Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was tested at concentrations between 0 and 5000 ug/plate in an Ames test with and without metabolic activation. Both a plate incorporation and pre-incubation assay were performed. The substance did not induce mutations in Salmonella Typhimurium strains TA 98, TA100, TA1535 and TA1537. Therefore it is concluded that the substance is not mutagenic in bacterial cells (BASF 1988)

The substance was tested in the L5178Y TK +/- Mouse  Lymphoma Assay according to OECD 490 up to precipitation level. Exposure was either 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). No increase in the mutant frequency at the TK +/- locus was reported (evaluation with the Global Evaluation Factor (GEF) of 126 E-06). Therefore it is concluded that the substance is non-mutagenic in this assay (Envigo 2017).

The substance was tested in an in vitro micronucleus test (OECD 487). Human lymphocytes were exposed for 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). After the exposure cytokinesis was inhibited by treatment with Cytochalasin B. There was no inhibition of the CBPI and the concentrations to be evaluated were limited by precipitate at 40 ug/mL (4 hour exposures) or 20 ug/mL (24 hours exposure). No increase in the percentage of micronucleated binuclear cells was observed in any of the treatments. Therefore it can be concluded that the substance is not clastogenic (Envigo 2017).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, non-GLP, strain for AT substitution was not included
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
No strain to detect AT substitution is included in the study design
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from Aroclor 1254 induced rat liver (prepared on Dec 7 1987)
Test concentrations with justification for top dose:
exp 1 (plate incorporation): S. typhimurium TA 1535, TA 1537, TA 98 and TA 100: 0, 20, 100, 500, 2500 and 5000 ug/plate
exp 2 (preincubation) S. typhimurium TA 1535, TA 1537, TA 98 and TA 100: 0, 20, 100, 500, 2500 and 5000 ug/plate
exp 3 (preincubation) S. typhimurium TA 98: 0, 20, 100, 500, 2500 and 5000 ug/plate
Vehicle / solvent:
aqua dist.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
other: N-methyl-N-nitro-N-nitrosoguanidine
Remarks:
for TA 100 and TA 1535 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
for TA98 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for TA 1537 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
for all strains with S9 (no additional control is included)
Details on test system and experimental conditions:
METHOD OF APPLICATION: exp 1 plate incorporation; exp 2 and 3 preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: titer/bacterial backgound lawn

S-9 fraction: ratio 3/7 as S9 fraction/cofactors prepared on the day of the experiment
concentration of cofactors:
MgCl 8 mM
KC1 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer(
Evaluation criteria:
positive if:
-doubling of the spontaneous mutation rate (control)
-dose-response relationship
-reproducibility of the results
Statistics:
NA
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight reduction of the titer in some of the strains at 5000 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The 3rd experiment was performed because in the 2nd experiment a decrease in bacterial background lawn was observed at all concentrations.
Conclusions:
negative without metabolic activation
negative with metabolic activation

The substance is negative in an Ames test with and without metabolic activation
Executive summary:

The substance was tested at concentrations between 0 and 5000 ug/plate in an Ames test with and without metabolic activation. Both a plate incorporation and pre-incubation assay were perfomed. The substance did not induce mutations in Salmonella Typhimurium strains TA 98, TA100, TA1535 and TA1537. Therefore it is concluded that the substance is not mutagenic in bacterial cells.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 October 2016 to 21 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
NA
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: blood of healthy non-snoking volunteers
- Cell cycle length: 16 hours
- Sex, age and number of blood donors: Preliminary Toxicity Test: female, aged 25 years; Main Experiment (4-hour with S9 and 24-hour): female, aged 24 years; Main Experiment (4-hour without S9): male, aged 33 years
- Cell type: lymphocytes of fresh heparinized whole blood (stimulated to divide by PHA)

CULTURE MEDIA USED: Whole blood cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 inhumidified air.

Culture medium lymphocytes:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood
Cytokinesis block (if used):
Cytochalasin B added to block actin polymerisation
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/beta-naphthaflavone induced rat liver S9
Test concentrations with justification for top dose:
4-hour without S9 0*, 1.25, 2.5, 5*, 10*, 20*, 40*, 80
4-hour with S9 (2%) 0*, 1.25, 2.5, 5*, 10*, 20*, 40*, 80
24-hour without S9 0*, 1.25, 2.5*, 5*, 10*, 20*, 40, 80


* evaluated concentration
Vehicle / solvent:
Minimal Essential Medium (MEM)
Untreated negative controls:
yes
Remarks:
MEM
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine
Details on test system and experimental conditions:
Cells were exposed to the substance in MEM in presence and/or absence of S9 for 4 or 24 hours at approximately 37 ºC. Thereafter the cultures were centrifuged, the treatment medium was replaced with original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.Thereafter cells were fixed with methanol/glacial acetic acid (19:1 v/v) and stored at 4 ºC prior to slide making. Slides were stained with Giemsa and dried. Thereafter cells (500 per culture) were scored for mono-, bi- and multinuclei and CPBI (plus cytostasis). In the main experiment 1000 cells per culture (2 cultures per concentration) were also scored for the number of micronucleated cells (and the number of micronuclei per cell)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: 2000 binucleated cells were analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

DETERMINATION OF CYTOTOXICITY: 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI (Cytokinesis Block Proliferation Index) value expressed as a percentage of the vehicle controls. Cytostasis was calculated from these values
Evaluation criteria:
Negative when:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.

Positive when:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
Statistics:
Chi-squared Test
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
concentrations limited by precipitate at 40 ug/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
concentrations limited by precipitate at 20 ug/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC FACTORS
- Effects of pH: 7.26-7.35
- Effects of osmolality: 276-296 mOsm

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: available in the report
- Negative (solvent/vehicle) historical control data: available in the report

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
Remarks on result:
other: 4 exposure

4-h without metabolic activation

Dose Level (µg/mL)

Mean CBPI

% Cytostasis

 

Mean % Binucleated cells with MN

 

 

 

 

0

1.52

0

0.25

5

1.48

9

0.50

10

1.50

4

0.50

20

1.52

0

0.25

40 P

1.50

4

0.40

MMC 0.2

1.32

38

4.60***

 

4-h with metabolic activation

Dose Level (µg/mL)

Mean CBPI

% Cytostasis

 

Mean % Binucleated cells with MN

 

 

 

 

0

1.44

0

1.30

5

1.47

0

0.80

10

1.47

0

0.90

20

1.50

0

0.60

40 P

1.47

0

0.70

CP 5

1.26

41

3.55***

 

24-h without metabolic activation

Dose Level (µg/mL)

Mean CBPI

% Cytostasis

 

Mean % Binucleated cells with MN

 

 

 

 

0

1.70

0

0.45

2.5

1.71

0

0.20

5

1.71

0

0.50

10

1.69

1

0.40

20 P

1.71

0

0.60

DC 0.075

1.57

18

2.85***

 

*** p <0.001

P = precipitate

Conclusions:
In the in vitro micronucleus assay the substance did not show clastogenic effects
Executive summary:

The substance was tested in an in vitro micronucleus test (OECD 487). Human lymphocytes were exposed for 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). After the exposure cytokinesis was inhibited by treatment with Cytochalasin B. There was no inhibition of the CBPI and the concentrations to be evaluated were limited by precipitate at 40 ug/mL (4 hour exposures) or 20 ug/mL (24 hours exposure). No increase in the percentage of micronucleated binuclear cells was observed in any of the treatments. Therefore it can be concluded that the substance is not clastogenic.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 October 2016 to 15 November 2016
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)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
``Kanpoan No. 287 - - Environment Protection Agency``
``Eisei No. 127 - - Ministry of Health and Welfare``
``Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry``
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro gene mutation study in mammalian cells
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED: L5178Y TK+/- 3.7.2c mouse lymphoma cell line
- Source of cells: MRC Cell Mutation Unit at the University of Sussex, Brighton, UK
- Cell cycle length:ca 12 hours
- Methods for maintenance in cell culture if applicable: stored in liquid nitrogen at approximately -196°C

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes, before freezing
Additional strain / cell type characteristics:
other: TK+/-
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/beta-naphthaflavone induced rat liver S9
Test concentrations with justification for top dose:
4 h exposure: 4.88, 9.77, 19.53, 39.06, 78.13, 156.25 µg/mL
24 h exposure: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5 µg/mL

Top dose based on presence of precipitate
Vehicle / solvent:
RPMI 1640 medium (R0)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
A pretest was conducted on cell cultures at 5 x 10 E5 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E5 cells/mL using a 24-hour exposure period without S9. The dose range was set at 7.81 to 2000 µg/mL. After exposure cells were washed twice, resuspended and counted with a Coulter counter. The cultures were serially diluted to 2 x 10E5 cells/mL. and incubated at 37 C with 5% CO2 in air and sub-cultured after 24 hours. After a further 24 hours the cultures were counted to assess Suspension Growth (SG)

In the main study exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) in R0 or R10 medium (total volume to 20 mL). The solutions were incubated at 37°C for 4 or 24 hours with continuous shaking. The initial cell number was 1 x 10E6 cells/mL for 4 hour exposures and 0.3 x 10E6 cells/mL for 24 hour exposure.
At the end of the treatment period, for each experiment, the cells were washed twice, resuspended in R20 medium and incubated at 37°C with 5% CO2 (subcultured and counted every 24 hours -> Relative Suspension Growth (%RSG)) for 2 days.

On Day 2 of the experiment, the cells were counted again, diluted to 10E4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. These plates were scored after incubation at 37°C with 5% CO2 for the presence of large and small colonies. On the same day additional cells were diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Statistics:
Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
concentrations tested based on presence of precipitate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
concentrations tested based on presence of precipitate
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC FACTORS
- Effects of pH: no (7.26-7.35)
- Effects of osmolality: no (276-296 mOs)
- Precipitation:
main study: at 156.25 µg/mL in the 4-hour cultures and at and above 156.25 µg/mL in the 24-hour culture

RANGE-FINDING/SCREENING STUDIES:
4 h without S9: no cytotoxicity, precipitate at at 156.25 µg/mL
4 h with S9: no cytotoxicity, precipitate at at 156.25 µg/mL
24 h without S9: cytotoxicity at 5000 µg/mL, precipitate at at 156.25 µg/mL and above

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) are reported in an annex to the report
Remarks on result:
other: 4 hour exposure

Treatment

(µg/ml)

4-hours -S-9 Treatment

 

Treatment

(µg/ml)

4-hours +S-9 Treatment

 

 

Treatment

(µg/ml)

 

24-hours -S-9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

%RSG

RTG

MF§

 

 

 

%RSG

RTG

MF§

 

 

 

%RSG

RTG

MF§

0

 

100

1.00

125.29

 

0

 

100

1.00

121.98

 

0

 

100

1.00

167.03

1.22

Ø

102

 

 

 

1.22

Ø

90

 

 

 

2.44

Ø

106

 

 

2.44

Ø

95

 

 

 

2.44

Ø

91

 

 

 

4.88

Ø

96

 

 

4.88

 

101

1.04

117.93

 

4.88

 

100

1.01

124.72

 

9.77

 

102

0.99

163.62

9.77

 

99

1.30

91.19

 

9.77

 

97

0.88

143.50

 

19.53

 

89

1.03

147.55

19.53

 

101

1.28

86.37

 

19.53

 

95

1.06

93.13

 

39.06

 

89

0.80

140.91

39.06

 

96

1.24

101.83

 

39.06

 

102

0.95

121.80

 

78.13

 

92

0.81

187.85

78.13

 

96

1.00

142.70

 

78.13

 

92

0.83

135.64

 

156.25

 

81

1.03

133.44

156.25

 

93

1.12

121.10

 

156.25

 

92

0.98

91.35

 

312.5

 

80

1.00

139.62

 

Linear

trend

 

NS

 

 

Linear

trend

 

NS

 

 

Linear

trend

 

NS

EMS

 

 

 

 

 

CP

 

 

 

 

 

EMS

 

 

 

 

400

 

80

0.61

977.54

 

1.5

 

75

0.49

1231.42

 

150

 

40

0.36

1813.54

  § Positive wells per tray, 96 wells plated

 

Conclusions:
The substance is considered non-mutagenic in the L5178Y TK +/- Mouse Lymphoma Assay
Executive summary:

The substance was tested in the L5178Y TK +/- Mouse  Lymphoma Assay according to OECD 490 up to precipitation level. Exposure was either 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). No increase in the mutant frequency at the TK +/- locus was reported (evaluation with the Global Evaluation Factor (GEF) of 126 E-06). Therefore it is concluded that the substance is non-mutagenic in this assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the outcome of the in vitro testing the substance does not need to be classified according to EC No 1272/2008 (CLP Regulation)