Amides, C16-18 (even numbered) saturated and C18 unsaturated, N,N'-[[(2-hydroxyethyl)imino]di-2,1-ethanediyl]bis-, ethoxylated (1-2.5 mol)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The reported data show that the test item C16-18,18'DETA-Amide_ethoxylated did not induce gene mutations in the S. typhimurium tester strains with and without mammalian metabolic activation. In conclusion the results of this bacterial reverse mutation assay were considered negative.

The reported data show that the test item C16-18,18'DETA-Amide_ethoxylated did not induce gene mutations in a chromosome aberration test on peripheral human lymphocytes with and without mammalian metabolic activation. In conclusion the results of this chromosome aberration assay were considered negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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:

other: GLP-Guideline study.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: GB/T21794-2008 Chemicals-Test method of in vitro mammalian chromosome aberration

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

purity 86.8%

Target gene:

not applicable

Species / strain / cell type:

other: peripheral human lymphocytes (isolated from the blood of a healthy adult, non-smoking, male volunteers (26-31 years old))

Details on mammalian cell type (if applicable):

- Type and identity of media: culture medium consisted of RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated foetal bovine serum, heparine, Colchicine, NADP and G-6-P, Mitomycin and Cyclophosphamide

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Without and with S9-mix: 62.5, 31.2 and 15.6 µg/mL culture medium (4 h exposure time, 24 h fixation time)

Vehicle / solvent:

- Culture medium: RPMI 1640 (85%); FBS (15%), PHA (200 µg/L), heparin (10U/mL), penicillin (100U/mL), strptomycin (100 U/mL).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

mitomycin C (-S9): 0.25 µg/mL (4 h exposure period), 0.2 and 0.3 µg/mL (24 h exposure period) and 0.1 and 0.15 µg/mL (48 h exposure period ), cyclophosphamide (+S9): 9.4 µg/mL for the 4 h exposure period (24 and 48 h fixation time)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h; 24h
- Fixation time (start of exposure up to fixation or harvest of cells): 24h

SPINDLE INHIBITOR (cytogenetic assays): colchicine (1 µg/mL medium, 4h)
STAIN (for cytogenetic assays): Giemsa (5% (v/v))

NUMBER OF REPLICATIONS: duplicate in two independent experiments

NUMBER OF CELLS EVALUATED: 200 per concentration and solvent control group, 100 for positive control group.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The chromosome aberration test was considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the range of the laboratory historical control data.
b) The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures was observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Species / strain:

other: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the continuous experiment at the highest dose of 300 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test.
The cytotoxicity result showed that no cell survived and no mitotic phase occured at the concentrations tested over 62.5 µg/mL, and the mitotic index showed a significant reduction at the concentration of 62.5 µg/mL (>50%); There were no cytotoxicity at the concentration of 15.6 µg/mL.
According to the above results of cytotoxicity test, three doses of 62.5, 31.2, 15.6 µg/ml were used.

Remarks on result:

other: all strains/cell types tested

Table 1: Test results of the experiment

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 4h, fixation time 24h, without S9 mix
dist.water	10	14.8	0	1
MMC	0.25	8.9	1	19
Test substance	15.6	14.8	0.5	0.5
	31.2	12.4	0.5	0.5
	62.5	9.2	0.5	0
Exposure period 4h, fixation time 24h, with S9 mix
dist.water	10	15.5	0	1.5
CP	4	8.5	0	21
Test substance	15.6	14.4	1	0.5
	31.2	11.6	0	0.5
	62.5	7.7	0	1.5
Exposure period 24h, fixation time 24h, without S9 mix
dist.water	10	16.1	0	1
MMC	0.25	7.8	2	23
Test substance	15.6	13.3	0.5	0
	31.2	10.8	0.5	0
	62.5	5.7	0.5	0.5

- MMC: Mitomycin C; CP: Cyclophosphamide

- *: CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

Conclusions:

negative

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-06-2013 to 08-07-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Lot number: STO26977671

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat; Arochlor 1254 induced)

Test concentrations with justification for top dose:

10.0, 31.6, 100, 316, 1000 and 3160 µg / plate

Vehicle / solvent:

distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation: TA 1535; TA 100; 1.5 µg/vessel

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 1,8-dihydroxy anthraquinone

Remarks:

without metabolic activation: TA 1537 and TA 102; 50 µg/vessel

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Remarks:

with metabolic activation: TA 97, TA 98, TA 100; 10 µg/vessel

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Dexon

Remarks:

without metabolic activation: TA 97, TA 98, TA 102, 50 µg/vessel

Details on test system and experimental conditions:

The test was performed both in existence and absence of the S9mix by adopting plate incorporation method,

Evaluation criteria:

A test substance producing no biologically relevant positive response in any one of the bacterial strains tested is considered to be non-mutagenic in this system.
A biologically relevant response is described as follows:
If the number of revertants is at least twice the spontaneous reversion rate and shows a dose-response relationship, the result is positive.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the substance was solved in distilled water.
- Precipitation: no precipitation was noted at all.


COMPARISON WITH HISTORICAL CONTROL DATA: The results of the negative and positive control cultures were within the range of the historical data generated by the testing lab.

Conclusions:

Interpretation of results:
negative

The reported data show that the test item C16-18,18'DETA-Amide_ethoxylated did not induce gene mutations in the S. typhimurium tester strains with and without mammalian metabolic activation. In conclusion the results of this bacterial reverse mutation assay were considered negative.

Executive summary:

The potential of C16-18,18'DETA-Amide_ethoxylated to induce gene mutations was examined in 5 Salmonella typhimurium strains TA 97, TA98, TA100, TA102 and TA1535 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The experiments were carried out as a plate incorporation test.

Six concentrations ranging from 62.5 to 1000 μg C16-18,18'DETA-Amide_ethoxylated/vessel were employed in the plate incorporation tests, each carried out without and with metabolic activation.

No cytotoxicity was noted in the plate incorporation tests, each carried out without and with metabolic activation

No increase in revertant colony numbers as compared with control counts was observed for C16-18,18'DETA-Amide_ethoxylated, tested up to 1000 µg/vessel, in any of the 5 test strains in two independent experiments without and with metabolic activation.

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

In conclusion, under the present test conditions, C16-18,18'DETA-Amide_ethoxylated caused no mutagenic effect in the Salmonella typhimurium strains TA 97, TA98, TA100, TA102 and TA1535 in the plate incorporation tests each carried out without and with metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

It was concluded that the test substance was negative in the mammalian erythrocyte micronucleus test in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-25 until 2013-09-30

Reliability:

1 (reliable without restriction)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

other: GB/T 21773-2008 Chemicals

GLP compliance:

yes

Type of assay:

other: mammalian bone marrow cells

Specific details on test material used for the study:

Lot: ST02697671
purity: 86.8%

Species:

mouse

Strain:

other: KM mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Changsa Tianqin biotechnology Ltd.
- Age at study initiation:
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: SPF barrier environment
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25
- Humidity (%): 54-60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
weight 3.75 g test substance and add distilled water to 30 mL to prepare the stock solution with the concentration of 125 mg/mL for high dose group (2500 mg/kg). Then draw 4.0 mL and 8.0 mL high dose group solution, add distilled water to 16 mL, to prepare stock solution for low dose group (625 mg/kg) and medium dose group (1259 mg/kg) animals respectively. The concentrations were 31.2, 62.5 mg/mL respectively.

Duration of treatment / exposure:

The test substance was administered to mice at a volume of 0.2 mL/10 g bw by gavage twice during the period of 24h (at the beginning and at the 24h respectively). The solvent control group was treated by the same method. The positive control group was treated with cyclophosphamid (0.2%) by celiac injection at a volume of 0.15 ml/10 g bw.

Frequency of treatment:

twice, at 0 and 24 hours

Post exposure period:

18 hours

Dose / conc.:

2 500 mg/kg bw (total dose)

Dose / conc.:

1 250 mg/kg bw (total dose)

Dose / conc.:

625 mg/kg bw (total dose)

Dose / conc.:

30 mg/kg bw (total dose)

Remarks:

positive control, Cyclophosphamide

No. of animals per sex per dose:

5

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: celiac injection
- Doses / concentrations: 30m/kg bw

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

Bone marrow cells were harvested from the sternum and cell smears were prepared and stained according to conventional procedures. The region of cell-completely, well-distributed and well-colored up was selected and examined under a microscope. 2000 polychromatic erythrocytes per animal were scored for the incidence of micronclei. The ratio of PCEs to NCEs was also determined for each animal by counting a total of 200 erythrocytes, as an indication of cytotoxicity to the bone marrow cells.

Statistics:

The micronucleated PCE ratios for all dose-groups and solvent control group were analyzed using the poisson distribution and that of positive control group was handled with Gaussion distribution. Each sex was analyzed respectively. The parameters and data were analyzed statistically by SPSS 13.0.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No clinical signs were observed in any test animal. And no death occurred in any test animal during the period of the test.
The incidence of MNPCE in each treatment group were summarized Table 1. The incidence of MNPCE in solventr control groups were 1.4% and 1.6% for female and male mice respectively. The incidence of MNPCE were 1.0%, 1.7%, 1.5% for female mice and 0.8%, 1.3%, 0.9% for male mice in low, medium and high dose groups respectively, there were no significant differences compared with the solvent control (p>0.05). The incidence of MNPCE in positive control group were 24.9% and 22.6% for female and male mice respectively, there were significant differences compared with the solvent control group (p<0.01)
The result indicated, that the test substance could not increase the micronuclear rates of KM mice obviously.

Conclusions:

It was concluded that the test substance was negative in the mammalian erythrocyte micronucleus test in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data on genetic toxicity, there is no indication that C16 -18, 18'DETA-Amide ethoxylated induces mutagenic properties in bacterial cells or mutagenic and clastogenic properties in mammalian cells, respectively. In conclusion,

C16 -18, 18'DETA-Amide ethoxylated

does not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.