A mixture of: 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C16)alkylacetamide; 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C18)alkylacetamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

strain to detect crosslinking/oxidizing agents was not included

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from Aroclor 1254 induced rat livers

Test concentrations with justification for top dose:

20 µg - 5000 µg/plate

Vehicle / solvent:

Tetrahydrofurane (THF)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S-9 mix 10 µg 2-aminoanthracene; without S-9 mix 5 µg N-methyl-N -nitro-N-nitrosoguanidine, 10 µg 4-nitro-o-phenylendiamine, 100 µg 9-aminoacridine chloride monohydrate

Details on test system and experimental conditions:

Standard plate test
Test tubes containing 2 ml soft agar kept in a water bath at 45°C, and remaining components added in the following order:
0 .1 ml test solution or vehicle
0 .1 ml bacterial suspension
0 .5 ml S-9 mix (in tests with metabolic activation) or 0 .5 ml phosphate buffer (in tests without metabolic activation).
After mixing, the samples are poured onto Vogel-Bonner agar plates.

Preincubation assay
0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0 .5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates.

Standard plate test and Preincubation assay
In each experiment 3 test plates per dose or per control used; after incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted. The titer was determined and in regularly measurements the strain characteristics were checked. Sterility control was performed.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfil the following requirements: doubling of the spontaneous mutation rate (control), dose-response relationship, reproducibility of the results.

Statistics:

none

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility: Incomplete solubility of the test substance in tetrahydrofurane (THF) from about 2500 µg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Standard plate test (20 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	27	1.0	no	negative
	yes	36	1.0	no	negative
TA 100	no	98	1.1	no	negative
	yes	130	1.2	no	negative
TA 1535	no	16	1.0	no	negative
	yes	19	0.9	no	negative
TA 1537	no	13	1.0	no	negative
	yes	14	1.1	no	negative
Preincubation test (20 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	18	1.3	no	negative
	yes	30	1.3	no	negative
TA 100	no	98	1.2	no	negative
	yes	95	1.4	no	negative
TA 1535	no	15	1.2	no	negative
	yes	13	1.3	no	negative
TA 1537	no	10	1.2	no	negative
	yes	14	1.0	no	negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, Sulzfeld, D
- Weight at study initiation: ca. 29 g
- Housing: single in Makrolon cages, type I
- Assigned to test groups randomly: yes, under following basis: appropriate computer program
- Diet: Standardized pelleted feed (Kliba Haltungsdiaet, Klingentalmuehle AG, Kaiseraugst, CH; ad libitum
- Water: drinking water; ad libitum
- Acclimation period: ca. 1 week (housing 5 per cage (Makrolon cage, type M III))


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

The dose leveis were set as follows: Due to technical reasons the highest applicable amount was 3000 mg/kg body weight at a temperature of not less than 42°C in order to guarantee that the test substance stayed liquid and did not become solid for administration. Higher doses were only possible at a temperature of > 60°C which could not be applicated any longer. Therefore, a dose of 3000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1500 mg/kg and 750 mg/kg body weight were administered as further doses.

Duration of treatment / exposure:

Sacrifice after 16 h (3000 mg/kg bw); 24 h (0-3000 mg/kg bw and ctrl.); 48 h (3000 mg/kg bw) exposure

Frequency of treatment:

single administration

Post exposure period:

not applicable

Remarks:

Doses / Concentrations:
3000 mg/kg, 1500 mg/kg, 750 mg/kg and 0 mg/kg body weight in a volume of 10 ml/kg body weight in each case
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

As positive control for clastogenicity, 20 mg of cyclophosphamide/kg body weight, dissolved in aqua dest., was administered once orally to male and female animals in a volume of 10 ml/kg body weight.
As positive control for spindle poison effects, 0.15 mg of vincristine/kg body weight, dissolved in aqua dest., was administered once intraperitoneally to male and female animals in a volume of 10 ml/kg body weight.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

The two femora were prepared from the animals, and all soft tissues were removed. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/ femur). The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.

Evaluation criteria:

In general, 1000 polychromatic erythrocytes from each of the male and female animais of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes, which occur, are also scored.

Statistics:

A statistical evaluation was not necessary to perform.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Irregular respiration was observed in all treatment groups 15 -30 min after treatment.

Substance	Dose (mg/kg)	Sacrifice interval (h)	PCE with micronuclei (‰)	NCE with micronuclei (‰)	Ratio NCE/PCE
olive oil	solvent	24	1.7	1.54	0.45
test substance	750	24	1.9	1.54	0.39
test substance	1500	24	1.5	1.84	0.44
test substance	3000	16	1.6	0.84	0.48
test substance	3000	24	1.9	0.93	0.43
test substance	3000	48	2.0	1.51	0.46
cyclophosphamide	20	24	10.4	3.03	0.20
vincristine	0.15	24	35.8	2.15	0.33
PCE = polychromatic erythrocyts (10,000 were scored for micronuclei)
NCE = normochromatic erythrocyts
The NCE/PCE ratio is based on the scoring of 10,000 erythrocyts

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach, D
- Mean weight at study initiation: about 248.3 g
- Assigned to test groups randomly: yes, under following basis: appropriate computer program
- Housing: individually in Makrolon cages, type III
- Diet: Standardized pelleted feed (Kliba Haltungsdiaet, Klingentalmuehle AG, Kaiseraugst, CH; ad libitum
- Water: drinking water; ad libitum
- Acclimation period: about one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
To achieve homogeneity of the test substance in the vehicle, the test substance formulation was stirred constantly during removal and administrationat about 60°C.

Duration of treatment / exposure:

Perfusion was performed 3 and 14 hours after treatment, respectively

Frequency of treatment:

single administration

Post exposure period:

Perfusion was performed 3 and 14 hours after treatment, respectively

Remarks:

Doses / Concentrations:
1,000 mg/kg and 2,000 mg/kg body weight in a volume of 10 ml/kg body weight in each case
Basis:
actual ingested

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Positive control(s):

As a positive control, 50 mg of 2-acetylaminofluorene (2-AFF)/kg body weight, dissolved in corn oil, was administered to the animals once orally in a volume of 10 ml/kg body weight.

Tissues and cell types examined:

rat hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The acute oral toxicity LD50 is > 2,200 mg/kg body weight in rats. Therefore, a dose of 2,000 mg/kg body weight was selected as the highest dose in the present study; 1,000 mg/kg body weight was administered as further dose.

DETAILS OF SLIDE PREPARATION:
For the determination of the test substance concentrations in the vehicle, 3 samples of each dose were taken from the test substance preparations, kept at room temperature until the treatment of the last animal and then deep-frozen until they were determined analytically.

Evaluation criteria:

A test result is considered positive if a dose-dependent increase in the mean number of net nuclear grains is demonstrated and is > 5 per nucleus at one of the test points. An increase in a group average between 0 and 5 net nuclear grains is considered to be a marginal response. A test article producing net nuclear grain counts < 0 at any test point is considered to be negative in this test system.

Statistics:

Due to very clear negative findings, a statistical evaluation was not carried out.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

As a negative control, male rats were administered merely the vehicle by the same route, which gave frequencies of mean nuclear net grain counts within the historical control range. The positive control chemical 2-acetylaminofluorene (2-AAF) administered once orally in a dose of 50 mg/kg body weight demonstrated the expected increase in unscheduled DNA synthesis.

Harvest time: 3 hours
Concentration (mg/kg bw)	No. of cells	Mean NG counts +- SD	Mean CG counts +- SD	Mean NNG counts +- SD	Mean % cells in repair NNG > 0 +- SD	Mean % cells in repair NNG > 5 +- SD
Vehicle Ctrl.	100	6.21 +- 0.25	10.01+- 0.46	-3.79 +- 0.28	8.33 +- 3.21	0 +- 0
1000	100	5.87 +- 0.43	9.81+- 1.22	-3.94 +- 0.84	6.33 +- 1.15	0 +- 0
2000	100	5.78 +- 0.74	9.95 +- 1.04	-4.17 +- 0.32	8.33 +- 2.08	0.33 +- 0.58
Positive Ctrl.	100	18.21+- 3.55	10.02 +- 1.35	8.19 +- 2.35	91.00+- 4.36	68.00+- 5.29
Harvest time: 14 hours
Concentration (mg/kg bw)	No. of cells	Mean NG counts +- SD	Mean CG counts +- SD	Mean NNG counts +- SD	Mean % cells in repair NNG > 0 +- SD	Mean % cells in repair NNG > 5 +- SD
Vehicle Ctrl.	100	6.31 +- 0.21	10.23+- 0.47	-4.10 +- 0.38	8.33 +- 0.58	0 +- 0
1000	100	6.01 +- 0.65	9.55 +- 1.09	-3.54 +- 0.45	11.67 +- 4.04	0.33 +- 0.58
2000	100	6.20 +- 1.22	9.82 +- 0.73	-3.62 +- 0.49	9.67 +- 2.52	0.33 +- 0.58
Positive Ctrl.	100	19.13+- 1.22	10.48 +- 0.74	8.67 +- 1.04	93.67+- 2.52	72.33+- 4.93
NG = nuclear grains
CG =cytoplasmic grains
NNG = net nuclear grains

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

in vitro

Gene mutation

In an Ames test according to OECD guideline No. 471 and GLP requirements, S. typhimurium tester strains TA97, TA100, TA1535 and TA1537 were used in both a Standard Plate Test and a Preincubation Test, both with and without metabolic activation (BASF AG 1991). The test substance was dissolved in tetrahydrofurane (THF). No mutagenic activity was found in concentrations ranging from 20 ug - 5000 µg/plate. Precipitation was observed in doses >= 2500 µg/plate. Positive and negative/ vehicle control results were valid.

Cytogenicity

An in vitro mammalian chromosome aberration test that conforms to GLP was performed in accordance with the OECD guideline 473 with and without metabolic activation in V79 cells (BASF AG 1995). Test concentrations ranged from 0.8 - 20 ug/ml. The test substance is considered to be a chromosome-damaging (clastogenic) agent in V79 cells since the test substance caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after adding a metabolizing system in two experiments independent of each other. An increase in the frequency of cells containing numerical aberrations was not demonstrated. Positive and negative/ vehicle control results were valid.

in vivo

Cytogenicity

A micronucleus test was performed with NMRI mice according to OECD guideline 474 and GLP requirements (BASF AG 1991). The test substance was applied once via gavage in concentrations of 3000 mg/kg, 1500 mg/kg and 750 mg/kg body weight in a volume of 10 ml/kg body weight. 24 hrs after application, the test substance showed no chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis. Negative results were also provided after high dose treatment and sacrifice intervals of 16 and 48 hrs. Positive and negative control results were valid.

An unscheduled DNA synthesis (UDS) test according to OECD guideline 486 and GLP requirements was performed in Wistar rats (BASF AG 1997). The test substance was administered as single gavage administration of 1,000 mg/kg and 2,000 mg/kg body weight in a volume of 10 ml/kg body weight. Hepatocytes were harvested after 3 and 14 hrs. In each case the treatment did not lead to an increase in the mean number of net nuclear grain counts at any dose level or exposure time in rat hepatocytes. Positive and negative control results were valid.

Short description of key information:
in vitro
Ames (TA97, TA100, TA1535, TA1537): negativ w/ and w/out S9 (GLP, OECD471, BASF 1991)
Chrom. aberration: positiv w/S9, negativ w/out S9 (GLP, OECD 473, BASF 1995)
in vivo
MNT: negative (GLP, OECD 474, BASF 1991)
UDS: negative (GLP, OECD 486, BASF 1997)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Keroflux ES 3241 showed a chromosome-damaging (clastogenic) potential in V79 cells in a chromosome aberration test. This potential was not expressed in vivo, since two guideline cytogenicity assays produced negative results. A classification of the genotoxic potential of the test substance is therefore not warranted according ot 67/548/EEC and UN-GHS.