Ethanol, 2-amino-, reaction products with ammonia, by-products from, distn. Residues

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic.

Link to relevant study records

Reference

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:

2010-08-14 until 2010-09-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to OECD 476

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: Clone 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 45.7; 91.3; 182.5; 365; 730; and 1460 µg/mL
with S9 mix: 45.7; 91.3; 182.5; 365; 730; and 1460 µg/mL
Experiment II:
without S9 mix: 91.3; 182.5; 365; 730; 1095; and 1460 µg/mL
with S9 mix: 91.3; 182.5; 365; 730; 1095; and 1460 µg/mL
Following the expression phase of 48 hours the cultures at the lowest concentrations in experiment I and II with and without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water (10 %)
- Justification for choice of solvent/vehicle: solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells (global evaluation factor, GEF) above the corresponding solvent control. The GEF was introduced by the International Workshop on Genotoxicity Testing (IWGT) to replace the former threshold of twice the mutation frequency of the corresponding solvent control (12).
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent con¬trols of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

Linear regression analysis (least squares) using SYSTAT(R) 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Effects of pH: the pH was adjusted with 2N HCl at the maximum test item concentration in the pre-experiment
- Effects of osmolality: Not increased
- Evaporation from medium: Not examined
- Water solubility: --
- Precipitation: none
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 11.4 µg/mL and 1460 µg/mL were used. The highest concentration in the pre-experiment was about 10 mM based on the
purity (100 % active ingredient) and the molecular weight (approx. 146 g/mol) of the test item.
Relevant toxic effect (relative suspension growth, RSG below 50% of the solvent control) occurred at 365 µg/mL and above following 24h
treatment without metabolic activation.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. No
precipitation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
In the pre-experiment the three highest concentrations were neutralised with 1 N sodium hydroxide. There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item.
Therefore, the maximum concentration of the main experiments was adjusted according to the results of the pre-experiment. In both main
experiments the individual concentrations were generally spaced by a factor of 2.0 in the lower range. In the second experiment a narrower
spacing was used at the highest concentrations to cover the cytotoxic range more closely.
To overcome problems with possible deviations in toxicity or solubility both main experiments were started with more than four concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: strain/cell type: in vitro gene mutation assay with L5178Y cells

Remarks:

Migrated from field 'Test system'.

Summary Table

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	10^6cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with water		-	100.0	123	249	100.0	89	215
Pos. control with MMS	19.5	-	37.3	373	249	17.2	950	215
Test item	45.7	-	culture was not continued^#			culture was not continued^#
Test item	91.3	-	118.5	94	249	93.7	101	215
Test item	182.5	-	116.5	120	249	93.2	113	215
Test item	365.0	-	104.5	95	249	108.9	115	215
Test item	730.0	-	104.9	89	249	120.9	97	215
Test item	1460.0	-	86.4	123	249	93.7	101	215
Solv. control with water		+	100.0	131	257	100.0	66	192
Pos. control with CPA	3.0	+	72.2	190	257	28.9	211	192
Pos. control with CPA	4.5	+	30.1	446	257	28.3	252	192
Test item	45.7	+	culture was not continued^#			culture was not continued^#
Test item	91.3	+	128.5	114	257	66.5	118	192
Test item	182.5	+	125.7	97	257	42.9	124	192
Test item	365.0	+	170.7	98	257	83.2	71	192
Test item	730.0	+	176.8	73	257	44.7	140	192
Test item	1460.0	+	149.5	85	257	48.2	106	192
Experiment II / 24 h treatment			culture I			culture II
Solv. control with water		-	100.0	59	185	100.0	113	239
Pos. control with MMS	13.0	-	19.2	336	185	14.8	564	239
Test item	91.3	-	culture was not continued^#			culture was not continued^#
Test item	182.5	-	114.3	41	185	61.0	105	239
Test item	365.0	-	53.5	100	185	53.0	95	239
Test item	730.0	-	29.9	114	185	26.7	112	239
Test item	1095.0	-	16.0	123	185	20.9	111	239
Test item	1460.0	-	7.7	153	185	7.3	228	239
Experiment II / 4 h treatment			culture I			culture II
Solv. control with water		+	100.0	59	185	100.0	92	218
Pos. control with CPA	3.0	+	57.0	133	185	88.2	135	218
Pos. control with CPA	4.5	+	9.7	303	185	53.9	313	218
Test item	91.3	+	culture was not continued^#			culture was not continued^#
Test item	182.5	+	91.2	63	185	105.9	67	218
Test item	365.0	+	105.3	57	185	112.8	80	218
Test item	730.0	+	98.1	79	185	122.6	72	218
Test item	1095.0	+	65.9	79	185	121.6	55	218
Test item	1460.0	+	68.5	99	185	133.5	178	218

Threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

# culture was not continued since a minimum of only 4 analysable concentrations is required

The values printed in bold are judged as invalid, since the acceptance criteria are not met (RTG < 10%).

 

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of Ethanol, 2-amino-, reaction products with ammonia, by-products from (CAS No.: 68910-05-4) to induce mutations at the mouse lymphoma thymidine kinase locus using the cellline L5178Y.

The study was conducted according to the procedures indicated by the following internationally accepted guidelines and recommendations:

Ninth Addendum to the OECD Guidelines for Testing of Chemicals, February 1998,
adopted, Guideline No. 476 “In vitro Mammalian Cell Gene Mutation Test”.

Commission Regulation (EC) No. 440/2008, B17: “Mutagenicity – In vitro Mammalian Cell Gene Mutation Test“, dated.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 4 h with and

24 h without metabolic activation.

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix:             91.3; 182.5; 365; 730; and 1460 µg/mL
with S9 mix:                  91.3; 182.5; 365; 730; and 1460 µg/mL

Experiment II:

without S9 mix:             182.5; 365; 730; 1095; and 1460 µg/mL
with S9 mix:                  182.5; 365; 730; 1095; and 1460 µg/mL

No precipitation of the test item visible to the naked eye was determined in both experiments up to the maximum concentration with and without metabolic activation.

No relevant toxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed in both main experiments up to the maximum concentration with and without metabolic activation, following 4 hours of treatment. After 24 hours of treatment (second experiment without metabolic activation) cytotoxic effects as described above occurred at 730 µg/mL and above. The data generated in both cultures of experiment II at 1460 µg/mL without metabolic activation are not considered valid since the relative total growth fell short of the threshold of 10%. The recommended cytotoxic range of approximately 10-20% RTG was covered in the second experiment without metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments with and without metabolic activation. The mutation frequency did not reach or exceed the threshold of 126 plus each solvent control count.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency using SYSTAT (R) 11 statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the first culture of experiment II with metabolic activation. This trend however, was not considered relevant since it was not reproduced in the parallel culture and all of the absolute values of the mutation frequency remained within the historical range of solvent controls.

In this study the range of the solvent controls was from 59 up to 131 mutant colonies per 10⁶cells; the range of the acceptable groups treated with the test item was from 41 up to 178 mutant colonies per 10⁶cells.

The cloning efficiency exceeded the upper limit of 120% in the second culture of the first experiment and the first culture of the second experiment with metabolic activation. The data are acceptable however, since the cloning efficiency of the parallel culture remained within the acceptable range. Cloning efficiency values above 100% occasionally occur since even suspension cell cultures do not form an ideal solution in medium. The cells tend to form transient aggregates that are counted as single cells during determination of the cell density. The aggregation does not compromise the validity of the data however, since the absolute values of the cloning efficiency are used to calculate the mutation frequency.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies. The CPA-control of the second culture of the second experiment did not quite meet the acceptance criteria (at least 150 induced small colonies) even though the total number of mutant colonies showed a substantial increase from 92 to 313 colonies per 10⁶cells at 4.5 µg/mL. However, more than half of the induced colonies were large ones. The data are acceptable since the corresponding positive control of the parallel culture met the acceptance criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

I Triethylenetetramine, which is a major constituent of this multiconstituent substace, has been found positive in Ames in vitro tests, but has given negative results in an in-vivo Mammalian Erythrocyte Micronucleus Test, according to OECD Guideline 474.

The substance has been tested in an Ames test, and found positive. The test item was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in an experiments with and without liver microsomal activation. Substantial and dose dependent increases in revertant colony numbers were observed following treatment with the substance in strains TA 98,100, and TA 102 in the presence and absence of metabolic activation (S9 mix).

Further in-vitro testing in a mammalian cell gene mutation test gave a negative response. The study was performed to investigate the potential of Ethanol, 2-amino-, reaction products with ammonia, by-products from (CAS No.:

68910-05-4) to induce mutations at the mouse lymphoma thymidine kinase locus using the cellline L5178Y. The study was conducted according to the procedures indicated by the following internationally accepted guidelines and recommendations:

Ninth Addendum to the GECD Guidelines for Testing of Chemicals, February 1998,adopted, Guideline No. 476 "In vitro Mammalian Cell Gene Mutation Test. Commission Regulation (EC) No. 440/2008, B17: "Mutagenicity - In vitro Mammalian Cell Gene Mutation Test. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 4 h with and 24 h without metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments with and without metabolic activation. The mutation frequency did not reach or exceed the threshold of 126 plus each solvent control count. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency using SYSTAT (R) 11 statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the first culture of experiment II with metabolic activation. This trend however, was not considered relevant since it was not reproduced in the parallel culture and all of the absolute values of the mutation frequency remained within the historical range of solvent controls.

Based on the results in the draft in-vitro mammalian cell gene mutation test and the in-vivo test performed on the major constituent triethylenetetramine, the substance is considered to be not mutagenic.