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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether was considered to be

non-mutagenic nor clastrogenic under the conditions of an Ames test and a Chromosome Abberation Test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
uvrB-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
The test item was tested using the following method. Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were initially assayed
in triplicate against each tester strain, using the direct plate incorporation method. However, bacterial tester strains TA100 and TA1535 dosed in the absence of
S9-mix showed excessive toxicity after the first experiment (resulting in an insufficient number of non-toxic doses) and, therefore these particular strains had
to be repeated employing an amended test item dose range as follows:
Salmonella strains TA100 and TA1535 (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.

The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains TA100, TA1535 and TA1537 (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
Vehicle / solvent:
Dimethyl sulphoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
The five strains of bacteria used, and their mutations, are defined above.
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the
histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to
larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the
strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers
ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA
repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would
normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in
nutrient broth (Oxoid Limited; lot number 1712138 07/20) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity
with titres determined by viable count analysis on nutrient agar plates.

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
Dimethyl sulphoxide was therefore selected as the vehicle.
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No
correction was made for purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement
of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains TA100, TA1535 and TA1537 (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.

Salmonella strains TA100, TA1535 and TA1537 (presence of S9) and TA98 (absence and presence of S9) and E.coli strain WP2uvrA (absence of S9):
0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
E.coli strain WP2uvrA (presence of S9):
0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.

Eight test item dose levels per bacterial tester strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item
following the change in test methodology from plate incorporation to pre-incubation.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer. 3.3.2.4 Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were
required, predominantly due to revertant colonies spreading slightly, thus distorting the actual
plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 μg/plate.
Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates.
Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
Combined historical negative and solvent control ranges for 2014 and 2015 are presented in Appendix 1.
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix.
All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.
Evaluation criteria:
Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and
Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello and
Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and
the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

Experiment 2.
In the initial first experiment the maximum dose level of the test item was selected as the maximum recommended dose level of 5000 μg/plate. However, two of the Salmonella
strains showed excessive toxicity after the first experiment (resulting in an insufficient number of non-toxic doses) and had to be repeated employing the toxic limit of test item as
the maximum dose. In the first mutation test (plate incorporation method) the test item induced toxicity evident as visible reductions in the growth of the bacterial background lawns
of all of the tester strains, initially from 50 μg/plate in the absence of metabolic activation (S9-mix) and 150 μg/plate in the presence of S9-mix. Consequently the toxic limit of the test
item was employed as the maximum dose level in the second mutation test. The test item induced a similar toxic response after employing the pre-incubation method in the second
mutation test with weakened bacterial background lawns noted in the absence of S9-mix from 50 μg/plate (TA100, TA1535, TA98 and TA1537) and 150 μg/plate (WP2uvrA). In the
presence of S9-mix, weakened bacterial lawns were noted from 150 μg/plate (TA100, TA1535 and TA1537) and 500 μg/plate (TA98 and WP2uvrA). No test item precipitate was
observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without
metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the
bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases
in revertant colony frequency were observed in the first mutation test at 50 μg/plate (TA100) and 15 μg/plate (TA1535) in the presence of S9-mix only. These increases were considered
to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant
dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.3 times the concurrent vehicle controls.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally within the normal range. A single count for WP2uvrA (vehicle control dosed in the absence
of S9-mix after the second mutation test) was just below the minimum level. This count was still considered acceptable as the other vehicle and untreated control counts were within
expected range and the tester strain responded very well with the respective positive controls in both the presence and absence of S9-mix. All of the positive control chemicals used in the
test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were
validated.
Conclusions:
1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl etherr using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Mammalian Chromosome Aberration Test
Details on test system and experimental conditions:
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability.
Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to
calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the
experiments for 1.5 x AGT is 24 hours.

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin,
amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to
divide by the addition of phytohaemagglutinin (PHA).

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 10/04/2016 was used in this study.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM),
glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test Item Preparation and Analysis
The test item was considered to be a UVCB* and, therefore, the maximum recommended dose was initially set at 5000 μg/mL. The purity of the test item was 100% and was not
accounted for in the test item formulations. The test item was insoluble in aqueous media at 50 mg/mL but was miscible in DMSO at 500 mg/mL in solubility checks performed in-house. Prior to each experiment, the test item
was accurately weighed, formulated in DMSO and appropriate serial dilutions prepared. There was small but significant change when the test item was dosed into media as the pH
increased by 1.5 units at 5000 μg/mL. Therefore, an intermediate dose level was assessed. This increase in pH restricted the maximum concentration to 3750 μg/mL. The osmolality
did not increase by more than 50 mOsm (Scott et al., 1991).
The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the
homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been
reflected in the GLP compliance statement.

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 mL MEM, 10% (FBS) 0.1 mL Li-heparin, 0.1 mL phytohaemagglutinin, 0.75 mL heparinized whole blood.
4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture
medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to
each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was
added to the cultures of the Preliminary Toxicity Test and Main Experiment. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were
centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by
suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.
4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the
culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle
control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash
of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned
to the incubator for a further 20 hours.
24-Hour Exposure Without Metabolic Activation (S9)
As the exposure was continuous the cultures were established, at a nominal volume of 9.9 mL. After approximately 48 hours incubation the cultures were removed from the
incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures
were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours. The preliminary toxicity test was performed using all three of the exposure conditions as
described for the Main Experiment but using single cultures only.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery
period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery
period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500
and 3750 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made.
Precipitate observations were recorded at the beginning and end of the exposure periods. Using a qualitative microscopic evaluation of the microscope slide preparations from each
treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the
main test.

Main Experiment
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in
treatment-free media prior to cell harvest. The dose range of test item used was 0,
2.5, 5, 10, 15, 20, 30 and 60 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in
treatment-free media prior to cell harvest. The dose range of test item used was 0,
2.5, 5, 10, 20, 30, 40, 50 and 60 μg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest.
The dose range of test item used was 0, 1.25, 2.5, 5, 10, 15, 20 and 30 μg/mL.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the
culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the
hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The
fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped
onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation of Response

Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used
to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerized random number generator.

Scoring of Chromosome Damage
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding
gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976)
recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985) . Cells with chromosome aberrations were reviewed as necessary by a senior
cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated
chromosomes) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal
volunteer donors.

Evaluation criteria:
See test system and conditions section
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case
by case basis.
Species / strain:
lymphocytes: Primary culture
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was initially set as 19.53 to 5000 μg/mL. The maximum dose was the maximum recommended dose level. There was small but
significant change in pH when the test item was dosed into media which increased by 1.5 units at 5000 μg/mL. Therefore, this increase in pH restricted the maximum concentration
tested to 3750 μg/mL.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 39.06 μg/mL in the 4(20)-hour exposure groups and at and above
78.13 μg/mL in the 24-hour continuous exposure group.
Hemolysis was observed following exposure to the test item at and above 39.06 μg/mL in all three exposure groups. Hemolysis is an indication of a toxic response to the erythrocytes and
not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 19.53 μg/mL in the exposure groups in the absence of
metabolic activation (S9). The maximum dose with metaphases present in the exposure
group in the presence of S9 was 39.06 μg/mL.
The test item induced extreme evidence of toxicity in all of the exposure groups. The selection of the maximum dose level for the Main Experiment was based on toxicity in
all three exposure group.

Chromosome Aberration Test – Main Experiment
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether (μg/mL)
4(20)-hour without S9 0*, 2.5, 5, 10*, 15*, 20*, 30*, 60, MMC 0.2*
4(20)-hour with S9 (2%) 0*, 2.5, 5, 10, 20*, 30*, 40*, 50*, 60, CP 1*
24-hour without S9 0*, 1.25, 2.5*, 5*, 10*, 15*, 20, 30, MMC 0.1*
* = Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

The qualitative assessment of the slides determined that the toxicity was similar to that
observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring
present up to 30 μg/mL in the 4(20)-hour exposure group in the absence of S9, up to 50 μg/mL in the presence of metabolic activation (S9) and up to 20 μg/mL in the 24-hour
continuous exposure group.
There were no precipitate was observed at the end of exposure in any of the exposure groups tested. Haemolysis was observed at 60 μg/mL in the in the 4(20)-hour exposure groups only.
The mitotic index data confirm the qualitative observations in that a moderate dose-related inhibition of mitotic index was observed in all three exposure groups.
In the 4(20)-hour exposure group in the absence of S9, 42% mitotic inhibition was achieved at 30 μg/mL. Above this dose level, no metaphases were present for analysis. Therefore, the
maximum dose level selected for chromosomal analysis was 30 μg/mL, which was slightly below optimum toxicity as stated in the OCED 473 test guideline (55±5%).
In the 24-hour continuous exposure group (in the absence of S9), 52% and 58% mitotic inhibition was observed at 15 and 20 μg/mL, respectively. Again, above this dose level were
no scorable metaphases present for analysis. Therefore, the maximum dose level selected for metaphase analysis was the lowest concentration with optimum toxicity (15 μg/mL).
In the presence of S9, an inhibition of mitotic index of 20% and 66% was noted at 40 and 50 μg/mL, respectively. Above these dose levels, there were no metaphases present. Due to
the tight dose range selected, the maximum concentration selected for metaphase analysis (50 μg/mL) exceeded optimum toxicity because the dose levels either side were either not
toxic (40 μg/mL) or were excessively toxic (60 μg/mL).
In all three exposure groups, the test item was considered to have been adequately tested despite the excessively toxic nature of the test item.
The chromosome aberration data are given in Table 4, Table 5 and Table 6. The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control
cultures were within the current historical control data range.
All the positive control chemicals induced a demonstrable positive response (p≤0.01) and
confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
The study was performed using all three exposure conditions using a top concentration which
meets the requirements of the current testing guideline.
The required number of cells and concentrations were analyzed.
The test item did not induce any statistically significant increases in the frequency of cells
with aberrations either in the absence or presence of metabolic activation.
The polyploid cell frequency data are given in Table 7. The test item did not induce a
statistically significant increase in the numbers of polyploid cells at any dose level in either of
the exposure groups.
Conclusions:
1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro gene mutation study in mammalian cells does not need to be conducted because adequate data from a reliable in vivo mammalian gene mutation test are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether was considered to be

non-mutagenic under the conditions of the in vivo micronucleus test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
ICR
Details on species / strain selection:
There was no marked difference in the toxicity of the test item between the sexes in the range finding study; therefore the main test was performed using only male mice.
Sex:
male
Details on test animals or test system and environmental conditions:
Sufficient albino Hsd: ICR (CD-1®) strain mice were obtained from Envigo, B.V., Horst, The
Netherlands. At the start of the main test the mice weighed 23.8 to 29.9 g and were
approximately six to ten weeks old. After a minimum acclimatization period of five days the
animals were selected at random and given a number unique within the study by tail marking
and a number written on a colour coded cage card.
The animals were housed in groups of five in solid-floor polypropylene cages with
wood-flake bedding. Free access to mains drinking water and food (Envigo Teklad 2014C
Global Certified Rodent Diet supplied by Envigo Laboratories UK Ltd., Oxon, UK) was
allowed throughout the study. Representative analyses of food and water quality are retained
in the laboratory archive.
The temperature and relative humidity were set to achieve limits of 19 to 25ºC and 30 to
70%, respectively. The rate of air exchange was approximately fifteen changes per hour and
the lighting was controlled by a time switch to give twelve hours light and twelve hours
darkness. The Delta Building Monitoring system was used during the course of the study.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Dose / conc.:
1 000 mg/kg bw (total dose)
Remarks:
Range finfing study
Dose / conc.:
1 200 mg/kg bw (total dose)
Remarks:
Range finfing study
Dose / conc.:
1 600 mg/kg bw (total dose)
Remarks:
Range finfing study
Dose / conc.:
2 000 mg/kg bw (total dose)
Remarks:
Range finfing study
Dose / conc.:
1 200 mg/kg bw (total dose)
Remarks:
Final test
Dose / conc.:
600 mg/kg bw (total dose)
Remarks:
Final test
Dose / conc.:
300 mg/kg bw (total dose)
Remarks:
Final test
Details of tissue and slide preparation:
Slide Preparation
Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were
dissected from each animal, aspirated with foetal bovine serum and bone marrow smears
prepared following centrifugation and re-suspension. The smears were air-dried, fixed in
absolute methanol, stained in May-Grünwald / Giemsa, allowed to air-dry and a cover slip
applied using mounting medium.
Evaluation criteria:
Slide Evaluation
Stained bone marrow smears were coded and examined blind using light microscopy at
x1000 magnification. The incidence of micronucleated cells per 4000 polychromatic
erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are
normally circular in shape, although occasionally they may be oval or half-moon shaped, and
have a sharp contour with even staining. In addition, the number of normochromatic
erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted;
these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with
appropriate group mean values and standard deviations.
Data Evaluation
Comparison was made between the number of micronucleated polychromatic erythrocytes
occurring in each of the test item groups and the number occurring in the vehicle control
group.
A positive mutagenic response is demonstrated when a statistically significant,
dose-responsive, toxicologically relevant increase in the number of micronucleated
polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared
to the vehicle control group.
If these criteria were not fulfilled, then the test item was considered to be non-genotoxic
under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean
polychromatic to normochromatic ratio was shown to be statistically significantly lower than
the vehicle control group.
All data were statistically analysed using appropriate statistical methods as recommended by
the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989).
The data was analysed following a transformation using Student's t-test (two tailed).
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The test item, 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether, was considered to be non-genotoxic under the conditions of the test.
Executive summary:

The test item, 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether, was considered to be non-genotoxic under the conditions of the test.

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

Additional information

Justification for classification or non-classification

Based on the results of the in vitro and in vivo genotoxicity studies and according tothe criteria of EC Regulation 1272/2008the test item 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether is not mutagenic and therefore must not be classified.