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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is considered to be non-mutagenic, based on the results of an Ames test with AMPD and read-acrosse from an in vitro chromosome aberration study and an in vitro gene mutation study with AEPD.

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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:
29 Sep - 09 Oct 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Basic data given: TA102 or E. coli WP2 are not included in the testing. According to OECD 471, at least five strains of bacteria should be included in the testing. In this Ames test, only four S. typhimurium strains (TA1535, TA1537, TA98, TA100) were used. It is known that these strains have not the potential to detect certain types of mutagens, such as cross-linking agents or oxidising mutagens. In order to detect such mutagens it is required to include S. typhimurium TA102 or to add a DNA repair-proficient strain of E.coli (e.g. E.coli WP2 uvrA or E.coli WP2 uvrA (pKM101)).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
- only 4 strains tested
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
- only 4 strains tested
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Strains are regularly checked for their histidine-requirement, crystal violet sensitivity, UV-sensitivity, ampicillin resistance (TA98 and TA100) and the frequency of spontaneous revertants.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat livers from adult males (Wistar or Sprague Dawley) intraperitoneally injected with a solution (20% w/v) of Aroclor 1254 (500 mg/kg bw) in corn oil
Test concentrations with justification for top dose:
with S9-mix: 100, 300, 1000, 3333, 5000
without S9-mix: 100, 300, 1000, 3333, 5000
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: milli-Q water (Millipore corp., Bedford, Mass., USA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9-mix: sodium azide (TA1535, 1 µg in saline), 9-aminoacridine (TA1537, 60 µg in saline, daunomycine (TA98, 2 µg in saline), methylmethanesulfonate (TA100, 650 µg in DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9-mix: 2-aminoanthracene (TA1535, TA1537: 0.5 µg in DMSO; TA98, TA100: 5 µg in DMSO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2 experiments with and without S9-mix, each experiment with 3 replicate plates

DETERMINATION OF CYTOTOXICITY
- Method: viability determination
Evaluation criteria:
When the number of reverse mutation colonies increased by almost twice the solvent control or more, and reproducibilityor dependence on the dose of the test substance was observed, the result was considered positive.
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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:
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity determination of the test substance in TA100 was done. The percentage survival of the TA100 culture was determined by comparing the number of colonies on the solvent control plate with those on the plates containing the test substance. Based on these data, the AMPD was tested in the main test up to a concentration of 5000 µg/plate in the absence and presence of S9-mix

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain -specific positive control values fell within the laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

Table 1: Results of the main test

1st experiment

number of revertants/plate: mean value of vehicle/solvent control ± SD

number of revertants/plate: mean value of positive control ± SD

max. number of revertants/plate: mean value of test material ± SD [concentration in experiment (µg/plate) resulting in max. number of revertants/plate]

without S9-mix

TA 98

17 ± 1

1621 ± 85

25 ± 1 [100]

TA 100

94 ± 11

673 ± 51

95 ± 16 [5000]

TA 1535

12 ± 3

195 ± 20

10 ± 3 [100]

TA 1537

12 ± 3

463 ± 35

9 ± 3 [100]

with S9-mix

TA 98

22 ± 2

440 ± 26

27 ± 5 [3330]

TA 100

85 ± 3

725 ± 83

95 ± 6 [100]

TA 1535

11 ± 3

358 ± 17

12 ± 4 [100]

TA 1537

7 ± 1

931 ± 59

10 ± 4 [3330]

2nd experiment

number of revertants/plate: mean value of vehicle/solvent control ± SD

number of revertants/plate: mean value of positive control ± SD

max. number of revertants/plate: mean value of test material ± SD [concentration in experiment (µg/plate) resulting in max. number of revertants/plate]

without S9-mix

TA 98

30 ± 3

1033 ± 83

46 ± 4 [1000]

TA 100

127 ± 16

883 ± 15

152 ± 17 [3330]

TA 1535

20 ± 2

210 ± 45

18 ± 8 [1000]

TA 1537

8 ± 2

273 ± 55

8 ± 3 [5000]

with S9-mix

TA 98

36 ± 5

904 ± 5

42 ± 4 [333]

TA 100

121 ± 4

1059 ± 73

139 ± 14 [100]

TA 1535

15 ± 5

203 ± 22

14 ± 3 [333]

TA 1537

6 ± 1

748 ± 23

9 ± 4 [333]

 

 

 

Conclusions:
Interpretation of results (migrated information): negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
February 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
See attached (in chapter 13 of IUCLID) document with the justification for the category/read-across approach.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
other: In vitro chromosome aberration assay
Specific details on test material used for the study:
- Name: 2-amino-2-ethyl-1,3-propanediol
- CAS No.: 115-70-8
- Structural formula: C2H5C(CH2OH)2NH2
- Purity: 99.4%
- Characteristics: slightly yellow, transparent viscous liquid.
- Molecular weight: 119.16
- Stability: it is judged to be stable during the experimental period and no problems with quality were observed based on analysis of the experimental material following the experiment
- Storage method: a cold dark place
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung (CHL/IU) cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 of rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
-S9 mix(short-term treatment); 0, 75, 150, 300, 600 and 1200 μg/mL
+S9 mix(short-term treatment); 0, 75, 150, 300, 600 and 1200 μg/mL
-S9 mix(continuous treatment 24 hr); 0, 75, 150, 300, 600 and 1200 μg/mL
-S9 mix(continuous treatment 48 hr); 0, 75, 150, 300, 600 and 1200 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiol. saline
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Key result
Species / strain:
mammalian cell line, other: Chinese hamster lung (CHL/IU) cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No increase in chromosomal aberrations was observed in the test with either the short- term treatment (-S9 and +S9) or continuous treatment.
Polyploidy: Negative with and without metabolic activation
Conclusions:
No increase in chromosomal aberrations was observed in the test with either the short- term treatment (-S9 and +S9) or continuous treatment.
Executive summary:

An in vitro chromosome aberration test using cultured Chinese hamster lung cells (CHL/IU) was conducted to study the clastogenicity of 2-amino-2-ethyl-1,3-propanediol.The maximum concentration used was 1200 µg/ml, determined on the basis of a cytotoxicity test. In the main test, the following concentrations were used: 75, 150, 300, 600 and 1200 µg/ml. Treatment times were 24 (short-term treatment) or 48 hours (continueous treatment). The test substance did not induce an increase in the number of structural chromosome aberrations, either in the absence or in the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See attached (in chapter 13 of IUCLID) document with the justification for the category/read-across approach.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro gene mutation study in mammalian cells
Specific details on test material used for the study:
- Test Article Name: XU-12399.00
- Chemical Name: 2-Amino-2-ethyl-1,3-propanediol
- Supplied by: ANGUS Chemical Company, a wholly owned subsidiary of The Dow Chemical Company, Buffalo Grove, Illinois USA
- Physical Appearance: yellow viscous liquid to semi solid
- Purity as per Certificate of Analysis: the purity of the test material was determined to be 99.2% ± 0.06% wt./wt. by gas chromatography with identification by gas chromatography mass spectrometry and nuclear magnetic resonance (Megregian and Pell, 2010).
- Lot No.: 201000687-21
- Molecular Weight: 119.2
- Manufacture Date: not applicable
- Recertification Date: 14 October 2012
- Recommended Storage Condition: ambient (+18 to +36 ºC)
Target gene:
This mammalian cell mutation assay system detects point mutations involving base substitutions, deletions, frameshifts and rearrangements within the locus. The enzyme hypoxanthine guanine phosphoribosyl transferase (hprt) catalyses phosphorylation of purines in one of the purine salvage pathways. The selective agent used in this assay, 6-Thioguanine (6-TG), is also a substrate for this enzyme and cells that retain the functional hprt enzyme are susceptible to the cytotoxic effects of 6-TG. Forward mutations that result in the loss of the functional hprt gene render cells resistant to 6-TG. These mutant cells can be quantitated after an expression period by cloning in culture medium supplemented with 6-TG, the selective agent.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a model chromosome number 20 and a population doubling time of 10 to 14 hours was used. Batch No. 2 of this CHO-K1 cell line was tested for the absence of mycoplasma contamination at Mycoplasma Laboratory, Statens Serum Institut, Denmark and certified free of mycoplasma contamination on July 8, 2010. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals. Cells were grown in tissue culture flasks at 37 ± 1 ⁰C in a carbon dioxide incubator (5 ± 0.2 % carbon dioxide in air).

Source of the Test System:
American Type Culture Collection, P.O.Box 1549, Manassas, VA 20108, USA

Storage of Test System:
Stock cultures of the CHO-K1 cell line were stored at the test facility as frozen permanents in liquid nitrogen.

Test Medium, Solutions and other Chemicals:
- Ham’s F-12 medium supplemented with sodium bicarbonate, antibiotics and L-glutamine was the basic medium.
- Basic medium supplemented with 10% fetal bovine serum (FBS) was the complete medium and was used for the growth and multiplication of cells as well as in detaching and diluting the cells.
- Basic medium without serum was the treatment medium and was used for target cell exposure to the test article and controls.
- Cloning medium was basic medium supplemented with 20% FBS and was used for the determination of cell viability or plating/cloning efficiency.
- Selective medium was basic medium supplemented with 20% FBS and the selective agent 6-Thioguanine (6-TG) at 35 μM and was used for the selection of mutants.
- Dulbecco’s Phosphate buffered saline (PBS) (pH: 7.4)
- Trypsin: EDTA solution
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 homogenate
Test concentrations with justification for top dose:
Based on the results of the cytotoxicity test, the following test doses were selected for testing in the initial gene mutation assay in the presence and absence of metabolic activation: 12, 38, 119, 377, and 1192 μg/mL (factor of √10).
Vehicle / solvent:
Sterile distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The results of the forward gene mutation assay at the hprt locus with XU-12399.00 (AEPD) indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the presence or absence of an externally supplied metabolic activation (S9) system.
Executive summary:

The genotoxic potential of the test article XU-12399.00 to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells. The study consisted of a preliminary toxicity test, an initial gene mutation assay and a confirmatory gene mutation assay. Each of these mutation assays comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver). XU-12399.00 formed a solution in sterile distilled water (SDW) at 500 mg/mL and was found to be stable in water at concentrations of 15 and 50000 μg/mL for 24 hours. In a preliminary cytotoxicity test XU-12399.00 did not cause a significant cell growth inhibition as evaluated by Relative Cloning Efficiency (RCE) up to the highest tested concentration of 1192 μg/mL (equivalent to 10 mM) in the presence as well as in the absence of metabolic activation. The test article did not precipitate in the treatment medium up to 1192 μg/mL, and did not cause any appreciable change in the osmolarity of the test solutions at the end of 4-hour exposure to treatment either in the presence or in the absence of metabolic activation. However, the test article altered the pH of the test solutions at and above 298 μg/mL in the presence of metabolic activation and at and above 149 μg/mL in the absence of metabolic activation, therefore; the pH of the test solutions of these concentrations were adjusted to neutrality before exposure to the cells. In the initial gene mutation assay, CHO cells were exposed to the test article in duplicate at concentrations of 12, 38, 119, 377, and 1192 μg/mL of the medium for 4 hours in the presence and absence of metabolic activation. In the confirmatory gene mutation assay, CHO cells were exposed to the test article in duplicate at concentrations of 15, 44, 132, 397 and 1192 μg/mL of the medium for 4 hours in the presence and absence of metabolic activation.

There was no evidence of induction of gene mutations in any of the test material treated cultures either in the presence or absence of metabolic activation. In each of these experiments, the respective positive controls produced a statistically significant increase in the frequencies of mutants, under identical conditions and concurrent solvent control cultures values were within laboratory historical controls. The results of the forward gene mutation assay at the hprt locus with XU-12399.00 indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the presence or absence of an externally supplied metabolic activation (S9) system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The in vitro genetic toxicity of 2-amino-2-methyl-1,3-propanediol (AMPD) was investigated in a bacterial reverse mutation assay (Ames test) according to OECD 471 (Enninga, 1988). The plate incorporation method was conducted with S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations up to 5000 µg/plate. AMPD did not induce mutations in the S. typhimurium strains in the presence and in the absence of a metabolic activation system. The positive and negative controls included in the experiment showed the expected results. No cytotoxic effects were observed in the concentrations tested with and without metabolic activation. However, only four test strains were used. According to OECD 471, at least five strains of bacteria should be included in the testing. It is known that the four S. typhimurium strains (TA1535, TA1537, TA98, TA100) tested have not the potential to detect certain types of mutagens, such as cross-linking agents or oxidising mutagens. In order to detect such mutagens it is demanded to include S. typhimurium TA102 or to add a DNA repair-proficient strain of E.coli (e.g. E.coli WP2 uvrA or E.coli WP2 uvrA (pKM101)). Therefore, only basic data given and further reliable data are required. However, there are no further data available on the genotoxicity potential of AMPD. Reliable data exist only for another member of the chemical category. Therefore, read-across was conducted based on a category approach.

Within the chemical category AMPD belongs to, data are available for 2-amino-2-ethyl-1,3-propanediol (AEPD; CAS 115-70-8).The in vitro genetic toxicity of AEPD was investigated in a bacterial reverse mutation assay (Ames test) according to OECD 471 (Mochizuki, 2004). The preincubation method was conducted with S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA at concentrations up to 5000 µg/plate. AEPD did not induce mutations in any of the S. typhimurium strains or in E. coli WP2 uvrA in the presence or in the absence of a metabolic activation system.No cytotoxic effects were observed in the concentrations tested with and without metabolic activation.The positive controls were valid.

According to OECD 473, the clastogenic potential of AEPD to induce chromosomal aberrations was tested in cultured Chinese hamster lung (CHL) cells (Sono, 2004). CHL cells were exposed to AEPD at concentrations up to 1200 µg/mL both with and without metabolic activation. No increase in chromosomal aberrations was observed in the experiments with short-term treatment (6 h) in presence or absence of metabolic activation. The test substance was not cytotoxic in the tested concentration range both with and without metabolic activation. The positive controls showed the expected results. Because of the negative results of the short-term treatment, an additional testing without activation was done with continuous treatment (24 and 48 h). After continuous treatment, AEPD did also not induce chromosomal aberrations in CHL cells.

AEPD was also tested for its mutagenic potential in another in vitro mammalian cell mutation assay (HGPRT) in CHO cells according to OECD 476. Both in the presence and in the absence of a metabolic activation system, no increases in mutant frequency were observed in any of the experiments. The positive controls induced highly significant increases in mutant frequency. No cytotoxic effects were observed for the concentrations tested with and without metabolic activation.


Short description of key information:
AMPD:
Negative Ames test with TA1535, TA1537, TA98 and TA100 both in the presence and in the absence of a metabolic activation system.

Read across from a structurally related substance within a category approach:
Negative Ames test with TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA both in the presence and in the absence of a metabolic activation system.
Negative results in a mammalian chromosomal aberration test with cultured Chinese hamster lung cells.
Negative results in a mammalian cell gene mutation test using CHO cells with and without metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification