EPOFLOC L-1R

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February 2014 to 17 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of N-(2-aminoethyl)-Ń-{2-[(2-aminoethyl)amino]ethyl}ethane-1,2-diamine,N-(2-aminoethyl)-Ń–[2-(piperazin-1-yl)ethyl]ethane-1,2-diamine,N-{2-[4-(2-aminoethyl)piperazin-1-yl]ethyl}ethane-1,2-diamine,N,N,Ń-tris(2-aminoethyl)ethane-1,2-diamine,carbon disulphide and sodium hydroxide.

IUPAC Name:

Reaction products of N-(2-aminoethyl)-Ń-{2-[(2-aminoethyl)amino]ethyl}ethane-1,2-diamine,N-(2-aminoethyl)-Ń–[2-(piperazin-1-yl)ethyl]ethane-1,2-diamine,N-{2-[4-(2-aminoethyl)piperazin-1-yl]ethyl}ethane-1,2-diamine,N,N,Ń-tris(2-aminoethyl)ethane-1,2-diamine,carbon disulphide and sodium hydroxide.

Constituent 2

Reference substance name:

Reaction product of tetraethylene pentamine, carbon disulphide and sodium hydroxide

Cas Number:

2365035-33-0

IUPAC Name:

Reaction product of tetraethylene pentamine, carbon disulphide and sodium hydroxide

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): Reaction products of N-(2-aminoethyl)-Ń-{2-[(2-aminoethyl)amino]ethyl}ethane-1,2-diamine,N-(2-aminoethyl)-Ń–[2-(piperazin-1-yl)ethyl]ethane-1,2-diamine,N-{2-[4-(2-aminoethyl)piperazin-1-yl]ethyl}ethane-1,2-diamine,N,N,Ń-tris(2-aminoethyl)ethane-1,2-diamine,carbon disulphide and sodium hydroxide. (Epofloc L-1R).
- Substance type: Industrial Chemical
- Physical state: Brown Liquid
- Analytical purity: 40.5%
- Lot/batch No.: 311045D
- Expiration date of the lot/batch: 22 November 2014
- Storage Temperature: Ambient temperature (in original container) indoor in dry and well-ventilated area.

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (induced using Aroclor 1254)

Test concentrations with justification for top dose:

- Cytotoxicity Test: 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL in the absence and presence (2% v/v final concentration S9) of metabolic activation
- Mutagenicity Test (2 independent experiments in the absence and presence (2% v/v final concentration S9) of metabolic activation: Trial 1: 312.5, 625, 1250, 2500 and 5000 μg/ml, Trial 2 (confirmation test): 500, 1000, 2000 4000 and 5000 μg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: Epofloc L-1R was soluble in sterile distilled water at 500 mg/mL.

Controls

Untreated negative controls:

yes

Remarks:

Sterile Distilled Water

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Remarks:

Ethyl Methanesulfonate (0.4 µL/mL final concentration in the absence of metabolic activation); Benzo(a)pyrene (6 µL/mL final concentration in the presence of metabolic activation).

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: approximately 24 hours prior to treatment
- Exposure duration: Mutagenicity Test Trial I: 3 hours 45 minutes; Trial II: 3 hours 45 minutes, both in the absence and presence (2% v/v final concentration S9) of metabolic activation
- Expression time (cells in growth medium): 7-9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days post treatment

SELECTION AGENT (mutation assays): 2-amino-6-mercaptopurine (6-thioguanine) at a final concentration of 5 µg/ml.

NUMBER OF REPLICATIONS: 2 per treatment concentration, positive and negative control.

NUMBER OF CELLS EVALUATED: 10000000 cells/culture for each treatment and negative and positive controls.

DETERMINATION OF CYTOTOXICITY
- Method: Other: assessed by calculating the percent relative cloning efficiency following treatment.

OTHER EXAMINATIONS:
- Determination of polyploidy: No
- Determination of endoreplication: No

Evaluation criteria:

Epofloc L-1R was considered positive if the following criteria were met:
- A concentration-related biologically significant increase in mutant frequency in comparison with concurrent negative control and the test item caused a three-fold increase (Nestmann, E.R. et al., 1991) in the number of 6-thioguanine resistant colonies relative to concurrent negative control with statistically significant increases outside the laboratory historical negative control range.
- A net increase in mutant colonies of treated cultures above the concurrent control in at least two of the concentrations tested. Negative results were confirmed by a repeat test (short duration, 3 hour 45 mins), using same experimental conditions considering the toxicity pattern achieved in the trial I mutagenicity experiment.

The analytical acceptance criteria for the concentration of the test item in the vehicle was ± 20% deviation from nominal value and CV% < 10%.

Statistics:

Weighted regression analysis was performed to evaluate the dose response relationship (Li, A.P. et al., 1987; Hsie, A.W. et al., 1981) on Epofloc-L-1R treatment groups against negative control.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No cytotoxicity of biological relevance at the limit concentration.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- No significant change in pH (± 1 pH unit) or osmolality (≥ 50 mOsm/kg H2O) was observed in culture medium at any tested concentration up to 5000 μg Epofloc-L-1R/ml in the absence or presence (2% v/v final concentration S9) of metabolic activation.
- Water solubility: Soluble in sterile distilled water at 500 mg/mL.
- Precipitation: None observed in culture medium up to 5000 μg Epofloc-L-1R/mL in either the absence or presence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: No significant reduction/toxicity in cell count was observed at the end of treatment in cultures treated at 5000 μg/mL both in the absence (relative cloning efficiency 86.82%) and presence (relative cloning efficiency 90.58%) of metabolic activation (2% v/v final volume S9) at 5000 μg/mL, the guideline limit dose. This was therefore the highest concentration tested in the absence and presence of metabolic activation for the main study.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The absolute cloning efficiency of the CHO-K1 cell line (negative control) was above 60% in both the trials. The spontaneous mutation level (negative control) was within the acceptable limit [less than 20 per 10⁶ clonable cells (Li, A.P. et al., 1987)] in both the trials, validating the acceptability of the test system. The increased mutant frequency in positive controls in trial I and II demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative

It is concluded that Epofloc-L-1R does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells, both in the absence and presence of metabolic activation under the present experimental conditions.