1-(N,N-bis(2-hydroxyethyl)amino)propan-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a GLP study (reliability 1), conducted according to OECD guideline 422, DEIPA showed no convincing evidence of mutagenic activity when tested at concentrations of up to 5000 µg/plate in a bacterial reverse mutation assay with S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2uvrA, with and without S9 (Mecchi, 1999). In a GLP study (reliability 1), conducted according to OECD guideline 473, DEIPA was considered to be negative in an in vitro chromosome aberration assay using rat lymphocytes, with and without S9 (Linscombe et al., 1999). In a GLP study (reliability 1), conducted according to OECD 476, TIPA 99 failed to induce a reproducible, statistically significant and dose-related increase in mutation frequency in CHO cells in vitro when tested at up to 5000 ug/mL in the presence or absence of S9 (Sitek Research Laboratories, 1993).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jan to 12 Feb 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine locus
E. coli WP2 uvr A: Tryptophan locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from Aroclor-induced rat liver

Test concentrations with justification for top dose:

100, 330, 1000, 3330, 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Not specified [OECD guideline recommends the use of aqueous solvent/vehicle]

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene and ICR-191

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

Preincubated in the presence of the tester strain for 20 mins at 37oC before adding the overlay agar and pouring onto the surface of minimal agar plates. The plates were incubated for 52 +/- 4 hours at 37 +/- 2oC.

DURATION
- Preincubation period:20 +/- 2 minutes
- Exposure duration: 48-56 hours

SELECTION AGENT (mutation assays): Deficiency in histidine or tryphtophan for selection of non-amino acid requiring mutants.

NUMBER OF REPLICATIONS: 2 independant assays

DETERMINATION OF CYTOTOXICITY
- Method: A decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn indicates cytotoxicity

Evaluation criteria:

The test article had to produce at least a 3-fold (TA98, TA1535, TA1537 and WP2uvr A) or 2-fold (TA100) dose related and reproducible increase in the mean revertants per plate of at least one tester strain over the mean revertants per plate of the appropriate vehicle control

Statistics:

None reported

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Not applicable

RANGE-FINDING/SCREENING STUDIES: Ten doses of test article from 6.67 to 5000 ul per plate were tested. No cytotoxicity observed with TA100 and WP2uvrA either in presence or absence of metabolic activation system, normal backgroud lawn evident and no decrease in number of revertants per plate. No test article precipitate observed at any of the doses tested.

COMPARISON WITH HISTORICAL CONTROL DATA: Revertants were within the range of historical data for the vehicle and positive controls with each of the strains tested, both with and without S9.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None reported

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In a GLP study, conducted according to OECD guideline 471, DEIPA showed no convincing evidence of mutagenic activity when tested at concentrations of up to 5000 µg/plate in a bacterial reverse mutation assay with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2uvrA, with and without metabolic activation (S9).

Executive summary:

In a GLP study, conducted according to OECD guideline 471, DEIPA was assessed for mutagenicity in a bacterial reverse mutation assay.

Two independent studies using the preincubation method were performed using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2uvrA in the presence and absence of a rat liver metabolic activation (S9 mix). Concentrations of 100, 333, 1000, 3330, and 5000 µg/plate of DEIPA were tested, along with concurrent positive and vehicle controls, all plated in triplicate.The positive control substances all showed mutagenic activity, demonstrating that the test was valid.

Following incubation of the test material, tester strain and S9 mix (or phosphate buffer), the number of revertant colonies per plate was counted. Cytotoxicity was evaluated based on the condition of the bacterial background lawn. No precipitation of the test article was obseved at any dose level.

Under the conditions of this study, DEIPA did not cause a positive increase in the number of revertant colonies per plate and no cytotoxicity was evident, with any of the tester strains, at any concentration, either in the presence or absence S9.

DEIPA showed no convincing evidence of mutagenic activity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In a GLP study, conducted according to OECD Guideline 471, DEIPA showed no convincing evidence of mutagenic activity when tested at concentrations of up to 5000 µg/plate in a bacterial reverse mutation assay with S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2uvrA, in the presence or absence of a mammalian metabolic activation (S9) system (Mecchi, 1999).

In a GLP study, conducted according to OECD Guideline 473, DEIPA was assessed for its potential to induce chromosome damage in anin vitrochromosome aberration assay utilising rat lymphocytes. In an initial assay (assay 1), approximately 48 hr after the initiation of whole blood cultures, cells were treated for 4 hr with the test material in culture medium at concentrations of 1.63, 5.44, 16.32, 54.4, 163.2, 544 and 1632 µg/ml, with and without S9. After washing to remove the test material the cells were incubated for a further 20 hr before harvesting. In a confirmatory assay (assay 2), cultures were treated as in assay 1 except when in absence of S9, cultures were treated continuously for 24 hr before being harvested. One hundred metaphases/replicate were scored for chromosome aberrations (a total of 200 metaphases/dose). The incidence of polyploid cells was also evaluated. The mitotic index was used to determine cytotoxicity. In assay 1 and 2, there were no statistical differences in aberrant cell frequency between the negative control and any of the test material treated cultures, with or without S9, with the exception of the highest dose (1632 µg/ml) with S9 in assay 2. However this was not considered biologically significant. Instead, it was thought to be a chance occurrence unrelated to treatment since the increase only occurred in one replicate and no increase in aberration frequency occurred in assay 1 at the high dose. There was no significant increase in the incidence of polyploid cells as compared to the negative control values, with or without S9. Under the conditions of this study, DEIPA did not induce a significant increase in the frequency of cells with chromosomal abnormalities at any concentration tested, with or without S9.

In summarising these studies, an expert group concluded that DEIPA "was found to be non-mutagenic in a bacterial reverse mutation assay, and non-clastogenic in a chromosomal aberration test". Therefore, DEIPA "is not expected to be a mutagen or clastogen in vivo" (NICNAS, 2009).

No gene mutation data in mammalian cells were identified for DEIPA. However, TIPA 99 was evaluated in an in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay, conducted according to OECD Guideline 476 and to GLP. The test material was assayed at concentrations ranging from 50 to 5000 μg/ml in the absence and presence of S9. In experiment 1, a statistically significant increase in gene mutations was observed at doses of 50 and 500 μg/ml without S9 and at 167 μg/ml with S9. However, there was no dose-response relationship seen and these increases were not reproducible (i.e. no statistically significant increases were seen in experiment 2). In conclusion, TIPA 99 failed to induce a reproducible, statistically significant and dose-related increase in mutation frequency in CHO cells in vitro when tested at up to 5000 μg/mL in the presence or absence of S9, and is therefore considered to not be mutagenic in this test system. Indeed, in reviewing this study for SIAM 29, experts at the OECD concluded that TIPA did not cause gene mutations in CHO cells (OECD, 2009).

References

NICNAS (2009). Australian National Industrial Chemicals Notification and Assessment Scheme. Full Public Report. DEIPA. File No: STD/1344. December 2009.http://www.nicnas.gov.au/publications/CAR/new/Std/StdFULLR/std1000FR/std1344FR.pdf 

OECD (2009). Organisation for Economic Co-operation and Development.SIDS Initial Assessment Report for SIAM 29. The Hague, Netherlands, 20-23 October 2009. Propanolamines category. July 2009.

Justification for selection of genetic toxicity endpoint
GLP, OECD guideline study (reliability 1).

Justification for classification or non-classification

Based on the lack of mutagenic potential observed in guideline studies (with DEIPA and TIPA), classification of DEIPA as mutagenic according to EU CLP regulations is not warranted.