Ethanol, 2-amino-, reaction products with ammonia, by-products from

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Nov 2013 - 06 Feb 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol, 2-amino-, reaction products with ammonia, by-products from

EC Number:

272-729-4

EC Name:

Ethanol, 2-amino-, reaction products with ammonia, by-products from

Cas Number:

68910-05-4

Molecular formula:

Unspecified

IUPAC Name:

Ethanol, 2-amino-, reaction products with ammonia, by-products from

Details on test material:

- Name of test material (as cited in study report: Amix 1000
- Physical state: Liquid, red brown, turbide
- Analytical purity: 100 area-% UVCB substance
- Lot/batch No.: AMF24390H0
- Expiration date of the lot/batch:The stability of the test substance under storage conditions throughout the study period was guaranteed until
24 Jun 2015 as indicated by the sponsor.
- Storage condition of test material: Room temperature
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Culture medium: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS).
- Treatment medium (without S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf
serum.
- Treatment medium (with S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine.
- Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with: hypoxanthine (13.6 x 10-3 mg/mL), aminopterin (0.18 x 10-3 mg/ mL), thymidine (3.88 x 10-3 mg/mL), 10% (v/v) fetal calf serum (FCS).
- Selection medium ("TG" medium): Hypoxanthine-free Ham's F12 medium supplemented with: 6-thioguanine (10 μg/mL), 1% (v/v) stable glutamine (200 mM), 10% (v/v) fetal calf serum (FCS).
- All media were supplemented with: 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes; During the week prior to treatment, any spontaneous HPRT-deficient mutants w ere eliminated by pretreatment with "HAT" medium.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

1st Experiment
without S9 mix: 0; 625.0; 1250.0; 2500.0; 5000.0 μg/mL
with S9 mix: 0; 625.0; 1250.0; 2500.0; 5000.0 μg/mL
2nd Experiment
without S9 mix: 0; 937.5; 1875.0; 3750.0; 5000.0 μg/mL
with S9 mix :0; 937.5; 1875.0; 3750.0; 5000.0 μg/mL
Concentrations in parentheses were not analyzed.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: The test substance was an aqueous solution, therefore culture medium (Ham's F12) was used as most suitable vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

300 μg/mL EMS , 1.25 μg/mL DMBA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9)
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures were used for all experimental groups.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range of 0.00 – 16.43 mutants per 10E6 clonable cells
• The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies (historical positive control data).
• At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment criteria
A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10E6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.0 5 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest requiried test substance concentration

RANGE-FINDING/SCREENING STUDIES:
In the pretest the parameters pH value and osmolarity were not relevantly influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.However, a slight pH shift was observed at the highest required concentration prior to testing.
Therefore, the pH of the stock solution was adjusted to a physiological value prior to application using small amounts of 32% (w/v) HCl.
In addition, in culture medium, no test substance precipitation occurred up to the highest applied concentration in the absence and presence of S9 mix.
After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival when tested up to the highest required concentration.

COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments after 4 hours treatment with the test the values for the corrected mutation frequencies were close to the respective vehicle
control values and clearly within the range of our historical negative control data.. The positive control substances induced a clear increase in
mutation frequencies, as expected. The values of the corrected mutant frequencies were clearly within the historical positive control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were
obser ed in all experiments under all test conditions up to the highest required concentration. The cell densities were not distinctly reduced.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

EXPERIMENTAL RESULT

						Cytotoxicity***
Exp	Exposure period [h]	Test groups [μg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	CE1 [%]	CE2 [%]
1	4	Negative control	-	-	0.00	100.0	100.0
		625.0	-	-	0.88	97.2	97.1
		1250.0	-	-	0.34	99.4	90.2
		2500.0	-	-	3.91	103.9	93.4
		5000.0	-	-	1.96	78.3	96.0
		Positive Control (EMS)	-	-	209.61	104.7	88.6
2	4	Negative control	-	-	3.15	100.0	100.0
		937.5	-	-	1.36	109.4	96.0
		1875.0	-	-	2.85	99.7	93.9
		3750.0	-	-	1.00	108.0	119.6
		5000.0	-	-	2.90	116.2	104.0
		Positive Control (EMS)	-	-	83.11	116.7	96.5
1	4	Negative control	+	-	0.87	100.0	100.0
		625.0	+	-	0.70	106.5	88.1
		1250.0	+	-	2.79	108.3	88.8
		2500.0	+	-	1.25	107.6	91.8
		5000.0	+	-	0.53	113.5	94.1
		Positive Control (DMBA)	+	-	418.66	98.7	66.8
2	4	Negative control	+	-	4.26	100.0	100.0
		937.5	+	-	0.31	116.2	101.8
		1875.0	+	-	3.26	105.6	100.3
		375050	+	-	0.90	109.1	110.5
		5000.0	+	-	4.54	105.1	99.1
		Positive Control (DMBA)	+	-	240.74	92.7	65.4

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 10⁶ cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells
in the absence and the presence of metabolic activation.

Executive summary:

The test substance Amix 1000 was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested and evaluated.

1st Experiment

without S9 mix (4-hour exposure period)

0; 625.0; 1 250.0; 2 500.0; 5 000.0 μg/mL

with S9 mix (4-hour exposure period)

0; 625.0; 1 250.0; 2 500.0; 5 000.0 μg/mL

2nd Experiment

without S9 mix (4-hour exposure period)

0; 937.5; 1 875.0; 3 750.0; 5 000.0 μg/mL

with S9 mix (4-hour exposure period)

0; 937.5; 1 875.0; 3 750.0; 5 000.0 μg/mL

Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 hours in the absence and the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance Amix 1000 is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.