Reaction mass of hydroxyethyl-DETA, DETA and dihydroxyethyl-DETA

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro studies on genetic toxicity studies are available for DETA, which isconsidered to be the most toxic consitituent of hydroxyethyl-DETA-derivatives

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Not applicable

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to test guidelines and in accordance with GLP

Justification for type of information:

For the detailed read-across justification please see attached document in section 13

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine-operon

Species / strain / cell type:

other: TA 1535, TA 100, TA 1537, TA 98 and E .coli WP2 uvrA

Details on mammalian cell type (if applicable):

- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction (Aroclor 1254-induced)

Test concentrations with justification for top dose:

Experiment 1: 0; 20 ; 100 ; 500 ; 2500 and 5000 μg/plate (vehicle water)
Experiment 2: 0; 1,000 ; 2000 ; 3000 ; 4000 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S9: 2-aminoanthracene; without S9: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine, N-ethyl-N'-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

No data

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

Not applicable

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no toxicity observed, but mean number of revertants is slightly reduced at the highest dose selected, indicating beginning toxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Not applicable

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Standart plate test:  Dose (µg/plate)  TA1535     TA100     TA1537     TA98        E. coli WP2 uvrA    -S9  +S9  -S9  +S9  -S9  +S9 -S9  +S9 -S9  +S9   0 19±1 19±3  108±8 120±17 10±1 10±4 30±3 43±5 32±3 41±2 20  17±3 17±2 101±4 125±10 9±2 11±2 32±3 38±3 34±4 41±2  100 17±2 20±2 105±5 125±22 9±1 11±1 27±5 41±5 33±3 41±3  500 15±2 21±2 121±2 148±7 8±2 12±2 25±1 39±3 36±3 50±2  2500 16±2 17±3 141±7 191±7 9±1 11±1 25±5 35±5 73±8 66±2  5000 13±2 19±2 109±16 155±26 7±3 10±1 14±2 17±3 60±3 87±3  2-AA - 194±12 - 1192±183 - 870±93 -  880±53 -  209±12  MNNG 997±56 - 1346±108 - - - - - - -  AAC - - - - 546±106 - -  -  - -  NPD  - - - - - - 940±9  -  -  -  ENNG - - - - - - - - 927±31 - Mean ± SD Standart plate-test (replication):  Dose (µg/plate)  TA100       E. coli WP2 uvrA           -S9  +S9  -S9  +S9 0 107±11 115±4 28±3 39±3 20 102±11 123±28 52±9 40±5  100 114±15 146±8 113±12 68±3  500 120±9 152±22 123±5 75±5  2500 103±11 127±27 104±5 61±12  5000 71±10 84±12 75±8 37±6  2AA  - 1058±20 - - MNNG 1145±53 - - -  AAC -  - - -  NPD - - - - ENNG - - 873±42 - Mean ± SD 2-AA: 2-aminoanthracene; MNNG; N-methyl-N-nitro-N-nitrosoguanidine ENNG; N-ethyl-N-nitro-N-nitrosoguanidine NPD: 4-nitro-o-phenylendiamine AAC: 9-aminoacridine chloride monohydrate According to the results of the present study, the test substance leads to an increase in the number of revertant colonies using E. coli WP2 uvrA both without S-9 mix and after adding a metabolizing system in two experiments carried out independently of each other. Thus, under the experimental conditions chosen here, it is concluded that diethylentriamine is a mutagenic agent in the bacterial reverse mutation test in vitro.

Conclusions:

Interpretation of results (migrated information):
positive

Diethylentriamine is a mutagenic agent in the bacterial reverse mutation test in vitro.

Executive summary:

The mutagenicity potential of DETA was evaluated in the Ames test. According to the present study, the test substance leads to an increase in the number of revertant colonies using E. coli WP2 uvrA both without S-9 mix and after adding a metabolizing system in two experiments carried out independently of each other. Thus, under the experimental conditions chosen here, it is concluded that diethylentriamine is a mutagenic agent in the bacterial reverse mutation test in vitro.

Reference 2

Endpoint:

genetic toxicity in vitro

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Justification for type of information:

For the detailed read-across justification please see attached document in section 13

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Testing was performed in compliance with Standard Operating Procedures used for these tests a t the Bushy Run Research Center

GLP compliance:

not specified

Type of assay:

other: CHO mutation, SCE test, UDS

Test concentrations with justification for top dose:

Refer to method section below.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

hepatocytes: rat liver cells

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

DETA-HP was consistently inactive as a rnuzagenic agent for CHO cells when tested with or without an S9 metabolic activation system over a 32-fold range of concentrations. None of the mutation values were statistically significant from the concurrent solvent control and values were within the expected variation in mutant frequencies observed in historical control data.

Treatments of CHO cells with DETA-HP over a 16 to 32 -fold range of concentrations failed to indicate a potential mutagenic activity in tests either with or without addition of an active S9, metabolic activation system. No evidence of a dose related effect of exposue to DETA-HP on the SCE frequency was evident in tests with or without S9 metabolic activation and the test agent was considered to be inactive in the present in vitro assay.

DETA-HP failed to stimulate consistently a dose-related incorporation of radio active thymidine in cells treated over a 1000-fold range of test concentrations. Although values for the positive controls were low, in the assessment of UDS with nuclei, measurements of radioactive incorporation into the precipitated DNA from those nuclei verified the activity of the positive control agents and the inactivity of the DETA-HP sample. DETA-HP was considered inactive in the tests with hepatocytes.

Conclusions:

Interpretation of results (migrated information):
negative

The pattern of negative responses obtained in the 3-test battery of mutagenicity assays indicated that DETA-HP did not produce a mutagenic effect typical of identified chemical mutagens and appeared to lack significant mutagenic potential in the three in vitro tests performed.

Executive summary:

Diethylenetriamine - High Purity (DETA-HP) was evaluated for potential mutagenic activity with a

battery of three in vitro tests, which were: the Chinese Hamster Ovary (CHO) Mutation test, the Sister

Chromatid Exchange (SCE) test and an assay for induction of Unscheduled DNA Synthesis (UDS) in rat

Liver cells. The pattern of negative responses obtained in the 3-test battery of mutagenicity assays

indicated that DETA-HP did not produce a mutagenic effect typical of identified chemical mutagens and

appeared to lack significant mutagenic potential in the three in vitro tests performed.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Not applicable

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to test guidelines and in accordance with GLP

Justification for type of information:

For the detailed read-across justification please see attached document in section 13

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: EPA Office of Toxic Substances 560/6-82-001 Reference: E.P.A. (1985): Identification of specific chemical substance and mixture testing requirements; Diethylenetriamine. 40 CFR Part 799. Federal Register, 50, 21398-21416.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

other: CHO cells (CHO-K1, CCL61)

Details on mammalian cell type (if applicable):

The cell line CHO-K1 (CCL61). originally obtained from the American Type Culture Collection, Rockville, MO, was used in this study. This cell line was derived as a Subclone from a parental cell line initiated from the ovary of an adult Chinese hamster (American Type Culture Collection Catalog, 1981).

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate prepared from AROCLOR 1254 treated (500 mg/kg) male Sprague-Dawley rats was purchased from Sitek Research Laboratories, Rockville, MO.

Test concentrations with justification for top dose:

250, 833 AND 2500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Cyclophosphamide a promutagen requiring metabolic activation for its clastogenicity was used as positive control for the activation system

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Evaluation criteria:

Mitotic indices were determined as the number of cells in metaphase among 500 cells/replicate (a total of 1000 cells/treatment) and expressed as percentages. One hundred metaphase photomicrographs (50/replicate) were examined at each dose level for structural abnormalities. The microscopic coordinates of each of the scored metaphases were recorded. Only those metaphases that contained 20 +/-2 chromosomes were scored with the exception of severely damaged cells, in which case accurate counts of the chromosomes were not possible. Structural chromosomal abnormalities included chromatid and chromosome gaps, chromatid breaks and exchanges, chromosome breaks, rings, dicentrics, and chromosomal disintegration. Those cells having 10 or more aberrations/cell were classified as severely damaged cells. Chromatid gaps and chromosome gaps were not included in calculations of total aberrations.

Statistics:

The descriptive and comparative statistics on cytogenetic aberrations considered each of the analyzed cells as an observational unit. Data from replicate cultures were pooled to calculate average scores. The frequencies of cytogenetic abnormalities, such as gaps/cell, total aberrations excluding gaps/cell, miscellaneous aberrations/cell, cells with gaps and cells with aberrations excluding gaps, and cells with miscellaneous aberrations, were analyzed by constructing two dimensional contingency tables. The total Chi-square was partitioned into components of interest. Specifically, statistics were generated to test the two global hypotheses of (1) no difference in average scores among the dose groups, and (2) no linear trend of increasing scores with increasing dose (Bhapkar. 1968). If either statistic was found to be significant at alpha=0.01, pairwise tests (i.e., control vs. treatment) were also performed at each dose level and evaluated at alpha=0.01. The final interpretation of biological significance of the cytogenetic responses was based on both statistical outcome and sound scientific judgment.

Species / strain:

other: CHO cells (CHO-K1, CCL61)

Metabolic activation:

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:

The data clearly indicate that the test material was stable over a 4 h period (test material concentrations at 2 and 4 hours following preparation ranged from 103 - 108% of time zero concentrations)..

The highest dose level of the test material (i.e., 2500 mcg/ml) gave an RCS value of 23.2% in the non-activation assay, whereas the corresponding value for the activation assay was 73.2%.

There were no statistically significant increases in the incidence of cytogenetic abnormalities in cultures treated with any of the three concentrations of DETA, either in the presence or absence of S9, as compared to the negative controls (Tables 1 and 2). The positive controls (EMS and CP) induced statistically significant increases in total aberrations (excluding gaps) and number of cells with aberrations (excluding gaps) indicating the sensitivity of the assay conditions for detecting known clastogens.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1 RESULTS OF THE CHROMOSOMAL ABERRATION ASSAY IN THE ABSENCE OF S9

	Negative Control	250 mcg/ml	833 mcg/ml	2500 mcg/ml	Positive Control
Mitotic Index (%)	8.4	10.2	11.3	5.8	7.5
No. of cells scored	100	100	100	100	100
Chromatid gaps	10	7	13	12	25
Chromatid breaks	2	0	0	1	15
Chromatid exchanges	1	1	1	0	10
Chromosome breaks	0	0	1	2	0
Chromosome exchanges	0	2	0	0	1
Total aberrations (excluding gaps)	3	3	2	3	26*
No. of cells with gaps	10	6	13	12	21
No. of cells with aberrations	3	3	2	2	23*
Miscellaneous aberrations	0	1	2	2	1
Number of cells with miscellaneous aberrations	0	1	2	1	1

"Significantly (alpha <0.01) different from the negative control

Table 2: RESULTS OF THE CHROMOSOMAL ABERRATION ASSAY IN THE PRESENCE OF S9

	Negative control	250 mcg/ml	833 mcg/ml	2500 mcg/ml	Positive Control
Mitotic Index (%)	10.6	10.8	11.3	9.8	2.7
No. of cells scored	100	100	100	100	100
Chromatid gaps	18	24	8	25	24
Chromatid breaks	0	1	1	6	27
Chromatid exchanges	3	0	0	2	51
Chromosome breaks	1	0	1	2	4
Total aberrations (excluding gaps)	4	1	2	10	82*
No. of cells with gaps	13	17	8	20	20
No. of cells with aberrations (excluding gaps)	4	1	2	9	53*
Miscellaneous aberrations	0	0	0	0	1
No. of cells with miscellaneous aberrations	0	0	0	0	1

Table 6: RESULTS OF THE CHROMOSOMAL ABERRATION ASSAY IN THE PRESENCE OF S9

Conclusions:

Interpretation of results (migrated information):
negative

The experimental conditions used, DETA was considered to be non-clastogenic to CHO cells in culture.

Executive summary:

Diethylenetriamine (DETA) was evaluated in an in vitro chromosomal aberration assay utilizing Chinese hamster ovary (CHO) cells. The clastogenicity of the test material was assessed in the absence and presence of an externally supplied metabolic activation (S9) system at dose levels of 250, 833 and 2500 micrograms/ml (mcg/ml) of culture medium. The dose levels of the test material were based upon the results of preliminary cytotoxicity assays. Cultures treated with 1242 mcg/ml ethylmethanesulfonate and 14 mcg/ml cyclophosphamide served as positive controls for the non-activation and activation assays, respectively. Negative control cultures were treated with distilled water (the solvent used to dissolve the test material). There were no statistically significant increases in the frequencies of chromosomal aberrations in cultures treated with the test material either in the absence or presence of S9 as compared to the negative control cultures. The positive control chemicals induced the expected increases in aberration frequencies. Hence, under the experimental conditions used, DETA was considered to be non-clastogenic to CHO cells in culture.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)