2-(4-amino-2-nitroanilino)ethanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro transformation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

No GLP compliant, Comparable to OECD Guidance Document Method (No. 214, 2015), no details on the test substance was provided. The control results (benzo(a)pyrene) was not reported.

Data source

Reference

Reference Type:

publication

Title:

An Interlaboratory Evaluation of the Syrian Hamster Embryo Cell Transformation Assay using Eighteen Coded Chemicals

Author:

C.A. Jones, E. Huberman, M.F. Callaham, A. Tu, W. Halloween, S. Pallota, A. Sivak, R.A. Lubet, M.D. Avery, R.E. Kouri, J. Spalding, and R.W. Tennant,

Year:

1988

Bibliographic source:

Toxicol. In Vitro, 2(2): 103-116

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Fibroblasts isolated from Syrian golden hamster embryos on gestational day 13 were used in a cellular transformation assay. HC Red n° 3 was evaluated for cellular transformation capability using the Syrian Hamster Embryo cell (SHE) assay. Two independent laboratories tested HC Red n° 3 at five dosage levels up to 500 and 600 μg/ml, respectively. Duplicate assays utilizing a pH 7.4 culture protocol were performed for a duration of 7 days. Benzo[a]pyrene (B[a]P) was included as a positive control. A positive transformation response was determined using strict morphological guidelines. A positive assay response was defined as a three-fold elevation over the control level of transformation at two or more doses in at least two assays.

GLP compliance:

no

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-amino-2-nitroanilino)ethanol

EC Number:

220-701-7

EC Name:

2-(4-amino-2-nitroanilino)ethanol

Cas Number:

2871-01-4

Molecular formula:

C8H11N3O3

IUPAC Name:

2-(4-amino-2-nitroanilino)ethanol

Test material form:

solid: particulate/powder

Remarks:

crystalline powder

Specific details on test material used for the study:

No detail was provided on the test substance.

Method

Species / strain

Species / strain / cell type:

other: Syrian Hamster Embryo Cells (SHE)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Cells were sampled from Pregnant Syrian golden Hamsters (from Charles River Breeding Laboatory) killed at day 13 of pregnancy. No more details

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's modified Eagle's medium containing 1000 mg/liter 110 mg sodium pyruvate/liter , 584 mg L-glutamine/liter and 20% foetal calf serum was used for all cell preparation and assays.
- Properly maintained: not specified
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

Five concentrations were selected with the highest concentration giving not less than 10% survival, based on the results of clonal survival assay.
Selecred doses were : 0, 100, 200, 400, 600 µg/mL for the Laboratory A and 0, 16, 31 63, 125, 250, 500 µg/mL for the Laboratory C.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no justification was provided

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 300 target and 6x104 feeder cells in 8mL medium

DURATION
- Exposure duration: Laboratory A method : 7 days ; Laboratory C : 3 days
- Fixation time (start of exposure up to fixation or harvest of cells):7 Days

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: One replicate was used per dose level in two independant studies, results for a same dose level were pooled for assessment.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Plates were rinsed, fixed with methanol and stained with Giemsa

NUMBER OF CELLS EVALUATED: more than 100 cells per condition

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A positive response was defined as a level of transformation at two or more chemically treated doses that exceeded three times the level of spontaneous transformation. In addition morphological transformants had to be scored in more than a single assay. An assay in which this level of transformation was observed at only one dose was deemed equivocal. A lower level or no transformation in repeat was considered negative provided that the benzo(a)pyrene result was positive.

Results and discussion

Test results

Key result

Species / strain:

other: Syrian Hamster Embryo Cells (SHE)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

not specified

Remarks:

Data on the positive control was not reported

Additional information on results:

No morphological transformation was induced by the HC Red 3 in assays conducted by the Laboratory A and C. Dose dependent toxicity was observed in both laboratories for this chemical.

Any other information on results incl. tables

Table 1 :Summaryof theresults

		Assay1			Assay2
	Dose (µg/mL)	RCE	SC	T(MA)	RCE	SC	T(MA)	Overall%T
LaboratoryA	0	1	1795	0(0)	1	2061	0(0)	0
	100	0.78	755	0(0)	0.87	960	0(0)	0
	200	0.4	357	0(0)	0.72	803	0(0)	0
	400	0.07	50	0(0)	0.38	386	0(0)	0
	600	0.07	60	0(0)	0.14	130	0(0)	0
LaboratoryC	0	1	1024	0(0)	1	1804	0(1)	0
	16	1	122	0(0)	ND	ND		0
	31	0.95	464	0(0)	1.05	812	0(0)	0
	63	0.77	281	0(0)	1.21	928	0(2)	0
	125	0.44	194	0(0)	0.9	692	0(0)	0
	250	0.26	126	0(2)	0.18	140	0(0)	0
	500	Toxic			Toxic			0

 

RCE :Relative Cloning Efficiency

SC :Number ofscorablecolonies

MA :Number or morphologically altered colony

ND : Not determinable

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of the study, HC Red n° 3 did not induce transformation at any dosage level in the high pH SHE assay in either laboratory.

Executive summary:

This No-GLP compliant study was performed in order to evaluate HC Red n°3 for cellular transformation capability using the Syrian Hamster Embryo cell (SHE) assay.

Fibroblasts isolated from Syrian golden hamster embryos on gestational day 13 were used in a cellular transformation assay. Two independent laboratories tested HC Red n° 3 at five dosage levels up to 500 and 600 μg/ml, respectively. Duplicate assays utilizing a pH 7.4 culture protocol were performed for a duration of 7 days. Benzo[a]pyrene (B[a]P) was included as a positive control. A positive transformation response was determined using strict morphological guidelines. The transformation activity was determined by pooling the data for the same dose level in both assays. The response was first determined within each laboratory, then between the laboratories. A positive assay response was defined as a three-fold elevation over the control level of transformation at two or more doses in at least two assays.

Under the experimental conditions of the study, HC Red n° 3 did not induce transformation at any dosage level in the high pH SHE assay in either laboratory.