultured as described above at 2- to 3-day intervals for a period of 9 days in the first assay and 8 days in the confirmatory assay prior to selecting the mutant phenotypes. The cells were grown as attached cultures in T-75 cm2 tissue culture flasks.

After the expression period, the cells from each of the treatment replicates were harvested and seeded in five 100 mm2 tissue culture plates at a density of 2x10E5 cells/plate in 10 ml of cloning medium containing 10 µM of 6-tioguanine (TG). To determine the cloning efficiency of the cells at the time of selection, 200 cells/60 mm2 dish were plated in triplicate in the cloning medium. All clonable test doses and appropriate positive and solvent controls were cloned for mutant selection. The cultures were then incubated for 7 days without disturbing the plates to minimize the formation of satellite colonies. The colonies were then washed with PBS, fixed, stained and counted for cloning efficiency and mutant selection. The average number of clones from the triplicate plates was calculated and the percent clonable cells (cloning efficiency) for each treatment condition was determined. The number of TG-resistant mutants for 1x10E6 cells seeded was calculated by totaling the number of mutants from the five replicate plates. Based on the cloning efficiency, the number of TG-resistant mutants per 1x10E6 surviving cells was calculated for each dose.
Evaluation criteria:
The test article is considered positive in the assay if it induces a statistically significant and reproducible increase in mutation frequency at more than one of the dose levels tested. The final interpretation of the data also takes into consideration such factors as the mutation frequency and cloning efficiencies in the negative controls and dose-response relationships.
Statistics:
The following method of statistical analysis was performed: The frequencies of mutants per 1x10E6 clonable cells were analyzed by the Cochran-Armitage test for trend (≤ 0.025, one-sided, increasing) and the Fisher-Irwin exact test for group comparisons for proportions (≤ 0.01, one sided). The within-group replicates were pooled for the final analysis.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Range Finding Test: In the range finding test, the test article was tested at dose levels of 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500, 1000 and 5000 ug/ml.

The test cultures seeded approximately 18-24 hours earlier. Triplicate culture plates were used at each test article and solvent control dose level.

In the activated system, the medium was removed and 5 ml of serum-free culture medium containing appropriate concentrations of S-9 mixture and test article was added to each of the culture plates. The cells were exposed to the test article for 5 hours. After the exposure period, the cells were washed with HBSS and refed with complete Hx-free medium.

In the non-activated system, the medium was removed, and 5 ml of test article containing serum-free culture medium was added to each of the culture plates. The cells were then exposed to the test article for 5 hours, washed with HBSS, and refed with complete Hx-free medium.

The cells were allowed to grow for a period of 7 days without any disturbance to minimize the formation of satellite colonies. The colonies were then washed with phosphate buffered saline (PBS), fixed with methanol, stained with Giemsa and counted. A cluster of more than 50 cells growing within a confined area was considered a colony. The average number of colonies per plate was calculated, and the relative cell survival (RCS) was determined by the following formula:
RCS = Average No. of Colonies in Test Plates/Average No. of Colonies in Solvent Plates x 100


Analysis of Dose Solutions by HPLC with Refractive Index Detection
Expt. B1 Expt. B2
Target Observed Target Observed
mg/ml mg/ml mg/ml mg/ml
500 551 500 542
167 189 167 184
50 60.5 50 57.8
17 20.5 16.7 20.0
5 7.22 5 6.97

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

CHO/HGPRT Mutation Assay - Exp. 1

                Without Activation     With S-9 Activation
Conc. (ug/ml)
   Mutants/10-6 Cells     Mutants/10-6 Cells
 
Sol. A
                4                       3
Sol. B
               14                      25

50 A
                 21*                      5
50 B
                 27*                      8

167 A
                12                      28*
167 B
                13                      19*

500 A
                20*                      6
500 B
                51*                      4

1667 A
               14                      10
1667 B
                6                      17

5000 A
               13                       3
5000 B
                0                        2
 
* Statistically Significant Dose (p<0.01)

CHO/HGPRT Mutation Assay - Exp. 2

                Without Activation     With S-9 Activation
Conc. (ug/ml)
   Mutants/10-6 Cells     Mutants/10-6 Cells
 
Sol. A
                2                       4
Sol. B
                2                       1

50 A
                  9                       3
50 B
                  3                       1

167 A
                 6                       4
167 B
                 1                       7

500 A
                 4                       0
500 B
                 5                       0

1667 A
                8                       8
1667 B
                3                       2

5000 A
                3                       3
5000 B
                0                       1



Based upon the frequency of the TG-resistant mutants recovered in cultures treated with the test material, it was concluded that TIPA 99 did not induce a mutagenic response in the assay system employed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based upon the frequency of the TG-resistant mutants recovered in cultures treated with the test material, it was concluded that TIPA 99 did not induce a mutagenic response in the assay system employed.
Executive summary:

Triisopropanolamine 99 was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two separate assays. The test material was assayed at concentrations ranging from 50 to 5000 pg/ml in the absence and presence of an externally supplied metabolic activation (S-9) system. The adequacy of the experimental conditions for detection of induced mutations was confirmed by employing positive control chemicals (ethyl methane sulfonate for assay without S-9 and 7,12-dimethylbenz(a)anthracene for assays with S-9). Negative control cultures were treated with the solvent used to dissolve the test material. Based upon the frequency of TGr mutants recovered in cultures treated with the test material, it was concluded that triisopropanolamine 99 did not induce a mutagenic response in the assay system employed.