Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The test substance was not mutagenic in a bacterial reverse mutation assay. in vitro cytogenicity studies: The test item was not clastogenic in a mammalian chromosome aberration test but was tested positive in two in vitro micronucleus assays in V79 cells. In vitro HPRT: The test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

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:
2015-05-27 to 2015-07-29
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:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
experiment 1
4 h without metabolic activation: 13.8, 27.5, 55.0, 110.0, 220.0, 330.0, 440.0, 660.0 μg/mL
4 h with metabolic activation: 6.9, 13.8, 27.5, 55.0, 110.0, 165.0, 220.0 μg/mL
experiment 2
24 h without metabolic activation: 13.8, 27.5, 55.0, 110.0, 220.0, 330.0, 440.0 μg/mL
4 h with metabolic activation: 55.0, 110.0, 123.8, 137.5, 151.4, 165.0 μg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen 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:
ethylmethanesulphonate
Remarks:
(EMS) 0.15 mg/mL = 1.2 mM, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
(DMBA) 1.1 μg/mL = 4.3 μM (experiment I) 2.2 μg/mL = 8.6 μM (experiment II), in DMSO, with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 h
- Expression time: 7 days
- Selection time: 8 days
- Fixation time: fixation 7 days after treatment (colonies for cloning efficiency)

SELECTION AGENT: 11 μg/mL 6-thioguanine
STAIN: 10 % methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: 2 experiments with each 2 culture dishes

NUMBER OF CELLS EVALUATED:
The stained colonies with more than 50 cells were counted.

DETERMINATION OF CYTOTOXICITY
- Method: reduction of cloning efficiency

OTHER:
A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 13.8 μg/mL and 1760 μg/mL were tested. It was checked for cytotocix effects, precipitation or phase seperation, pH and osmolarity.

The dose range of experiment 1 were based on the pre-experiment and of experiment 2 on the pre-experiment (without metabolic activation) and experiment 1 (with metabolic activation).
Evaluation criteria:
Acceptability
The gene mutation assay was considered acceptable if it met the following criteria:
a) the numbers of mutant colonies per 10exp6 cells found in the solvent controls fell within the laboratory historical control data range
b) the positive control substances produced a significant increase in mutant colony frequencies and remained within the historical control range of positive controls
c) the cloning efficiency II (absolute value) of the solvent controls exceeded 50 %.
The study complied with those criteria.

Evaluation
A test item was classified as positive if it induced either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points was considered non-mutagenic in this system.
A positive response was described as follows:
A test item was classified as mutagenic if it reproducibly induced a mutation frequency that was three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item was classified as mutagenic if there was a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency was not observed.
However, in a case by case evaluation this decision depended on the level of the corresponding solvent control data. If there was by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range had to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares, calculated using Sum_neu_v2.xltm, version 2.0) 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 was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological and statistical significance was considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
experiment 1 at 110 μg/mL (without metabolic activation), experiment 2 ≥ 123.8 μg/mL (with metabolic activation)
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: no effects observed
- Effects of osmolality: no effects observed
- Other confounding effects: In the pre-experiment phase separation was observed at the two highest concentrations of 880.0 and 1760 μg/mL following 4 hours treatment with and without metabolic activation. After 24 hours treatment without metabolic activation phase separation was noted at 1760 μg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects were observed for experiment 1 at 110 μg/mL (without metabolic activation) and for experiment 2 ≥ 123.8 μg/mL (with metabolic activation). The cell cultures at these concentrations were terminated earlier.
  concentration [µg/mL] S9 mix relative cloning efficiency I [%] relative cell density [%] relative cloning efficiency II [%] mutant colonies/10exp6 cells induction factor relative cloning efficiency I [%] relative cell density [%] relative cloning efficiency II [%] mutant colonies/10exp6 cells induction factor
Experiment 1 (4 h treatment)     culture I culture II
Solvent control (DMSO)   - 100 100 100 12.9 1 100 100 100 16.2 1
Positive control (EMS) 150 - 93 72.7 92.1 181.4 14.1 95.9 100.7 97.5 168.7 10.4
Test item 13.8 - 99.7 131.3 92.6 23.3 1.8 105.2 130.3 101.6 9.8 0.6
27.5 - 95 109.2 90.9 14.4 1.1 98.7 86.3 100 15.1 0.9
55 - 88.8 108.1 96.1 14 1.1 90.8 131.5 99.1 12.1 0.7
110 - 17.6 22.1 95.5 18.2 1.4 12.8 19.8 99.6 13.6 0.8
220 - 0 culture was not continued 1) 0 culture was not continued 1)
330 - 0 culture was not continued 1) 0 culture was not continued 1)
440 - 0 culture was not continued 1) 0 culture was not continued 1)
660 - 0 culture was not continued 1) 0 culture was not continued 1)
Solvent control (DMSO)   + 100 100 100 7.6 1 100 100 100 16.4 1
Positive control (DMBA) 1.1 + 102.9 85.7 103.1 221.7 29 98.7 107.9 99.9 291.5 17.8
Test item 3.4 + 98.7 culture was not continued 2) 102.4 culture was not continued 2)
6.9 + 103.1 89.4 101.7 19.5 2.6 99.8 109.2 104 11.8 0.7
13.8 + 100.8 55.3 102.8 18 2.4 103.4 125.8 103.7 14.2 0.9
27.5 + 98.6 66 101.6 18.8 2.5 97.4 106.5 103.3 22 1.3
55 + 93.4 72.6 102.5 17.6 2.3 91.5 111.7 104.7 13 0.8
110 + 53.8 49.8 102.9 20.1 2.6 60.5 72.6 103.4 31.1 1.9
165 + 0 culture was not continued 1) 0 culture was not continued 1)
220 + 0 culture was not continued 1) 0 culture was not continued 1)
Experiment 2 (24 h treatment)     culture I culture II
Solvent control (DMSO)   - 100 100 100 14 1 100 100 100 9.6 1
Positive control (EMS) 150 - 93.1 80.3 69.8 370.1 26.4 72 79.5 99.2 350.9 36.6
Test item 6.9 -   culture was not continued 2) culture was not continued 2)  
13.8 - 99.4 110 76.3 18.9 1.3 97.5 111.5 95.7 21.9 2.3
27.5 - 93.4 92.6 90.5 11.9 0.9 95.1 96.3 101.3 9.9 1
55 - 94.5 80.5 83.5 17.1 1.2 84.4 82.1 99.4 28.6 3
110 - 96.1 76.7 73.2 23.1 1.6 70.8 70.1 99.1 10.4 1.1
220 - 0 8.4 culture was not continued 1) 0 8.8 culture was not continued 1)
330 - 0 0 culture was not continued 1) 0 3.9 culture was not continued 1)
440 - culture was not continued 1) culture was not continued 1)
Experiment 2 (4 h treatment)     culture I culture II
Solvent control (DMSO)   + 100 100 100 24.5 1 100 100 100 11 1
Positive control (DMBA) 2.2 + 79.9 85.7 104.2 236.3 9.6 98.3 97.2 78.8 258.8 23.6
Test item 13.8 + culture was not continued 2) culture was not continued 2)
27.5 + 84.9 culture was not continued 2) 88.6 culture was not continued 2)
55 + 80.2 81.2 102.3 25 1 86.9 135.8 92.5 7.2 0.7
110 + 55.6 101.7 101.5 21.4 0.9 63.2 114.5 84.8 13.5 1.2
123.8 + 19.6 93.6 100.9 25.9 1.1 29 78.2 93.9 18.9 1.7
137.5 + 7.4 85.8 99.1 26.7 1.1 6.1 59.4 77.8 24.1 2.2
151.4 + 5.3 59.3 101.6 25.8 1.1 3.8 54 76.2 32.3 2.9
165 + culture was not continued 2) 0 culture was not continued 2)

1) culture was not continued due to exceedingly severe cytotoxic effects

2) culture was not continued since a minimum of only four analysable concentrations is required

Conclusions:
An in vitro gene mutation study (HPRT) was conducted in V79 cells of the Chinese hamster. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
Executive summary:

An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 13.8 μg/mL and 1760 μg/mL were tested. For the main experiments a concentration range between 6.9 and 660 μg/mL was used. The main assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The used concentration range of the experiments was limited by cytotoxic effects. Cytotoxic effects were observed for experiment 1 at 110 μg/mL (without metabolic activation) and for experiment 2 ≥ 123.8 μg/mL (with metabolic activation). No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium: histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: All tester strains except the strain TA102 contain a uvrB- mutation, TA98 and TA100: R-factor; all of the strains contain an additional mutation (rfa), which causes the loss of the outer lipopolysaccharide barrier
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix, Aroclor 1254 induced
Test concentrations with justification for top dose:
with metabolic activation: 0, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
without metabolic activation: 0, 5, 15, 50, 150, 500, 1500 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (all strains)
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 h

SELECTION AGENT: minimal medium

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
The mean number of revertant colonies per plate from two independent experiments were compared to the mean numbers of revertants from the respective positive and solvent control groups. The statistical significance was estimated.
Statistics:
The mean number of revertant colonies per strain were compared to the positive and negative controls. The estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level was done using a X2-test.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity was observed in all strains at 1500 and 5000 µg/plate and at 500 µg/plate in strain TA102 and 1537
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: The historical control data of the revertant frequencies of the strains used were in the same range as the data obtained in the test.

Table 1: Number of revertants per plate (mean of three plates), Experiment 1

strain

TA 98

strain

TA 100

strain

TA 102

strain

TA 1535

strain

TA 1537

conc. [µg]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Solvent control

17

24

123

122

265

342

21

9

9

13

0

18

24

133

129

301

353

20

12

10

11

5

22

28

 

 

271

325

 

 

 

 

15

21

20

104

 

264

308

22

6

9

9

50

18

23

111

126

257

291

22

9

9

11

150

17

25

103

118

238

278

20

8

4

13

500

17

19

96

108

126*

231*

17

5

5*

14

1500

8*

15*

98*

111

4*

13*

1*

6

6*

7*

5000

 

 

 

26*

 

 

 

 

 

 

Sodium azide

0.7 µg

 

 

560

 

 

 

525

 

 

 

2-Nitrofluorene

2.5 µg

283

 

 

 

 

 

 

 

 

 

9-Aminoacridine

50 µg

 

 

 

 

 

 

 

 

583

 

Mitomycin C

0.15 µg

 

 

 

 

1001

 

 

179

 

 

2-aminoanthracene

0.8 µg

 

687

 

1035

 

 

 

 

 

 

2-aminoanthracene

0.9 µg

 

 

 

 

 

664

 

 

 

 

2-aminoanthracene

1.7 µg

 

 

 

 

 

 

 

 

 

231

*Toxicity

Table 2: Number of revertants per plate (mean of three plates), Experiment 2

strain

TA 98

strain

TA 100

strain

TA 102

strain

TA 1535

strain

TA 1537

conc. [µg]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Solvent control

21

22

106

106

239

360

20

10

16

15

0

24

24

115

123

306

390

16

9

14

15

5

14

25

 

 

255

357

17

 

21

11

15

14

18

89

122

238

353

20

 

20

14

50

17

17

100

116

249

357

17

10

17

16

150

15

13

105

128

253

369

22

11

15

17

500

12

19

89

107

188*

271*

16

11

10*

12

1500

2*

10*

84*

97

 

 

 

6

 

 

5000

 

 

 

 

 

 

 

0*

 

 

Sodium azide

0.7 µg

 

 

317

 

 

 

789

 

 

 

2-Nitrofluorene

2.5 µg

355

 

 

 

 

 

 

 

 

 

9-Aminoacridine

50 µg

 

 

 

 

 

 

 

 

333

 

Mitomycin C

0.15 µg

 

 

 

 

585

 

 

141

 

 

2-aminoanthracene

0.8 µg

 

453

 

735

 

 

 

 

 

 

2-aminoanthracene

0.9 µg

 

 

 

 

 

897

 

 

 

 

2-aminoanthracene

1.7 µg

 

 

 

 

 

 

 

 

 

197

*Toxicity

 

Conclusions:
The test substance was not mutagenic in this bacterial reverse mutation assay.
Executive summary:

The mutagenicity of the substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in DMSO and tested in concentrations of 5 to 5000 µg per plate in the presence and of 5 to 1500 µg per plate in the absence of S9. In the absence of S9-mix bacteriotoxicity was observed towards the strains TA102, and TA1537 at 500 μg/plate and towards the strains TA1535, TA98, and TA100 at 1500 µg/plate. In the presence of S9-mix the test item was bacteriotoxic towards the strains TA102 at 500 μg/plate and towards the strains TA1537 and TA98 at 1500 µg/plate and towards the strains TA1535 and TA100 at 5000 μg/plate. Precipitation of the test compound on the plates was not observed. Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system. In the concentration range investigated, the test substance did not induce a significant or dose related increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that the test compound under the experimental conditions described was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

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

Genetic toxicity in vivo

Description of key information

In vivo mutagenicity test: The test substance did not cause micronuclei in bone marrow cells of the mouse.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-08-15 to 2007-08-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, annex 4C, 19 May 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males 37 g (mean), females 29.6 g (mean)
- Assigned to test groups randomly: yes
- Housing: single
- Diet: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-72
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: corn oil
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Amount of vehicle: total amount: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was formulated in corn oil.

Duration of treatment / exposure:
single treatment, sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Frequency of treatment:
only one application
Post exposure period:
The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1 h, 2 - 4 h, 6 h, 24 h and 48 h after administration of the test item. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
24 h preparation interval
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
24 h preparation interval
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
24 h preparation interval
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
48 h preparation interval
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: orally
- Doses / concentrations: 40 mg/kg bw
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, D-64293 Darmstadt)/Giemsa (Merck, D-64293 Darmstadt). Cover slips were mounted with EUKITT (Kindler, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 500, 750 and 1000 mg/kg bw
- Clinical signs of toxicity in test animals: The animals treated with 750 and 1000 mg/kg bw expressed toxic reactions (ruffled fur, reduction of spontaneous activity, abdominal position, eyelid closure, apathy, tremor, death)

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group.
- Ratio of PCE/NCE: The ratio of PCE to NCE was not relevantly decreased after treatment with the test item as compared to the mean value of PCE/NCE of the vehicle control.

Table 1: Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 2000 total erythrocytes

Test group

Dose mg/kg bw

Sampling time

PCEs with micronuclei [%]

range

PCE per 2000 erythrocytes

vehicle

0

24

0.12

0-6

1137

Test item

125

24

0.1

1-4

1132

Test item

250

24

0.12

0-6

1146

Test item

500

24

0.155

1-6

986

Positive control

40

24

2.385

12-68

1025

Test item

500

48

0.115

1-5

1020

 

Conclusions:
The test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

The test item was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated: 24 h preparation interval: 125, 250 and 500 mg/kg bw. 48 h preparation interval: 500 mg/kg bw. The analysis of the formulations used for dosing the animals showed, that the obtained results for the formulation concentrations correspond to their respective nominal values. The recovery values ranged between 93.7 to 100.7 % of the nominal values. As estimated by a pre-experiment 500 mg test substance per kg bw was suitable. In the main experiment one male (animal no. 66) died after treatment with this dose. The mean number of polychromatic erythrocytes was not relevantly decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test substance did not have any cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test item were below or near to the value of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

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

Additional information

Genetic toxicity in vitro:

Key studies

Ames test

The mutagenicity of the substance was studied in Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without metabolic activation system. The test substance was tested in concentrations of 5 to 5000 µg per plate in the presence and of 5 to 1500 µg per plate in the absence of S9. In conclusion, the results indicate that the test compound was not mutagenic to Salmonella typhimurium strains in the presence and absence of a metabolizing system but bacteriotoxic in high concentrations.

HPRT assay

An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A concentration range between 6.9 and 660 μg/mL was used. The main assay was performed in two independent experiments. Cytotoxic effects were observed. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

   

Weight of evidence

Chromosome aberration test

An in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells was conducted using primary human lymphocytes. Four treatment conditions were used for the study. The test material was toxic and did not induce any statistically significant increases in the frequency of cells with aberrations using a dose range that induced approximately 50 % mitotic inhibition. As a conclusion, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

Micronucleus test

The test item was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and the presence of metabolic activation by S9 mix. The test substance concentrations were: 13.8, 27.6, 55.2, 110.3, 220.6, 441.3, 882.5, and 1765.0 µg/mL. In the absence of S9 mix, the values of the two highest scorable concentrations (55.2 and 110.3 µg/mL) clearly exceeded the laboratory's historical control data range (0.0-2.5 % micronucleated cells). In the presence of S9 mix, two statistically significant increases in the percentage of micro-nucleated cells (2.75 % and 2.45 % of control) clearly exceeding the laboratory's historical control data range (0.0-2.5 % micronucleated cells) were observed at the two highest scored concentrations of 110.3 and 220.6 µg/mL. Therefore, the test substance was considered to induce micronuclei in vitro in V79 cells.

 

Conclusion

Two studies (Ames = bacterial gene mutation, HPRT = mammalian gene mutation) were selected as key studies as the studies were conducted according to OECD guideline and the tests were performed using the test material provided by the registrant. The Chromosome aberration and Micronucleus test both cover the same mutagenic endpoint but with different methods of analysis and were conducted according to OECD guidelines. Therefore, they were considered as a weight of evidence approach. As the test conducted in human lymphocytes showed no mutagenic potential and the test in hamster cells showed mutagenic potential a further study in vivo was conducted to evaluate the chromosomal aberration potential of the test substance.

 

Genetic toxicity in vivo

Micronucleus test

The test item was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. 10 animals per test group were evaluated for the occurrence of micronuclei. The dose levels of the test item were: 24 h preparation interval: 125, 250 and 500 mg/kg bw; 48 h preparation interval: 500 mg/kg bw. One male died after treatment with 500 mg/kg bw. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. In conclusion the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

 

Overall conclusion

The tests investigating the gene mutation properties of the test substance (Ames test, HPRT assay) showed negative results. Therefore the test substance was considered to have no gene mutation potential. The in vitro chromosome aberration test in human lymphocytes did not show an increase in chromosomal aberrations after treatment with the test substance. The in vitro micronucleus test in V79 hamster cells to test for chromosomal aberrations on the other hand resulted in a formation of micronuclei and therefore was positive. Due to this finding an in vivo micronucleus test was performed as advised in ECHA Guidance (Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a, October 2015). The test was conducted in mice according to OECD Guideline 474 and showed no increase in formation of micronuclei in vivo. Therefore it was determined to be non clastogenic. Considering all available data it was concluded that the test substance is not to be classified for genetic toxicity.

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The data did not indicate genetic mutation properties of the test substance and was concluded to be non clastogenic. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.