Nα-acetyl-DL-tryptophan

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Oct - 22 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted Jul 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

other: In vitro Mammalian Cell Gene Mutation Test

Test material

Test material information

Constituent 1

Reference substance name:

Nα-acetyl-DL-tryptophan

EC Number:

201-739-3

EC Name:

Nα-acetyl-DL-tryptophan

Cas Number:

87-32-1

Molecular formula:

C13H14N2O3

IUPAC Name:

N-acetyltryptophan

Test material form:

solid

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing, Technical University, Darmstadt, Germany
- Suitability of cells: the V79 cell line has been used successfully in in vitro experiments for many years.
- Doubling time: 12 - 16 h
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C with 1.5% CO2 in 75 cm² plastic flasks. The cells were sub-cultured once or twice weekly.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with neomycin (5 μg/mL) and amphotericin B (1%). During treatment 10% fetal bovine serum (FBS) was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-test for toxicity
with and without S9 mix: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL (4 h)
Experiment I and II
with and without S9 mix: 62.5, 125, 250, 500, 1000 and 2000 µg/mL (4 h)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 0.7 - 1.2E+07 cells per plastic flask

DURATION
- Exposure duration: 4 h exposure with and without metabolic activation.
- Expression time: 7 days
- Selection time (if incubation with a selection agent): 7 - 9 days

SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine (6-TG)

STAIN: 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no relevant shift of pH in the medium. The pH was 7.39 (solvent control) and 7.18 (2000 µg/mL; adjusted with 2M sodium hydroxide), respectively.
- Effects of osmolality: There was no relevant shift of osmolality in the medium. Osmolality was 392 (solvent control) and 368 (2000 µg/mL), respectively.
- Precipitation: no precipitation occured up to the maximum concentration with and without metabolic activation

RANGE-FINDING/SCREENING STUDIES: a pre-test was performed in order to determine the concentration range for the mutagenicity experiment. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony-forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

VALIDITY OF THE ASSAY: The 95% confidence interval was slightly exceeded at 2000 μg/mL in the first culture without metabolic activation (30.9 versus an upper limit of 29.7 mutant colonies/106 cells), and with metabolic activation (36.4 versus an upper limit of 28.7 mutant colonies/106 cells). The 95% confidence interval was also exceeded with the second culture at 250.0 μg/mL (29.8 versus an upper limit of 28.7 mutant colonied/106 cells) with metabolic activation. These isolated increases were judged as irrelevant as they were not reproduced in the corresponding parallel cultures and there was no dose dependent increase as indicated by the not-significant linear regression analysis. As the "outliers" are near to the outer border of the historical control range, the assay is considered as acceptable.

Any other information on results incl. tables

Table 2: Results of the pre-experiment

Concentration [µg/mL]	Cloning efficiency [%]
	- S9	+ S9
0 (DMSO)	100	100
15.6	94.9	102.3
31.3	91.5	94.2
62.5	97.7	90.5
125	94.4	94.8
250	95.6	96.0
500	92.1	92.3
1000	88.6	94.6
2000	82.8	89.9

DMSO = Dimethylsulfoxide

Table 3: Experiment I - 4 h exposure - without metabolic activation

Concentration [µg/mL]	Cloning efficiency [%] Survival	Cloning efficiency [%] Viability	Mutation colonies per 10^6cells	95% confidence intervall
0 (DMSO)	100	100	13.7	0.2 - 29.7
62.5	95.0	#	#	0.2 - 29.7
125	105.4	84.4	24.1	0.2 - 29.7
250	98.7	93.9	13.4	0.2 - 29.7
500	111.9	90.9	25.5	0.2 - 29.7
1000	111.3	89.5	24.6	0.2 - 29.7
2000	114.4	93.3	30.9	0.2 - 29.7
EMS	99.0	98.2	218.2	0.2 - 29.7

EMS = Ethyl methane sulphonate

DMSO = Dimethylsulfoxide

# = culture was not continued as a minimum of only four analysable concentrations is required

Table 4: Experiment I - 4 h exposure - with metabolic activation

Concentration [µg/mL]	Cloning efficiency [%] Survival	Cloning efficiency [%] Viability	Mutation colonies per 10^6cells	95% confidence intervall
0 (DMSO)	100	100	13.6	0.6 - 28.7
62.5	93.2	#	#	0.6 - 28.7
125	85.6	84.4	15.2	0.6 - 28.7
250	89.3	81.3	19.0	0.6 - 28.7
500	91.6	75.5	25.3	0.6 - 28.7
1000	82.7	81.4	11.2	0.6 - 28.7
2000	92.7	95.7	36.4	0.6 - 28.7
DMBA	99.2	81.7	162.0	0.6 - 28.7

DMBA = 7,12-dimethylbenz(a)anthracene

DMSO = Dimethylsulfoxide

# = culture was not continued as a minimum of only four analysable concentrations is required

Table 5: Experiment II - 4 h exposure - without metabolic activation

Concentration [µg/mL]	Cloning efficiency [%] Survival	Cloning efficiency [%] Viability	Mutation colonies per 10^6cells	95% confidence intervall
0 (DMSO)	100	100	21.1	0.2 - 29.7
62.5	87.0	#	#	0.2 - 29.7
125	82.1	77.5	19.7	0.2 - 29.7
250	90.8	88.4	10.6	0.2 - 29.7
500	86.7	79.8	25.0	0.2 - 29.7
1000	93.7	74.6	26.1	0.2 - 29.7
2000	82.7	99.8	17.0	0.2 - 29.7
EMS	88.0	80.9	191.9	0.2 - 29.7

EMS = Ethyl methane sulphonate

DMSO = Dimethylsulfoxide

# = culture was not continued as a minimum of only four analysable concentrations is required

Table 6: Experiment II - 4 h exposure - with metabolic activation

Concentration [µg/mL]	Cloning efficiency [%] Survival	Cloning efficiency [%] Viability	Mutation colonies per 10^6cells	95% confidence intervall
0 (DMSO)	100	100	21.5	0.6 - 28.7
62.5	97.1	#	#	0.6 - 28.7
125	104.2	97.8	18.1	0.6 - 28.7
250	93.5	107.8	29.8	0.6 - 28.7
500	105.5	97.6	22.3	0.6 - 28.7
1000	99.6	95.4	27.4	0.6 - 28.7
2000	106.1	94.3	26.7	0.6 - 28.7
DMBA	101.3	102.1	187.9	0.6 - 28.7

DMBA = 7,12-dimethylbenz(a)anthracene

DMSO = Dimethylsulfoxide

# = culture was not continued as a minimum of only four analysable concentrations is required

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative