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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in bacteria (OECD 471, GLP): negative in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and E. coli WP2 hcr- with and without metabolic activation

In vitro cytogenicity (OECD 473, GLP): negative in human lymphocytes with and without metaolic activation

In vitro gene mutation study (OECD 476, GLP): negative in V79 cells with and without metaolic activation

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted Jul 2016
Deviations:
no
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
Target gene:
HPRT locus
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.
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.
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.

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 106 cells 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 106 cells 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 106 cells 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 106 cells 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

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Nov 2016 - 08 Mar 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted Jul 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
other: In vitro chromosome aberration assay
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Suitability of cells: The cells can be stimulated to provide large numbers of rapidly dividing cells and metaphases. They have a stable karyotype and an aberration rate of about 0 - 10% of the metaphases.
- Sex, age and number of blood donors: Two female donors, aged 18 - 35
- Separated lymphocytes were used.
- Methods for maintenance in cell culture: 96 µg phytohaemagglutinin E per 10 mL whole blood was added and the samples were incubated for 1.5 h in the refrigerator. After that, chromosome medium B containing phytohaemagglutinin L was added to the cell suspension for 48 h at 37 °C and 5% CO2 leading to mitotic stimulation of lymphocytes.
- Modal number of chromosomes: 46

MEDIA USED
- Type and identity of media: Chromosome medium B
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital (80 mg/kg bw/day) and β-naphtoflavone (100 mg/kg bw/day).
Test concentrations with justification for top dose:
Experiment I
5 h treatment without metabolic activation: 500, 1000 and 2000 µg/mL
5 h treatment with metabolic activation: 250, 500, 1000 and 2000 µg/mL

Experiment II
29 h without metabolic activation: 125, 250, 500, 1000 and 2000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 5 and 29 h
- Fixation time (start of exposure up to fixation or harvest of cells): 29 h

SPINDLE INHIBITOR: Colchicine (0.1 µg/mL)

STAIN: Aceto-orcein

NUMBER OF REPLICATIONS: Duplicates each in 2 independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After treatment period, the cell cultures were placed into tubes, centrifuged and KCl solution (0.2%) was added to the cells. For fixation of cells, the tubes were placed into an ice bath and fixative (methanol:acetic acid, 3:1) was added. Cultures were incubated for 20 min at room temperature, then spun for 10 min/169xg. Two further fixation/centrifugation cycles followed. After the third fixation cycle, the supernatant was aspirated and the cell pellet carefully resuspended. From each parallel culture, two slides were prepared. The cell suspension was dropped onto wet, pre-cooled glass slides. The slides were dried for at least three days, then stained in aceto-orcein, immersed in xylene and made permanent with Entellan®.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: Parallel cultures were treated at each concentration; 150 metaphases per culture were scored, 300 in total.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

OTHER: Each S9 batch was tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The bacterial mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene was thus determined once for each S9 batch. Clear increases in the number of revertants for various bacterial strains with all positive controls were used as an acceptance criterion for each S9 batch.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test, or occurs across at least two concentrations
c) any of the results is outside the distribution of the historical negative control data (e.g. 95% control limits).
When all of these criteria are met, the test item is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. 95% control limits).
The test item is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.
Statistics:
For further statistical analysis, the numbers of aberrant metaphases (gaps excluded) were used.
Pairwise comparisons within each experiment were performed. Each treatment group was compared to the concurrent negative control. For these comparisons, the Fisher's Exact Test was performed against one-sided alternatives.
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in Experiment II without metabolic activation a 60% decrease of the mitotic index was noted at 2000 µg/mL (29 h exposure time).
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: No change in the pH value of the cell culture medium occured in the concentration range tested.
- Effects of osmolality: No change in the osmotic value of the cell culture medium was noted in the concentration range tested.
- Precipitation: No precipitation of the test substance was observed up to the highest concentration tested.
- Cytotoxicity: In the absence of metabolic activation after a prolonged exposure period, the mitotic index was reduced below 50% at the highest concentration of 2000 µg/mL indicating cytotoxicity of the test substance, therefore this concentration was not valid.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Aberrant metaphases (excl. gaps) fell within the range of the historical control data (please refer to Table 2 under "any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: Aberrant metaphases (excl. gaps) fell within the range of the historical control data (please refer to Table 2 under "any other information on results incl. tables").

Table 1: Summary of results

Treatment Group Concentration [µg/mL] Rel. Mitotic Index [%] Polyploid metaphases [%] Endomitotic metaphases [%] Aberrant metaphases [%]
Without S9 mix
Exposure time: 5 h		         incl. gaps excl. gaps exchanges
Solvent 100 0.025 0.00 2.83 2.33 0.00
500 103 0.150 0.00 1.33 1.00 ns 0.00
1000 89.3 0.050 0.00 0.67 0.67 ns 0.00
2000 81.4 0.050 0.00 2.67 2.33 ns 0.00
MMC (0.15 µg/mL) 74.0 0.100 0.00 10.3 10.0** 0.33
Without S9 mix
Exposure time: 29 h		 Solvent 100 0.050 0.00 2.83 2.67 0.00
125 103 0.100 0.00 2.00 1.33ns 0.00
250 67.3 0.000 0.10 2.33 2.33 ns 0.00
500 89.5 0.000 0.00 1.67 1.33 ns 0.00
1000 66.7 0.000 0.05 2.33 2.00 ns 0.00
2000 34.6 nd nd nd nd nd
MMC (0.15 µg/mL) 73.9 0.100 0.00 10.0 9.33** 1.67
With S9 mix
Exposure time: 5 h		 Solvent 100 0.050 0.125 2.17 1.83 0.00
250 96.1 0.100 0.50 2.33 2.00 ns 0.00
500 131 0.100 0.50 2.67 1.33 ns 0.00
1000 94.5 0.150 0.00 1.00 1.00 ns 0.00
2000 94.5 0.150 0.00 2.00 2.00 ns 0.00
CPA (3.0 µg/mL) 66.1 0.050 0.50 24.0 24.0** 9.67

ns: not significant

nd: not determined

* 0.01 < p ≤ 0.05

** p ≤ 0.01

MMC: Mitomycin C

CPA: Cyclophosphamide

Table 2: Historical control data

  Mean aberrant metaphases (excl. gaps) [%] *
  Solvent control ** Positive control ***
  Without metabolic activation With metabolic activation Without metabolic activation With metabolic activation
Mean ± SD 1.8 ± 0.9 2.7 ± 1.6 12.8 ± 5.2 14.8 ± 5.5
Range 0.0 - 4.0 0.3 - 5.8 4.5 - 30.0 6.5 - 32.0
95% Control limit -0.1 - 3.6 -0.4 - 5.9    

* Mean values from 2 to 4 individual cultures, for each culture 100 metaphases evaluated, of studies performed in the laboratory using the described protocol

** All solvent controls from different preparation times are pooled for each study

*** MMC (0.15, 0.25 or 0.30 μg/ml) in the absence and CPA (3, 4, 5, 14 or 20 μg/ml) in the presence of S9 mix

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
E. coli WP2
Remarks:
hcr-
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
Key result
Species / strain:
S. typhimurium, other: all strains
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:
other: Sd-4
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The number of revertant colonies could not be counted at 1000 and 10 000 µg/plate with metabolic activation due to over-growing of tryptophane requiring bacteria.
Remarks:
Source: CAS 1218-34-4
Conclusions:
The source substance did not exhibit mutagenic properties with and without metabolic activation in the conducted Ames test. The results are considered to be valid also for the target substance.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in bacteria (Ames)

The mutagenicity of N-acetyl-DL-tryptophan was tested in a bacterial reverse mutation assay similar to OECD guideline 471; as performed with a standard battery of Salmonella typhimurium tester strains including TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and Escherichia coli WP2 hcr-bacterial cells with and without metabolic activation (reference 7.6.1-1). The concentration range from 0.1 to 10000 µg/plate was tested in all bacterial strains. N-acetyl-DL-tryptophan did not exhibit mutagenic properties in the absence or presence of metabolic activation at any concentration levels in any of the tester strains. Cytotoxicity, as indicated by reduced number of revertants, was not observed in any of the tester strains up to and included the highest concentration levels tested. The positive control substances induced a significant increase in the number of revertants in all strains with and without metabolic activation, and the vehicle control was shown to be valid, thereby proving the validity of the assay. Based on the results of the conducted study, N-acetyl-DL-tryptophan is not considered to exhibit mutagenic properties in bacterial cells.

Genetic toxicity (cytogenicity) in mammalian cells in-vitro

The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test in cultured peripheral human lymphocytes performed according to OECD guideline 473 and under GLP conditions (reference 7.6.1-2). The test substance was dissolved in DMSO and two independent experiments were performed.

In the first experiment, the test substance was applied up to 2000 μg/mL for a 5 h exposure time with a 29 h fixation time in the absence and presence of metabolic activation (S9 mix). The test substance did not precipitate in the culture medium at any tested dose level. In the second experiment, the test substance was tested up to 2000 μg/mL for a 29 h continuous exposure time with a 29 h fixation time in the absence of S9 mix. Cytotoxicity (as reduced mitotic index) was observed at the highest tested dose of 2000 µg/mL after the prolonged incubation period. The number of cells with chromosome aberrations found in the solvent control cultures fell within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 mix) functioned properly. The solvent control was shown to be valid and the results fell within the historical control data range. The test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9 mix, in either of the two independently repeated experiments. No relevant effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed in the absence and presence of S9 mix. Therefore it can be concluded that the test substance is not clastogenic in human lymphocytes under the experimental conditions described.

 

Genetic toxicity (mutagenicity) in mammalian cells in-vitro

An in vitro mammalian cell gene mutation assay according to OECD guideline 476 and under GLP conditions was performed with the test substance in Chinese hamster lung fibroblasts (V79) (reference 7.6.1-3). The cells were treated with the test substance for 4 h with and without metabolic activation (cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with phenobarbital andβ-naphthoflavone). The test substance did not show cytotoxic properties (measured as cloning efficiency) in a preliminary cytotoxicity test, and a concentration range of 62.5 - 2000 µg/mL was selected for the two independent mutation experiments with and without metabolic activation. 7,12-dimethylbenzanthracene and ethylmethanesulphonate were used as positive controls with and without S9 mix, respectively and caused a distinct increase of the mutation colonies. The vehicle control results fell within the range of the 95% confidence interval and were considered to be valid. The mean number of mutant colonies observed after treatment with the test substance in both experiments fell within the range of the 95% confidence interval, with the exception of the highest tested dose of 2000 µg/mL in experiment I. In the experiment performed without metabolic activation, the mean number of colonies was 30.9, while the 95% confidence interval was 0.2-29.7. In the experiment performed with metabolic activation, the mean number of colonies was 36.4, while the 95% confidence interval was 0.6 - 28.7. As this effect was not concentration-related when evaluated with an appropriate trend test, it was considered to be incidental and not test item related. Therefore, no biological significant increase in the mutation frequency at the HPRT locus was observed after treatment either in the absence or in the presence of S9-mix and the test substance was not mutagenic in Chinese hamster lung fibroblasts (V79) under the experimental conditions described.

 

Conclusion for genetic toxicity

The available data show that N-Acetyl-DL-tryptophan (CAS 87-32-1) is not mutagenic to bacteria and toChinese hamster lung fibroblasts (V79) in vitro. In addition, the available data with N-Acetyl-DL-tryptophan (CAS 87-32-1) revealed no clastogenic properties incultured peripheral human lymphocytesin vitro.

Justification for classification or non-classification

The available data on genetic toxicity with N-Acetyl-DL-tryptophan (CAS 87-32-1) do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.