Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA with and without metabolic activation

Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): negative in human lymphocytes with and without metabolic activation

Gene mutation in mammalian cells (OECD 490, Thymidine kinase test): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Mar - 16 Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted May 2008
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S. typhimurium strains) and trp operon (E. coli strains)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
The dose-range finding test served as Experiment 1.
Experiment 1: 52, 164, 512, 1600 and 5000 µg/plate with and without metabolic activation
Experiment 2: 52, 164, 512, 1600 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: The test item was miscible in Milli-Q water at 25 °C and stable for at least 24 h at room temperature under normal laboratory light conditions, for at least 8 days in refrigerator (2-8°C) and for at least 3 weeks in the freezer (≤ -15°C), which was confirmed over the concentration range 1 to 60 mg/mL (project # 20144290 at the same testing facility).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: -S9: 4-nitroquinoline N-oxide 10 µg/plate for WP2uvrA and ICR-191 2.5 µg/plate for TA1537 (Exp. 1); +S9: 2-aminoanthracene 1, 2.5 or 15 µg/plate for all strains
Remarks:
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, Experiment 1); preincubation (Experiment 2)

DURATION
- Preincubation period: 30 ± 2 min (Experiment 2)
- Exposure duration: 48 ± 4 h (Experiment 1 and 2)

NUMBER OF REPLICATIONS: triplicates each in 2 independant experiments

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Key result
Species / strain:
other: S. typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2
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
- Precipitation: The test item did not precipitate in the incubation medium at the start or at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES: Experiment 1 served as range-finding study.

HISTORICAL CONTROL DATA
- Positive historical control data: The positive control values were within the range of the historical control data (please refer to table 4 under "any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: The negative control values were within the range of the historical control data (please refer to table 4 under "any other information on results incl. tables").

Table 1: Results of dose-range finding study/Experiment 1 (direct plate assay)

Mean number of revertant colonies/3 replicate plates (± S.D.) with oneSalmonella typhimuriumand oneEscherichia colistrain.
Without S9-mix TA100 WP2uvrA
Positive control 1040± 42 1093 ± 98
Solvent control 123 ± 13 36 ± 8
1.7 123 ± 4 31 ± 7
5.4 132 ± 18 26 ± 6
17 117 ± 5 36 ± 5
52 105 ± 7 34 ± 4
164 124 ± 23 32 ± 8
512 123 ± 12 41 ± 8
1600 123 ± 27 44 ± 6
5000 121 ± 8 33 ± 6
With S9-mix TA100 WP2uvrA
Positive control 1513 ± 3 401 ± 118
Solvent control 141 ± 12 40 ± 4
1.7 128 ± 14 41 ± 8
5.4 122 ± 13 45 ± 4
17 108 ± 11 47 ± 7
52 110 ± 16 36 ± 6
164 114 ± 13 32 ± 6
512 103 ± 11 34 ± 9
1600 116 ± 10 36 ± 3
5000 91 ± 8 31 ± 6

Table 2: Results of Experiment 1 (direct plate assay)

Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
Without S9-mix TA1535  TA1537 Ta98
Positive control 1073 ± 97 1036 ± 50 1058 ± 40
Solvent control 8 ± 2 3 ± 2 15 ± 4
52 8 ± 1 6 ± 3 16 ± 1
164 12 ± 5 5 ± 5 20 ± 6
512 11 ± 4 4 ± 1 15 ± 6
1600 8 ± 7 6 ± 2 21 ± 2
5000 8 ± 4 7 ± 5 16 ± 6
With S9-mix TA1535  TA1537 Ta98
Positive control 349 ± 22 471 ± 37 1181 ± 40
Solvent control 10 ± 2 3 ± 2 20 ± 5
52 12 ± 2 11 ± 4 30 ± 11
164 12 ± 7 9 ± 9 26 ± 3
512 14 ± 2 7 ± 0 23 ± 8
1600 7 ± 7 4 ± 5 22 ± 3
5000 10 ± 1 5 ± 2 18 ± 2 

Table 3: Results of Experiment 2 (pre-incubation assay)

Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
Without S9-mix TA1535 TA1537 TA98 TA100 WP2uvrA
Positive control 1164 ± 79 145 ± 15 1147 ± 80 765 ± 93 217 ± 85
Solvent control 8 ± 4 3 ± 2 18 ± 2 109 ± 15 29 ± 6
52 12 ± 6 3 ± 1 11 ± 4 104 ± 14 32 ± 5
164 11 ± 4 6 ± 3 22 ± 19 105 ± 12 36 ± 8
512 15 ± 4 5 ± 5 17 ± 5 110 ± 17 31 ± 3
1600 17 ± 13 5 ± 2 16 ± 6 106 ± 17 34 ± 6
5000 4 ± 0 5 ± 2 15 ± 5 114 ± 9 39 ±8
With S9-mix          
Positive control 283 ± 20 217 ± 21 898 ± 85 1801 ± 423 477 ± 5
Solvent control 21 ± 9 12 ± 2 35 ± 6 110 ± 0 39 ± 8
52 20 ± 5 12 ± 4 32 ± 4 128 ± 14 38 ± 4
164 16 ± 2 18 ± 4 29 ± 2 106 ± 9 43 ± 5
512 19 ± 4 13 ± 4 34 ± 5 106 ± 20 39 ± 8
1600 14 ± 4 15 ± 4 29 ± 6 104 ± 2 46 ± 6
5000 21 ± 1 12 ± 3 27 ± 3  97 ± 13 38 ± 4

Table 4: Historical control data of the positive and negative control

  TA1535 TA1537 TA98 TA100 WP2uvrA
Positive control
S9-mix - + - + - + - + - +
Range 125 – 1248 73 – 1206 55 – 1353 54 – 1051 365 – 1995 250 – 1977 439 – 1848 408 - 2651 93 – 1951 111 - 1359
Mean 846 219 787 353 1406 887 901 1232 1094 437
SD 146 119 345 162 258 349 168 343 477 149
n 2348 2229 2003 2234 2200 2276 2335 2327 2021 2085
Negative control
S9-mix - + - + - + - + - +
Range 3 – 29 3 - 27 3 – 20 3 – 23 8 - 41 8 - 55 63 – 176 54 - 160 10 – 59 9 - 69
Mean 10 11 6 7 16 23 108 107 25 32
SD 3 4 2 3 5 7 19 20 7 8
n 2356 2336 2264 2235 2319 2360 2341 2336 2075 2078
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:
13 Apr - 27 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Sex, age and number of blood donors: Blood was collected from three donors with an age of 30, 30 and 32 years, respectively. Average generation time was 14.0, 14.8 and 14.8 h, respectively.
- Whether whole blood or separated lymphocytes were used: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.
- Methods for maintenance in cell culture: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 54 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.6 - 37.1°C). Temperature and humidity were continuously monitored throughout the experiment.

MEDIA USED
- Type and identity of media: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) and 30 U/mL heparin.
- 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 a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
Test concentrations with justification for top dose:
Dose-range finding test:
3, 24 and 48 h treatment without metabolic activation, respectively: 62.5, 125, 250, 500, 1000 and 1412 µg/mL

Main experiment:
3 h treatment with and without metabolic activation: 500, 1000 and 1412 µg/mL
24 h treatment without metabolic activation: 10, 1000 and 1412 µg/mL
48 h treatment without metabolic activation: 10, 750 and 1200 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
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
- Exposure duration: 3, 24 and 48 h
- Expression time (cells in growth medium): 3 h treatment: 20 - 22 h
- Fixation time: 3 h treatment: 24 h; 24 and 48 h treatment: 24 and 48 h, respectively

SPINDLE INHIBITOR: Colchicine (0.5 μg/mL medium)

STAIN: 5% (v/v) Giemsa

NUMBER OF REPLICATIONS: duplicate cultures each in 2 independent experiments

NUMBER OF CELLS EVALUATED: 300 (150 cells per culture)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 75

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
Evaluation criteria:
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
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:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A concentration of 0.01 M (highest tested dose level, 1412 μg/mL) showed no precipitation in the culture medium.

RANGE-FINDING/SCREENING STUDIES: In the dose-range finding test blood cultures were treated with 62.5, 125, 250, 500, 1000 and 1412 μg N-ethylcaprolactam/mL culture medium for 3 h with and without S9-mix, and for 24 and 48 h without S9 mix, respectively. The test item did not show cytotoxic properties in the short-term experiment of 3 h with and without metabolic activation system. In the long-term experiment a decrease of the mitotic index was observed at the highest tested dose level of 1412 µg/mL after 24 and the 48 h.

HISTORICAL CONTROL DATA
- Positive historical control data: The positive controls both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- Negative (solvent/vehicle) historical control data: The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.

Table 1: Dose-range finding study

Concentration (µg/mL) Number of metaphases
  Absolute Number of cells scored % control
Without metabolic activation (-S9-mix) 3 h exposure time, 24 h fixation time      
Control a) 82 1002 100
62.5 88 1024 107
125 76 1017 93
250 78 1012 95
500 72 1014 88
1000 85 1006 104
1412 64 1010 78
Without metabolic activation (-S9-mix) 24 h exposure time, 24 h fixation time
Control a) 73 1014 100
62.5 48 1016 66
125 56 1012 77
250 34 1009 47
500 40 1011 55
1000 39 1003 53
1412 18 1005 25
Without metabolic activation (-S9-mix) 48 h exposure time, 48 h fixation time      
Control a) 63 1013 100
62.5 47 1018 75
125 45 1013 71
250 40 1011 63
500 41 1003 65
1000 34 1022 54
1412 17 1016 27
With metabolic activation (+S9-mix) 3 h exposure time, 24 h fixation time      
Control a) 85 1013 100
62.5 78 1021 92
125 66 1017 78
250 69 1009 81
500 71 1009 84
1000 51 1007 60
1412 61 1012 72

a) RPMI 1640 medium

Table 2: Chromosome aberrations in human lymphocyte cultures treated with the test item in the absence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

  RPMI-C 500 µg/mL 1000 µg/mL 1412 µg/mL 0.5 µg/mL MMC-C
Culture A B A + B A B A + B A B A + B A B A + B A B A + B
Mitotic Index (%) 100 92 87 80 62
No. of Cells scored 150 150 300 150 150 300 150 150 300 150 150 300 150 150 300
No. of Cells with aberrations (+ gaps) a) 2 4 6 1 2 3 3 0 3 0 2 2 39 25  64***
No. of Cells with aberrations (- gaps) 2 4 6 1 2 3 3 0 3 0 2 2 39 25  64***
Chromatid break  2     2    1       34 17  
Chromosome break          2         2 3  
Exchange figure                         8 6  
 Miscellanous              2 endo             3 intra   intra
 

total aberrations (+ gaps)

 
 2   2    3  0    0  2    47  27
 total aberrations (- gap)  2  4    2  2    3  0    0  2    47  27  

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

MMC-C: Mitomycin C

endo: endoreduplication

intra: chromosome intrachange

Table 3: Chromosome aberrations in human lymphocyte cultures treated with the test item in the presence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

  RPMI-C 500 µg/mL 1000 µg/mL 1412 µg/mL 10 µg/mL CP
  A B A + B A B A + B A B A + B A B A + B A B A + B
Mitotic Index (%) 100 90 81 77 48
No. of Cells scored 150 150 300 150 150 300 150 150 300 150 150 300 150 150 300
No. of Cells with aberrations (+ gaps) a) 2 2 4 1 2 3 3 3 6 2 0 2 26 30  56***
No. of Cells with aberrations (- gaps) 2 2 4 1 2 3 3 3 6 2 0 2 26 30  56***
Chromatid break  1  2   1  2    3  1    1     18 38  
Chromosome break  1              1     8 7  
Exchange figute              1    1      3  4  
Miscellanous              endo  2 endo      endo   intra  
total aberr (+ gaps) 2 2   1 2   3 3   2 0   30 49  
total aberr (- gaps) 2 2   1 2   3 3   2 0   30 49  

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

CP: Cylcophosphamide

endo: endoreduplication

intra: chromosome intrachange

Table 4: Chromosome aberrations in human lymphocyte cultures treated with the test item in the absence of S9-mix in the second cytogenetic assay (24 h exposure time, 24 h fixation time)

  RPMI-C 10 µg/mL 1000 µg/mL 1412 µg/mL 0.2 µg/mL MMC-C
Culture A B A + B A B A + B A B A + B A B A + B A B A + B
Mitotic Index (%) 100 95 61 47 34
No. of Cells scored 150 150 300 150 150 300 150 150 300 150 150 300 150 150 300
No. of Cells with aberrations (+ gaps) a) 1 0 1 1 0 1 0 0 0 0 0 0 24 24 48***
No. of Cells with aberrations (- gaps) 1 0 1 1 0 1 0 0 0 0 0 0 23 24 48***
Chromatid break 1   1           11 9  
Chromosome break                       5

7

 

Exchange figure

 

 

 

 

 

 

 

 

 

 

 

 

7

 9

 

Dicentric chromosome

 

 

 

 

 

 

 

 

 

 

 

 

 1

 

Miscellanous

 

 

 

 

 poly

 

 

poly

 

 

 p

 

 

total aberr (+ gaps)

1

0

 

1

0

 

0

0

 

0

0

 

25

26

 

total aberr (- gaps)

1

0

 

1

0

 

0

0

 

0

0

 

24

26

 

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

MMC-C: Mitomycin C

p: pulverized chromosome

poly: polyploidy

Table 5: Chromosome aberrations in human lymphocyte cultures treated with the test item in the absence of S9-mix in the second cytogenetic assay (48 h exposure time, 48 h fixation time)

Conc

RPMI-C

10 µg/mL

750 µg/mL

1200 µg/mL

0.1 µg/mL MMC-C

Culture

A

B

A + B

A

B

A + B

A

B

A + B

A

B

A + B

A

B

A + B

Mitotic Index (%)

100

107

65

50

50

No. of Cells scored

150

150

300

150

150

300

150

150

300

150

150

300

150

150

300

No. of Cells with aberrations (+ gaps) a)

1

0

1

1

0

1

0

2

2

0

1

1

29

34

63***

No. of Cells with aberrations (- gaps)

1

0

1

0

0

0

0

2

2

0

0

0

27

34

61***

Chromatid gap

 

 

1

 

 

 

 

1

 

1

 

Chromsome gap

 

 

 

 

 

 

 

 

 

 

1

 

Chromatid break

 

 

 

 

 

 

 

 1

 

 

 

 

8

21

 

Chromosome break

 

 

 

 

 

 

 

 1

 

 

 

 

9

5

 

Exchange figure

 

 

 

 

 

 

 

 

 

 

 


13

8

 

chromatid deletion

1

 

 

 

 

Miscellanous

 

 

 

 

p

p

 

 total aberrations (+ gaps)  1  0    1  0    0  2    0  1    32  35  
 total aberrations (- gaps)  1  0    0  0    0  2    0  0    30  35  

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

MMC-C: Mitomycin C

p: pulverized chromosome

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Aug - 08 Oct 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
adopted Jul 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
tk locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA)
- Suitability of cells: recommended test system in international guidelines
- Methods for maintenance in cell culture: Cells were cultured in a humid atmosphere (actual range 77 - 99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 37.5°C).

MEDIA USED
- Type and identity of media: Basic medium was a RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin. Basis medium supplemented with 5, 10 or 20% heat-inactivated horse medium was used as exposure, growth, selective and non-selective medium, respectively.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: 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 a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
Test concentrations with justification for top dose:
Dose-range finding study
With and without S9 mix: 125, 250, 500, 1000 and 1412 µg/mL (3 h)
Without S9 mix: 125, 250, 500, 1000 and 1412 µg/mL (24 h)

Experiment I
With and without S9 mix: 16, 31, 63, 125, 250, 500, 1000 and 1412 µg/mL (3 h)

Experiment II
Without S9 mix: 62.5, 125, 250, 500, 750, 1000, 1250 and 1412 µg/mL (24 h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q-water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 h
- Expression time (cells in growth medium): 2 days
- Selection time: 11 or 12 days

SELECTION AGENT (mutation assays):
5 μg/mL trifluorothymidine

STAIN: 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
Duplicate measurements each of 5 wells for mutant frequency and duplicate measurements each of 2 wells for cloning efficiency, respectively, in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range. The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro-well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH at a concentration of 1412 μg/mL was 8.11 compared to 8.10 in the solvent control.
- Effects of osmolality: The osmolarity at a concentration of 1412 μg/mL was 0.299 Osm/kg compared to 0.288 Osm/kg in the solvent control.
- Precipitation: The test substance did not precipitate in the exposure medium up to and including the highest concentration of 1412 μg/mL (= 10 mM).

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 125 to 1412 μg/mL in the absence of S9-mix with 3 and 24 h treatment periods and in the presence of S9-mix with a 3 h treatment period. After 3 h of exposure, both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 1412 μg/mL compared to the suspension growth of the solvent control. After 24 h of exposure, the relative suspension growth was 35% at the test item concentration of 1412 μg/mL compared to the relative suspension growth of the solvent control.

HISTORICAL CONTROL DATA
- Positive historical control data: The mutation frequencies of both positive controls were within the range of the historical control data (please refer to Table 3 under 'any other information on results incl. tables).
- Negative (solvent/vehicle) historical control data: The mutation frequency of the solvent control was within the range of the historical control data (please refer to Table 2 under 'any other information on results incl. tables).

Table 1: Results of Experiment 1 (3 h incubation) with and without metabolic activation and Experiment 2 (24 h incubation) without metabolic activation

dose RSG CE day2 RCE RTG mutation frequency per 106survivors
(µg/ml) (%) (%) (%) (%) total small large
without metabolic activation: 3 h treatment
SC1 100 86 100 100 60 11 48
SC2 84 82 21 59
16 107 76 89 95 76 22 51
31 99 107 126 124 61 13 47
63 97 88 103 100 58 23 33
125 101 90 106 107 69 15 52
250 104 91 107 112 78 24 51
500 115 93 109 126 89 26 59
1000 108 98 115 124 78 21 55
1412 104 101 119 123 55 20 33
MMS 86 57 67 57 630 427 158
 
SC1 100 105 100 100 76 27 46
SC2 88 76 33 41
16 109 88 91 99 82 13 67
3 97 113 117 13 74 18 54
63 84 99 103 87 70 26 41
125 98 108 112 110 73 22 49
250 111 86 90 100 91 26 62
5000 74 150 155 115 65 18 45
1000 100 86 90 89 64 31 31
1412 91 131 135 123 64 20 41
CP 69 99 103 71 476 288 138
without metabolic activation: 24 h treatment
SC1 100 81 100 100 118 82 31
SC2 97 88 65 20
16 106 95 107 113 109 71 33
31 118 90 101 120 103 60 38
63 127 85 95 121 122 70 46
125 91 89 100 91 107 67 35
250 64 94 106 86 116 79 32
500 46 90 101 47 94 69 22
1000 32 79 89 29 109 74 31
1272 31 125 140 43 89 63 22
MMS 75 68 77 57 829 411 304

CP: Cylcophosphamide

MMS: Methylmethanesulfonate

Table 2: Historical control data of the solvent control

  Mutation frequency per 10E+6 survivors
  - S9-mix + S9-mix
  3 hour treatment 24 hour treatment 3 hour treatment
Mean 101 98 100
SD 30 31 30
n 279 262 293
Upper control limit (95% control limits) 170 162 165
Lower control limit (95% control limits) 31 34 36

Table 3: Historical control data of the positive control

  Mutation frequency per 10E+6 survivors
  - S9-mix + S9-mix
  3 hour treatment 24 hour treatment 3 hour treatment
Mean 803 695 1545
SD 253 223 887
n 142 132 151
Upper control limit (95% control limits) 1533 119 3954
Lower control limit (95% control limits) 72 1270 -864
Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation in bacteria:

N-ethylcaprolactam was tested for its mutagenic potential in bacteria (Ames test) according to OECD guideline 471 and in compliance with GLP (Charles River Laboratories, 2018). Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 and Escherichia colistrain WP2 uvrA were exposed to the test item in the presence and absence of metabolic activation (S9 mix). In two independent experiments, N-ethylcaprolactam was tested at a concentration range of 52 to 5000 µg/plate, using the plate incorporation (first experiment) and the pre-incubation method (second experiment). Vehicle (Milli Q water) and positive controls were included in each experiment. Precipitation of the test substance was not reported and no cytotoxicity occurred up to t The test item did not induce a biologically relevant increase in the number of revertant colonies in any of the tested strains either with or without metabolic he highest tested concentration in the presence and absence of S9 mix. activation. Positive and vehicle controls were in the range of historical control data and confirmed the validity of the test system.

Thus, based on the results of the present study and under the experimental conditions chosen, N-ethylcaprolactam is not mutagenic in bacteria with and without metabolic activation.

 

In vitro cytogenicity in mammalian cells:

In vitro cytogenicity in mammalian cells was investigated in a chromosome aberration test performed in accordance with OECD guideline 473 and in compliance with GLP (Charles River Laboratories, 2018). The induction of structural chromosomal damage was evaluated in vitro in cultured peripheral human lymphocytes. In the first experiment, the cells were treated with the test item (vehicle: culture medium) for 3 h at concentrations of 500, 1000 and 1412 µg/mL with and without metabolic activation (S9 mix), and in the second experiment, for 24 h at 10, 1000 and 1412 µg/mL and for 48 h at concentration of 10, 750 and 1200 µg/mL, in the absence of metabolic activation. For the 48 h exposure time, appropriate toxicity was reached at this dose levels. Vehicle control and positive control substances (mitomycin C and cyclophosphamide) were included. All control substances showed the expected results and were within the range of historical control data, thus demonstrating the validity of the test system and the functionality of the S9 mix.

N-ethylcaprolactam did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome abberations in the absence and presence of S9 mix. In addition, the frequency of polyploid cells and cells with endoreduplicated chromosomes were not affected with or without metabolic activation.

Therefore, under the conditions of the study, the test substance did not show clastogenic activity in the chromosomal aberration test with and without metabolic activation in human lymphocytes in vitro.

 

In vitro gene mutation in mammalian cells:

N-ethylcaprolactam was tested for its mutagenic potential in mammalian cells according to OECD guideline 490 and in compliance with GLP (Charles River Laboratories, 2018). Two independent experiments were performed in L5178Y mouse lymphoma cells (thymidine kinase locus). The cells were treated with the test item at eight concentrations up to 1412 µg/mL for 3 h (first experiment) with and without metabolic activation (S9 mix) and for 24 h (second experiment) in the absence of metabolic activation. No toxicity was observed in the first experiment at the top concentration. In the second experiment the relative total growth was reduced to 43% at the test item concentration of 1412 µg/mL. Concurrent vehicle controls (water) and positive control substances were included. The mutation frequency of the vehicle control was within the range of historical control data. The positive control substances cyclophosphamide (+S9 mix) and methylmethanesulfonate (–S9 mix) significantly increased the mutation frequency in L5178Y cells, thus demonstrating the sensitivity and validity of the test system.

Under the conditions of the test, the N-ethylcaprolactam did not induce a biologically relevant increase in mutation frequency in any of the tested concentrations, either in the presence or absence of metabolic activation. Thus, the test item was not considered to be mutagenic in mammalian cells.

 

Conclusion:

In conclusion, assessment of the available experimental data on gene mutation in bacteria, gene mutation in mammalian cells and chromosome aberration in mammalian cells suggests that N-ethylcaprolactam is neither mutagenic nor clastogenic in vitro.

Justification for classification or non-classification

The available data on genetic toxicity in vitro do not meet the criteria for classification according to Regulation No. (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.