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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is 1 study available on the target substance which was conducted to OECD TG 471. The test substance was negative for mutagenic activity in all strains tested, with and without metabolic activation, under the conditions of this assay.

There is data on the read across source 1 substance which includes:

- Chromosome aberration conducted to OECD TG 473, test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system under the experimental conditions described.

- Gene mutation conducted to OECD TG 476, test substance is considered to be non-mutagenic in this mouse lymphoma assay.

- Gene mutation conducted to OECD TG 471 under the test conditions employed, test substance showed no evidence of mutagenic activity in this bacterial system.

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
Study period:
from 15 August 2002 to 14 October 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production bureau, November 24, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
supplemented liver homogenate fraction (S9 mix)
Test concentrations with justification for top dose:
test 1 (range-finding): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
test 2: 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solubility of DV6850 was assessed at 50 mg/mL in water, in which it dissolved and formed a clear gel after sonication for 30 minutes. Water (purified in-house by reverse osmosis) was, therefore, used as the solvent for this study.
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Used in the absence of S9 mix
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Used in the absence of S9 mix
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Used in the absence of S9 mix
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2)
Remarks:
Used in the absence of S9 mix
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
Used in the presence of S9 mix
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Used in the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, test 1) and preincubation (30 min pre-incubation, test 2)

DURATION
- Preincubation period: 30 minutes in second test, no preincubation in first test
- Exposure duration: ca. 72 hours

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls with some evidence of a positive dose-relationship (increased revertant colony counts at concentrations below that at which the maximal increase is obtained), it will be considered to show evidence of mutagenic activity in this test system. No statistical analysis was performed.
If exposure to a test substance does not produce an increase in revertant colony numbers in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis was performed.

Statistics:
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after the additional testing outlined in the mutation test procedure, the test data may have been subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are ususally analysis of variance followed by Dunnett's test. Biological significance should always be considered along with statistical significance.
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic effects were observed at 5000 µg/mL without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
MAIN ASSAYS:
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to DV6850 at any concentration in either the presence or absence of S9 mix.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to DV6850.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the laboratory.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 - Results for test 1 (range-finding)

Metabolic activation

Test group

Dose level [µg/plate, for water: mL/plate]

Revertant colony counts (mean±SD)

TA98

TA100

TA1535

TA1537

WP2uvrA

yes

DV6850

5000

57±5

161±13

17±3

26±2

214±54

yes

DV6850

1500

66±5

169±5

18±7

27±5

180±28

yes

DV6850

500

55±15

182±16

14±4

26±5

181±17

yes

DV6850

150

57±2

172±21

14±5

21±4

186±4

yes

DV6850

50

62±13

165±13

16±3

24±6

199±16

yes

DV6850

15

61±16

158±10

17±3

27±9

169±22

yes

DV6850

5

59±8

174±14

20±2

23±7

166±15

yes

water

0.1

60±10

192±27

24±3

19±8

152±8

no

DV6850

5000

38±12

150±11

26±3

13±5

127±43

no

DV6850

1500

41±4

156±14

22±4

18±3

203±14

no

DV6850

500

44±12

149±4

19±2

24±2

184±40

no

DV6850

150

52±4

155±16

10±4

10±3

194±26

no

DV6850

50

56±7

145±6

14±10

10±4

159±11

no

DV6850

15

54±11

148±6

22±9

7±2

169±7

no

DV6850

5

50±14

119±18

22±6

13±5

135±56

no

water

0.1

46±8

157±12

19±4

22±6

199±20

yes

Benzo(a)pyrene

5

666±87

743±8

 

350±8

 

yes

2-Aminoanthracene

2

 

 

65±5

 

 

yes

2-Aminoanthracene

10

 

 

 

 

601±563

no

2- Nitrofluorene

1

208±14

 

 

 

 

no

Sodium azide

0.5

 

588±32

405±15

 

 

no

9-Aminoacridine

30

 

 

 

273±88

 

no

AF-2

0.05

 

 

 

 

841±80

Table 2 - Results for test 2, with pre-incubation

Metabolic activation

Test group

Dose level [µg/plate, for water: mL/plate]

Revertant colony counts (mean±SD)

TA98

TA100

TA1535

TA1537

WP2uvrA

yes

DV6850

5000

42±7

201±18

12±4

42±10

206±14

yes

DV6850

1500

39±11

187±31

23±1

42±12

140±56

yes

DV6850

500

49±13

161±11

19±3

31±3

215±9

yes

DV6850

150

47±5

173±16

21±2

23±12

211±36

yes

DV6850

50

51±12

191±21

17±2

49±10

232±21

yes

water

0.1

49±12

180±2

20±13

30±3

206±11

no

DV6850

5000

38±5

187±14

23±6

30±6

118±10

no

DV6850

1500

36±7

199±15

15±6

31±3

166±16

no

DV6850

500

33±4

182±21

20±5

27±3

136±3

no

DV6850

150

35±5

193±23

14±5

29±6

157±7

no

DV6850

50

39±4

172±12

14±4

24±5

162±27

no

water

0.1

34±10

173±18

12±3

31±7

135±23

yes

Benzo(a)pyrene

5

616±32

583±57

 

136±82

 

yes

2-Aminoanthracene

2

 

 

354±219

 

 

yes

2-Aminoanthracene

10

 

 

 

 

1149±119

no

2- Nitrofluorene

1

204±59

 

 

 

 

no

Sodium azide

0.5

 

369±49

111±79

 

 

no

9-Aminoacridine

30

 

 

 

690±175

 

no

AF-2

0.05

 

 

 

 

1566±121

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that, under the test conditions employed, DV6850 showed no evidence of mutagenic activity in this bacterial system.
Executive summary:

The mutagenic potential of DV6850 in a bacterial system was assessed in a valid GLP study. The study was conducted in compliance with the OECD Guideline 471: Bacterial Reverse Mutation Test.

In this in vitro assessment of the mutagenic potential of DV6850, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA 1535, TA 1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101 (CM891), were exposed to DV6850 diluted in water. Water was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254—treated rats (S9 mix). The first (range-finding) test was a standard plate incorporation assay, the second involved a pre-incubation stage.

Concentrations of DV6850 up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. Slight cytotoxic effects were observed for E. coli treated at 5000 µg/mL without metabolic activation. No other signs of toxicity were observed towards the tester strains in either mutation test.

No evidence of mutagenic activity was seen at any concentration of DV6850 in either mutation test. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, under the test conditions employed, DV6850 showed no evidence of mutagenic activity in this bacterial system.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 23 March 2010 to 21 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase, TK +/-, locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal activation (S9)
Test concentrations with justification for top dose:
Doses applied in the test (absolute values):
Experiment I, without S9 mix: 5.0, 10.0, 20.0, 40.0, 60.0, 80.0, 100.0 µg/mL
Experiment I, with S9 mix: 25.0, 50.0, 100.0, 200.0, 300.0, 350.0, 400.0 µg/mL
Experiment II, without S9 mix: 5.0, 10.0, 20.0, 40.0, 60.0, 80.0, 100.0 µg/mL
Experiment II, with S9 mix: 12.5, 25.0, 50.0, 100.0, 200.0, 250.0, 300.0, 320.0 µg/mL

The main experiments were evaluated at the following concentrations (absolute values):
Experiment I, without S9 mix: 20.0, 40.0, 60.0, 80.0, 100.0 µg/mL
Experiment I, with S9 mix: 25.0, 50.0, 100.0, 200.0, 300.0 µg/mL
Experiment II, without S9 mix: 20.0, 40.0, 60.0, 80.0, 100.0 µg/mL
Experiment II, with S9 mix: 25.0, 50.0, 100.0, 200.0, 250.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium (RPMI 1640)
- Justification for choice of solvent/vehicle: not reported
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Used without metabolic activation Migrated to IUCLID6: Experiment I: 19.5 µg/mL, Experiment II: 13 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Used with metabolic activation Migrated to IUCLID6: 3 µg/mL and 4 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 h with and without metaboilc activation,
Experiment 2: 24 h without metabolic activation, 4 h with metaboolic activation
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10-15 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 cultures

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 1E6 cells above the corresponding solvent control or negative control, respectively. A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures. However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls and the mutation rates of all negative and vehicle controls of this study are taken into consideration. Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the International Workshop on Genotoxicity Testing (IWGT) recommendations are fulfilled. Whenever a test item is considered genotoxic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase in the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase in mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Pre-experiment, 4 hour treatment: relevant toxic effects observed at >= 90.6 µg/mL without metabolic activation and at >= 362.5 µg/mL with metabolic activation. Pre-experiment, 24 hour treatment: strong toxic effects noted at >= 90.6 µg/mL.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: No relevant shift of osmolality of the medium at the maximum concentration of the test item
- Precipitation: No precipitation occurred up to the maximum concentration tested
- Others: Following 24 h treatment, strong toxic effects were noted at 90.6 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 45.3 µg/mL and 5800 µg/mL (as supplied) were used, equivalent to 40 µg and 5000 µg pure test substance/mL, respectively. The highest concentration in the pre-experiment was chosen with regard to the purity (87.2 %) of the test item.
Relevant toxic effects were observed at 90.6 µg/mL and above in the absence of metabolic activation and at 362.5 µg/mL and above in the presence of metabolic activation (4 h treatment). Following 24 h treatment strong toxic effects were noted at 90.6 µg/ml and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
In the second experiment (24 hours treatment, without S9 mix) the threshold (number of mutant colonies per 1E6 cells of each solvent control plus 126) and the historical range of solvent controls was exceeded in culture I at an intermediate concentration of 40.0 µg/mL without metabolic activation. The range of the historical solvent control data was exceeded at 20.0 and 60.0 µg/mL in the first culture of experiment II without metabolic activation (345 and 306 mutant colonies/1E6 cells, respectively). However, this increase was neither dose dependent as indicated by the lacking statistical significance nor reproduced in the parallel culture under identical conditions, and was therefore considered incidental.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

See "attached background material" for details on results.

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y up to the highest analysable concentrations of 100 µg/mL in the absence of metabolic activation in experiment 1 (4 hours treatment) and in experiment 2 (24 hours treatment) and at 400 µg/mL (experiment 1) and 320 µg/mL (experiment 2) in the presence of metabolic activation (4 hours treatment). Therefore, DV6850 is considered to be non-mutagenic in this mouse lymphoma assay.
Executive summary:

The potential of DV6850 to induce mutations at the mouse lymphoma thymidine kinase locus using the mouse lymphoma cell line L5178Y was assessed in a valid GLP study in accordance with the following guidelines:

- Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, February 1998, adopted July 21, 1997, Guideline No. 476 "In vitro Mammalian Cell Gene Mutation Test"

- Commission Regulation (EC) No. 440/2008 B.17: "Mutagenicity — In vitro Mammalian Cell Gene Mutation Test", dated May 30, 2008.

The assay was performed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation (S9) and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The highest concentration of 5800 µg/mL used in the pre-experiment was chosen with regard to the purity of the test item. The concentrations were corrected by a factor of 1.15 so, 5800 µg/mL correspond to 5000 µg/mL of the pure test item. The dose range of the main experiments was limited by cytotoxic effects.

No precipitation of the test item was observed in any experimental part. No substantial and reproducible dose dependent increase in the mutation frequency was observed up to the maximum concentrations with or without metabolic activation.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

The following concentrations were tested:

Experiment 1, without S9 mix, 4 hour treatment: 5, 10, 20, 40, 60, 80, 100 µg/mL

Experiment 1, with S9 mix, 4 hour treatment: 25, 50, 100, 200, 300, 350, 400 µg/mL

Experiment 2, without S9 mix, 24 hour treatment: 5, 10, 20, 40, 60, 80, 100 µg/mL

Experiment 2, with S9 mix, 4 hour treatment: 12.5, 25, 50, 100, 200, 250, 300, 320 µg/mL

The relative total growth and the number of mutant colonies per 1E6 cells were evaluated.

In conclusion it can be stated that during this mutagenicity test, the test item did not induce mutations in the thymidine kinase locus assay using the mouse lymphoma cell line L5178Y up to the highest analysable concentrations of 100 µg/mL in the absence of metabolic activation in experiment 1 (4 hours treatment) and in experiment 2 (24 hours treatment) and at 400 µg/mL (experiment 1) and 320 µg/mL (experiment 2) in the presence of metabolic activation (4 hours treatment).

Therefore, DV6850 is considered to be non-mutagenic in this mouse lymphoma assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 21 October 2002 to 6 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: whole blood culture
Test concentrations with justification for top dose:
Mitotic index:
First test (3 hours treatment + 17 hours recovery): 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500, 5000 µg/mL
First test, repetition (3 hours treatment + 17 hours recovery): 12.5, 25, 50, 75, 100, 150, 300 µg/mL
Second test, without S9 mix (20 hours continuous treatment): 6.25, 12.5, 25, 50, 75, 100, 125, 150 µg/mL
Second test, with S9 mix (3 hours treatment + 17 hours recovery): 25, 50, 75, 100, 125, 150, 175 µg/mL

Metaphase Analysis:
First test (3 hours treatment + 17 hours recovery): 75, 100 and 150 µg/mL
Second test, without S9 mix (20 hours continuous treatment): 12.5, 25, 150 µg/mL
Second test, with S9 mix (3 hours treatment + 17 hours recovery): 25, 100, 150 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test substance in solvents compatible with the test system was assessed. DV6850 was found to be insoluble in purified water, dimethyl sulphoxide, ethanol and acetone, however it formed a doseable suspension in culture medium at 10 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
used in the absence of S9 mix Migrated to IUCLID6: test 1: 0.2 µg/mL; test 2: 0.1 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
used in the presence of S9 mix Migrated to IUCLID6: 10 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
first test: 3 hours treatment + 17 hours recovery
second test, in absence of S9 mix: 20 hours (continuous treatment)
second test, in presence of S9 mix: 3 hours treatment + 17 hours recovery
- Fixation time (start of exposure up to fixation or harvest of cells)
first test: 18 hours
second test: 18 hours

NUMBER OF REPLICATIONS: 2 cultures

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
- Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
- The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit
- The increases are reproducible between replicate cultures.
- The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
- Evidence of a dose-relationship is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Mitiotix index <50% in metaphase test with exposure of 3 hours and 17 hours recovery at dose level of 150 µg/mL.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
MAIN ASSAYS:
First test (3 hours treatment + 17 hours recovery with and without S9 mix): Mitotic indices of cultured human lymphocytes treated with DV6850 at concentrations of up to 5000 µg/mL were calculated. Due to strong toxicity, this test was repeated at lower concentrations of up to 300 µg/mL. In the absence of S9 mix, DV6850 caused a reduction in the mitotic index to 35% of the solvent control value at 150 µg/mL. In the presence of S9 mix, DV6850 caused a reduction in the mitotic index to 40% of the solvent control at 150 µg/mL. The quantitative analysis for polyploidy showed a non-significant increase (4/500 versus 0 to 1/500 in controls) in the number of polyploid metaphase figures at 150 µg/mL in the absence of S9 mix. In the presence of S9 mix, a statistically significant increase in the number of polyploid meatphase figures was observed at 150 µg/mL when compared to solvent control.
Second test (3 hours treatment + 17 hours recovery with S9 mix, 20 hours continuous treatment without S9 mix): In the absence of S9 mix, DV6850 caused a reduction in the mitotic index to 56 % of the solvent control value at 150 µg/mL. In the presence of S9 mix, DV6850 caused a reduction in the mitotic index to 45% of the solvent control value at 150 µg/mL. The quantitative analysis for polyploidy showed a statistically significant increase in the number of polyploid metaphase figures at 150 µg/mL, in the absence and presence of S9 mic, when compared to the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA:
The frequency of aberrant metaphases for the positive and negative controls lie within lie within the current historical control range.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 - Summary of results of metaphase test 1

Exposure
period

(hours)

S9
mix

Concentration of
DV6850

(µg/mL)

Cells with aberrations
Excluding caps

Cells with aberrations
Including gaps

Relative
Mitotic

Index

(%)

Individual values (%)

Mean(%)

Individual values (%)

Mean (%)

3

-

0 (Culture medium)

0

2

1.0

0

2

1.0

100

3

-

75

0

2

1.0

0

2

1.0

87

3

-

100

1

1

1.0

1

1

1.0

67

3

-

150

2

4

3.0

2

4

3.0

35

3

-

0.2 (Mitomycin C)

22

24

23.0

22

24

23.0

-

3

+

0 (Culture medium)

1

0

0.5

1

0

0.5

100

3

+

75

2

1

1.5

2

1

1.5

77

3

+

100

2

2

2.0

2

2

2.0

59

3

+

150

1

0

0.5

1

1

1.0

40

3

+

10 (Cyclophosphamide)

24

22

23.0

24

22

23.0

-

Table 2 - Summary of results of metaphase test 2

Exposure
period

(hours)

S9
mix

Concentration of
DV6850

(µg/mL)

Cells with aberrations
Excluding caps

Cells with aberrations
Including gaps

Relative
Mitotic

Index

(%)

Individual values (%)

Mean(%)

Individual values (%)

Mean (%)

20

-

0 (Culture medium)

2

2

2.0

2

2

2.0

100

20

-

12.5

1

0

0.5

1

0

0.5

97

20

-

25

1

0

0.5

1

0

0.5

67

20

-

150

0

0

0.0

0

0

0.0

56

20

-

0.1 (Mitomycin C)

22

24

23.0

22

24

23.0

-

3

+

0 (Culture medium)

1

1

1.0

1

1

1.0

100

3

+

25

2

1

1.5

2

1

1.5

85

3

+

100

2

1

1.5

2

1

1.5

76

3

+

150

2

1

1.5

2

1

1.5

45

3

+

10 (Cyclophosphamide)

20

22

21.0

20

22

21.0

-

Table 3 – Mitotic index data, first test, repeat, without S9 mix, 3 hours treatment and 17 hours recovery

Concentration of DV6850
(µg/mL)

Mitotic indexa

Relative
mitotic indexa
(%)

Polyploidy

Incidence

% Mean

Incidence

% Mean

0 (Culture medium)

143/1000

15.5

100

0/500

0.1

167/1000

1/500

12.5

187/1000

18.5

119

 

 

182/1000

 

25

175/1000

17.1

110

 

 

166/1000

 

50

139/1000

13.9

90

 

 

138/1000

 

75

145/1000

13.5

87

 

 

124/1000

 

100

115/1000

10.4

67

 

 

93/1000

 

150

68/1000

5.5

35

4/500

0.8

42/1000

4/500

300

b

 

 

 

 

b

 

aCalculations have been made with rounded values

bFew cells, no metaphases present on the slide.

 

Table 4 – Mitotic index data, first test, repeat, with S9 mix, 3 hours treatment and 17 hours recovery

Concentration of DV6850
(µg/mL)

Mitotic indexa

Relative
mitotic indexa
(%)

Polyploidy

Incidence

% Mean

Incidence

% Mean

0 (Culture medium)

185/1000

18.4

100

3/500

0.4

183/1000

1/500

12.5

185/1000

18.1

98

 

 

176/1000

 

25

147/1000

15.6

85

 

 

164/1000

 

50

159/1000

15.4

84

 

 

148/1000

 

75

144/1000

14.2

77

 

 

140/1000

 

100

107/1000

10.8

59

 

 

109/1000

 

150

78/1000

7.4

40

9/500

1.4

69/1000

5/500

300

b

 

 

 

 

b

 

aCalculations have been made with rounded values

bFew cells, no metaphases present on the slide.

 

Table 5 – Mitotic index data, second test, without S9 mix, 20 hours continuous treatment

Concentration of DV6850
(µg/mL)

Mitotic indexa

Relative
mitotic indexa
(%)

Polyploidy

Incidence

% Mean

Incidence

% Mean

0 (Culture medium)

68/1000

6.6

100

0/500

0.0

63/1000

0/500

6.25

64/1000

6.8

103

 

 

71/1000

 

12.5

61/1000

6.4

97

 

 

66/1000

 

25

41/1000

4.4

67

 

 

47/1000

 

50

36/1000

3.9

59

 

 

41/1000

 

75

36/1000

3.2

48

 

 

28/1000

 

100

33/1000

3.0

45

 

 

26/1000

 

125

28/1000

3.7

56

 

 

46/1000

 

150

39/1000

3.7

56

22/500

3.7

35/1000

15/500

aCalculations have been made with rounded values

 

 Table 6 – Mitotic index data, second test, with S9 mix, 3 hours treatment and 17 hours recovery

Concentration of DV6850
(µg/mL)

Mitotic indexa

Relative
mitotic indexa
(%)

Polyploidy

Incidence

% Mean

Incidence

% Mean

0 (Culture medium)

80/1000

8.0

100

0/500

0.0

79/1000

0/500

25

69/1000

6.8

85

 

 

66/1000

 

50

59/1000

6.4

80

 

 

68/1000

 

75

57/1000

6.2

78

 

 

67/1000

 

100

52/1000

6.1

76

 

 

70/1000

 

125

35/1000

4.1

51

 

 

47/1000

 

150

36/1000

3.6

45

40/500

7.1

35/1000

31/500

175

26/1000

2.3

29

 

 

19/1000

 

aCalculations have been made with rounded values

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the test substance DV6850 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system under the experimental conditions described.
Executive summary:

The ability of DV6850 to induce chromosomal aberrations in human lymphocytes cultured in vitro was assessed in a valid GLP study in compliance with OECD Guideline No. 473.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the presence and absence of S9 mix derived from rat livers. Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

The cytotoxicity of DV6850 was assessed based on the mitotic index. On the basis of these data, the following concentrations were selected for metaphase analysis:

First test

With and without S9 mix - 3 hours treatment, 17 hours recovery: 75, 100 and 150 µg/mL.

Second test

Without S9 mix - 20 hours continuous treatment: 12.5, 25 and 150 µg/mL.

With S9 mix - 3 hours treatment, 17 hours recovery: 25, 100 and 150 µg/mL.

In both the absence and presence of S9 mix, DV6850 caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent controls in either test.

A quantitative analysis for polyploidy was made in cultures treated with the negative control and highest dose level. In the first test in the absence of S9 mix, a non-significant increase in the proportion of polyploid cells was observed at 150 µg/mL. In the presence of S9 mix, a statistically significant increase in the proportion of polyploid cells was observed at 150 µg/mL (p<0.01).

In the second test a statistically significant increase in the proportion of polyploid cells was observed at 150 µg/mL, in the absence and presence of S9 mix (p<0.001). However, the toxicological significance of the increases in polyploid cells remained doubtful.

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It is concluded that the test substance DV6850 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system under the experimental conditions described.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 March 2017 to 13 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted according to OECD test guidelines and was conducted to GLP. No deviations from the protocol were known to have occurred during the conduct of this study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test susbtance was received as a liquid at ambient temperature. The active substance was 31% and was diluted in water and propylene glycol micture with 4.2% sodium chloride. A correction factor of 3.22 was used in the preparation of all dose formulations to correct for purity.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Test concentrations with justification for top dose:
25, 50, 100, 250, 500, 1000, 2500 and 5000 μg/plate
Vehicle / solvent:
A stock solution of the test substance was prepared in DMSO at a concentration of 50 mg/mL on the day of each assay. The lower concentrations were prepared by serial dilutions with DMSO to attain the appropriate concentration for testing.
Untreated negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
Details on test system and experimental conditions:
The test substance was evaluated for mutagenic activity in the in vitro Salmonella E.Coli/mammalian microsome reverse mutation assay. The purity of the test susbtance was 31%. A correction factor of 3.22 was used in the preparation of all dose formulations to correct for purity. Four tester strains of Salmonella typhimurium (TA 1537, TA 98, TA100 and TA 1535) and 1 Exherichia Coli strain (WP2 urvA) were used for mutagenic testing. The test susbtance was prepared as a stock formulation in DMSO at concentrations of 50 mg/mL. Mutagenicity was performed in triplicate at each concentration with and without an Aroclor 1254-induced rat liver S9 metabolic activation system
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Based on the results of this study the test material is not considered to be mutagenic
Executive summary:

The test substance was evaluated for mutagenic potential in the in vitro salmonella E.Coli/mammalian microsome reverse mutation assay. The purity of the test substance was 31%. Acorrection factor of 3.22 was applied in the preapration of all dose formulations to correct for purity. Four tested strains of salmonella typhimurium (TA 1537, TA 98, TA 100 and TA 1535) and 1 Escherichia Coli strain (WP2 urvA) were used for mutagenicity testing. The test substance was prepared as a stock formulation in DMSO at concentrations of 50 mg/mL. Mutagenicity testing was performed in triplicate at each concentration with and without an Aroclor 1254 -induced rat liver S9 metabolic activation system.

In the initial assay the test susbtance was tested at 25, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate using the plate incorporation method. Precipitates were not observed at any dose level in all strains with or without metabolic activation.

Precipitates were not observed at any dose level in all strains with or without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or > 50% reduction in the mean number of revertant colonies compared to

the vehicle control) was observed at ≥ 50 μg/plate in strain TA1537 without metabolic activation, at ≥ 100 μg/plate in strain TA100 without metabolic activation, at ≥ 250 μg/plate in strains TA98 and TA1535 without metabolic activation and in strain TA100 with metabolic activation, and at ≥ 500 μg/plate in strains TA1537, TA98, and TA1535 with metabolic activation. Criteria for a negative response were met for all tester strains with and without metabolic activation. However, strain TA1537 and TA100 without metabolic activation contained only 1 and 2 non-cytotoxic concentrations respectively. None of the non-cytotoxic doses exhibited an increase above the historical vehicle control range for the analyzed concentrations in either strain. The confirmatory assay was adjusted to capture a larger range of non-cytotoxic concentrations and confirm the negative result.

In the confirmatory assay, Mirataine Bet O-30 was tested in Salmonella strains TA1537, TA98, TA100, and TA1535 at 1.0, 2.5, 5.0, 10, 25, 50, 100, 250, and 500 μg/plate in strains without metabolic activation and at 10, 25, 50, 100, 250, 500, 1000, and 1500 μg/plate with metabolic activation using the plate incorporation method. The E. coli strain WP2 uvrA was tested at concentrations of 100, 250, 500, 1000, 2500, and 5000 μg/plate in both with and without metabolic activation treatments using the plate incorporation method. Precipitates were not observed at any dose level in all strains with or without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or > 50% reduction in the mean number of revertant colonies compared to the vehicle control) was observed at ≥ 100 μg/plate in strains TA1537,

TA98, and TA100 without metabolic activation, at ≥ 250 μg/plate in strain TA1535 without metabolic activation and in strain TA100 with metabolic activation, at ≥ 500 μg/plate in strains TA98 and TA1535 with metabolic activation, and at ≥ 1000 μg/plate in strain TA1537 with metabolic activation. Criteria for a negative response were met for all tester strains with and without metabolic activation

The test substance did not induce a significant dose-related increase in the number of (His+) colonies in each of the four tester strains and in the number of revertant (Trp+) colonies in the tester strain Wp2urvA both in the absence and presence of S9 -metabolic activation. These results were confirmed in the follow up experiements.

These data support the conclusion that the test susbtance was negative for mutagenic activity in all strains tested, with and without metabolic activation, under the conditions of 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

Boundary composition.001 (Composition of C18:1 amidopropyl betaine containing ≥0.1% and < 10% C18:1 aminopropyl amide)

The target substance (TS) is not classified for genetic toxicity, according to Commission Regulation (EU) No 286/2011 of 10 March 2011 (second amendment of CLP regulation), based on the test data for the source 1 substance and target substance

 

Boundary composition.002 (Composition of C18:1 amidopropyl betaine containing < 0.1% C18:1 aminopropyl amide)

The target substance (TS) is not classified for genetic toxicity, according to Commission Regulation (EU) No 286/2011 of 10 March 2011 (second amendment of CLP regulation), based on the test data for the source 1 substance and target substance