Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test (GLP OECD 471 guideline study, Kr.1): No evidence of mutagenic activity in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA in the presence or the absence of S9-mix.

- In vitro mammalian gene mutation assay (GLP OECD 490 guideline study, Kr. 1): No relevant increase in mutation frequency reported in mouse lymphoma L5178Y cells in the presence or absence of metabolic activation.

- In vitro micronucleus test (GLP OECD 487 guideline study, Kr. 1): negative with and without metabolic activation activation in peripheral human lymphocytes.

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:
19-JAN-1995 (preliminary test-treatment) to 14-FEB-1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD guideline 471 (Bacterial Reverse Mutation Assay: Salmonella typhimurium) and OECD guideline 472 (Bacterial Reverse mutation Assay: Escherichia coli)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Histidine auxotrophy in S. typhimurium
Tryptophan auxotrophy in E.coli
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal preparation from 1254-aroclor induced rat (S9-mix).
Test concentrations with justification for top dose:
Preliminary toxicity test (S. typhimurium TA 98 and E. coli WP2 uvrA in the absence of S9-mix):
- 0 (DMSO) and 0 (top-agar and culture alone),
- 2.5, 25, 250, 2500 µg/plate (first test),
- 5, 50, 500 5000 µg/plate (second test)

Main test:
- 0, 50, 158, 500, 1580, 5000 µg/plate with and without metabolic activation in all bacteria strains (two independent tests)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO ( batch 04047HZ, Aldrich) for test substance dilution and all positive control compounds except sodium azide which was dissolved in purified water
- Justification for choice of solvent/vehicle: no data
- Volume of vehicle/solvent in the medium: 0.1 mL of DMSO (as vehicle negative control) or 0.1 mL of test substance concentrations (in DMSO)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: see "any other information on materials and methods"
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration: 48 hours at 37°C

SELECTION AGENT (mutation assays): histidine auxotrophy for S. typhymurium strains and tryptophan auxotrophy for E. coli

NUMBER OF REPLICATIONS: triplicates


DETERMINATION OF CYTOTOXICITY
- Method: observation of the visible thinning of the background lawn of non-revertant cells in following exposure to test substance


OTHER: no data
Evaluation criteria:
No data
Statistics:
No data
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: no data



RANGE-FINDING/SCREENING STUDIES: no visible thinning of the background lawn of non-revertant cells was obtained following exposure to RHODIASTAB X2 up to the highest tested concentrations in the preliminary range-finding test. Therefore,a top exposure level of 5000 µg/plate was selected for use in the main test.


COMPARISON WITH HISTORICAL CONTROL DATA: no


ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Table 7.6.1/1: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (test 1)

Conc.
(µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA/pKM101

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

23

1

114

5

16

1

17

4

45

8

50**

23

3

93

6

17

4

15

2

38

12

158**

21

5

99

2

16

1

16

2

48

1

500**

19

3

111

1

15

5

16

4

38

7

1580**

20

2

111

23

17

4

13

2

33

8

5000**

21

6

117

22

14

1

15

2

32

4

Positive controls***

143

10

851

47

561

45

588

66

846

47

34

6

110

10

24

4

15

2

42

3

Table 7.6.1/2: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (test 2)

Conc.
(µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA/pKM101

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

28

2

98

10

16

4

12

3

50

7

50**

26

9

106

9

12

3

9

2

45

6

158**

31

0

90

6

11

2

9

1

42

0

500**

27

5

104

11

9

3

12

2

45

6

1580**

21

7

86

7

12

5

11

1

42

11

5000**

21

8

74

5

9

3

13

2

29

6

Positive controls***

123

6

834

72

502

14

540

11

698

19

22

4

104

5

9

1

14

2

53

3

* Solvent control = negative control: 100 µL DMSO/plate

** Test substance solution volume: 100 µL/plate

*** Mutagens positive controls:

-Sodium azide (2 µg/plate) in TA1535 and TA 100 strains

-2-Aminoanthracene in TA1535 (2 µg/plate) and WP2 uvrA (10 µg/plate)

-9-aminoacridine (80 µg/plate) in TA1537 strain

-2-nitrofluorene (1 µg/plate) in TA 98 strain

-Benzo[a]pyrene (5 µg/plate) in TA1537, TA 100, TA 98 strains

-N-Ethyl-N’-Nitro-N-nitrosoguanidine (2 µg/plate) in WP2 uvrA

Table 7.6.1/3: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (test 1)

Conc.
(µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA/pKM101

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

27

1

125

11

21

4

18

1

43

3

50**

21

6

104

3

20

1

19

3

47

4

158**

23

2

109

4

18

2

18

3

43

8

500**

21

4

80

10

17

1

14

5

46

2

1580**

21

1

103

23

17

1

13

1

56

9

5000**

24

4

95

11

18

2

17

4

41

13

Positive controls***

478

34

598

43

486

78

114

1

553

18

Table 7.6.1/4: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (test 2)

Conc.
(µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA/pKM101

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

29

3

103

10

16

3

12

2

47

6

50**

33

7

113

16

11

4

12

3

52

7

158**

40

2

110

16

10

4

11

2

45

13

500**

29

2

100

12

8

2

11

1

49

7

1580**

35

7

89

9

11

7

8

3

51

4

5000**

24

2

82

3

7

1

8

1

50

2

Positive controls***

482

13

338

4

237

11

202

8

500

33

* Solvent control = negative control: 100 µL DMSO/plate

** Test substance solution volume: 100 µL/plate

*** Mutagens positive controls:

-2-Aminoanthracene in TA1535 (2 µg/plate) and WP2 uvrA (10 µg/plate)

-Benzo[a]pyrene (5 µg/plate) in TA1537, TA 100, TA 98 strains

Conclusions:
There was no evidence of mutagenic activity in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA at any dose level of Rhodiastab X2 in the presence and the absence od S9 -mix. Hence, Rhodiastab X2 was not mutagenic in these bacterial test systems.
Executive summary:

The mutagenic potential of Rhodiastab X2 (former commercial name of the registered substance) was assessed in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA, according to OECD guidelines 471-472, Japan MITI/MHW and US/EPA-TSCA 798.5265 and in compliance with Good Laboratory Practices. Tests were carried out in presence and in absence of liver preparations from Aroclor 1254-induced rats (S9-mix). Rhodiastab X2 was dissolved in dimethyl sulphoxide (DMSO) and tested at concentrations up to 5000 µg/plate without S9 -mix in a range-finding preliminary test to determine dose-level cytotoxicity. In the absence of cytotoxicity at the highest RHODIASTAB X2 concentrations, dose levels used in the main assays were: 50, 158, 500, 1580 and 5000 µg/plate with and without metabolic activation system. Two independent tests were performed .

Under conditions of both tests, no evidence of mutagenic activity was observed at any dose level of Rhodiastab X2 in the presence and the absence od S9 -mix. Hence, Rhodiastab X2 was not mutagenic in these bacterial test systems.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 10 September 2015 to 29 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: MR92143651
- Expiration date of the lot/batch: 01 January 2017
- Purity: 86.4% (No correction was made for purity of the test substance)
- Purity test date: 18 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Yes, maximum temperature: 50°C, maximum duration: unknown (until liquefaction).
- Solubility and stability of the test substance in the solvent/vehicle: unknown
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: unknown

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was heated to 37°C, to obtain a homogeneous liquid sample.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A
Species / strain:
lymphocytes: Human
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years)
- Suitability of cells: Peripheral human lymphocytes are recommended in the international OECD guideline.
- Cell cycle length, doubling time or proliferation index: Average Generation Time (AGT) of the cells: 12.8 to 12.9 hours.
- Sex, age and number of blood donors if applicable: Sex unknow. Age: from 25 to 35 years old. Number of donors: 4.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Number of passages if applicable: N/A (primary cells)
- Methods for maintenance in cell culture if applicable: N/A
- Modal number of chromosomes: Not mentioned
- Normal (negative control) cell cycle time: Not mentioned

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Life Technologies) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands). All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 56 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.9 - 37.4°C).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: N/A
- Periodically checked for karyotype stability: N/A
- Periodically 'cleansed' against high spontaneous background: N/A
Additional strain characteristics:
not specified
Cytokinesis block (if used):
Yes, using Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and ß-naphthoflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
- Dose range finding test: At the 24 hour exposure time single blood cultures were treated with 1.7, 5.4, 17, 52 and 164 μg 1-Phenyldecane-1,3-dione /ml culture medium without S9-mix.
- First cytogenetic assay: At the 3 h exposure time, blood cultures were treated in duplicate with 17, 52 and 164 μg test item/ml culture medium with and without S9-mix.
- Cytogenetic assay 1A: Based on the results of the first cytogenetic assay and solubility in the culture medium the following dose levels were selected for cytogenetic assay 1A (top dose based on the solubility of the test item in culture medium): Without & With S9-mix: 10, 50, 75 and 100 μg/ml culture medium
(3 hours exposure time, 27 hours harvest time).
- Cytogenetic assay 1B: Based on the results of the cytogenetic assay 1A and the solubility in the culture medium the following dose levels were selected for cytogenetic assay 1B (top dose based on the solubility of the test item in culture medium) in the presence of S9-mix: 10, 50, 70 and 80 μg/ml culture medium
(3 hours exposure time, 27 hours harvest time).
- Second cytogenetic assay: To obtain more information about the possible clastogenicity and aneugenicity of 1-Phenyldecane-1,3-dione, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay: Without S9-mix : 5, 10, 30, 40, 50, 60 and 70 μg 1-Phenyldecane-1,3-dione/ml culture medium (24 hours exposure time, 24 hours harvest time).
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO. The final concentration of the solvent in the culture medium was 1.0% (v/v).
- Justification for choice of solvent/vehicle: solvent in which the test item was found soluble.
Negative controls:
yes
Remarks:
= solvent control
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Details on test system and conditions:
METHOD OF APPLICATION: in medium.
- Cell density at seeding: N/A

DURATION
- Preincubation period: 46 ± 2 hours ((0.4 ml blood of a healthy donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin)
- Exposure duration: 3 hours or 24 hours
- After the exposure to 1-Phenyldecane-1,3-dione the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium with Cytochalasine B (5 μg/ml) and incubated for another 24 hours (1.5 times normal cell cycle).

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasine B

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: N/A

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were resuspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the WIL Research Europe study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensenbuffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Histolab, Gothenburg, Sweden) and mounted with a coverslip in an automated coverslipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

NUMBER OF CELLS EVALUATED:
- Cytotoxicity assessment: A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).
- Cytogenetic assessment/scoring of micronuclei: At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Not specified

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method:The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
Rationale for test conditions:
Acceptability of the assay:
An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analysed by the Chi-square test (one-sided, p < 0.05).
Evaluation criteria:
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided,
p < 0.05) increase compared with the concurrent negative control.
b) All results are inside the 95% control limits of the negative historical control data range.

Since the Chi-square test showed that there is a statistically significant difference between one of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.
Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.0.0 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not assessed
- Effects of osmolality: Not assessed
- Evaporation from medium: Not assessed
- Water solubility: Not assessed
- Precipitation: The test substance precipitated at concentrations of 164 μg/ml and upwards.
- Definition of acceptable cells for analysis: The following criteria for scoring of binucleated cells were used :
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
- Other confounding effects: The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).

RANGE-FINDING/SCREENING STUDIES:
In order to select the appropriate dose levels for the in vitro micronucleus test, cytotoxicity data was obtained in a dose range finding test. 1-Phenyldecane-1,3-dione was tested in the absence and presence of S9-mix.
The highest tested concentration was determined by the solubility of 1-Phenyldecane-1,3-dione in the culture medium.
Lymphocytes (0.4 ml blood of a healthy donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of 1-Phenyldecane-1,3-dione for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.
The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: see detailed results in section below.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see summary tables 7.6.1/10 and 7.6.1/11 in section below.
- Indication whether binucleate or mononucleate where appropriate: see summary tables 7.6.1/10 and 7.6.1/11 in section below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see summary table 7.6.1/12
- Negative (solvent/vehicle) historical control data: see summary table 7.6.1/13

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
- Other observations when applicable: N/A

Table 7.6.1/10: Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with 1-Phenyldecane-1,3-dione in the first cytogenetic assay (3 hours exposure time, 27 hours harvest time)

 

Metabolic activation (S9-mix)

Concentration (μg/ml)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

 

Number of binucleated cells with micronuclei

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

Without

0

0

0

1

1

3

3

6

10

12

3

0

3

5

0

5

50

22

0

0

0

2

1

3

75

61

3

0

3

3

0

3

0.25 MMC-C

25

1

3

4

45

40

85***

0.1 Colch

90

32

26

58***

162)

62)

22***

With

0

0

0

0

0

6

5

11

10

5

1

1

2

5

5

10

50

14

2

2

4*

4

0

4

803)

27

0

0

0

2

4

6

15 CP

71

1

0

1

24

23

47***

0

0

0

0

0

6

5

11

 

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1) 1000-1039 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

2) 472 and 212 binucleated cells respectively were scored for the presence of micronuclei

3) 1-Phenyldecane-1,3-dione precipitated in the culture medium.

Table 7.6.1/11: Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with 1-Phenyldecane-1,3-dione in the second cytogenetic assay (Without metabolic activation (-S9-mix); 24 hours exposure time, 24 hours harvest time)

Concentration (μg/ml)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

 

Number of binucleated cells with micronuclei

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

0

0

0

3

3

4

5

9

5

12

2

3

5

5

5

10

30

40

2

2

4

2

5

7

40

51

1

2

3

6

3

9

0.15 MMC-C

40

3

1

4

42

25

67***

0.05 Colch

96

63

55

118***

42)

32)

7***

 

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

2) 93 and 115 binucleated cells respectively were scored for the presence of micronuclei.

Table 7.6.1/12: HISTORICAL CONTROL DATA FOR IN VITRO MICRONUCLEUS STUDIES OF THE POSITIVE CONTROL SUBSTANCES

 

 

Mononucleated

Binucleated

 

- S9-mix

+ S9-mix

- S9-mix

 

3 hour exposure

24 hour exposure

3 hour exposure

3 hour exposure

24 hour exposure

Mean number of micronucleated cells

(per 1000 cells)

46.2

52.1

29.3

30.4

30.0

SD

28.1

25.0

14.9

16.4

10.1

n

54

56

58

60

60

Upper control limit

(95% control limits)

93.1

92.7

57.8

56.48

54.7

Lower control limit

(95% control limits)

-0.58

11.5

0.70

4.28

5.31

SD = Standard deviation

n = Number of observations

 

Table 7.6.1/13: HISTORICAL CONTROL DATA FOR IN VITRO MICRONUCLEUS STUDIES OF THE SOLVENT CONTROL

 

 

Mononucleated

Binucleated

 

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

 

3 hour exposure

3 hour exposure

24 hour exposure

3 hour exposure

3 hour exposure

24 hour exposure

Mean number of micronucleated cells

(per 1000 cells)

0.85

0.93

0.85

3.26

3.80

4.06

SD

0.88

0.97

1.13

2.17

2.63

2.47

n

54

56

55

54

56

55

Upper control limit

(95% control limits)

3.36

3.35

3.17

8.58

10.53

10.85

Lower control limit

(95% control limits)

-1.66

-1.49

-1.46

-2.06

-2.92

-2.74

 

SD = Standard deviation

n = Number of observations

Conclusions:
It is concluded that this test is valid and that 1-Phenyldecane-1,3-dione is not clastogenic or aneugenic in human lymphocytes in the absence and presence of S9-mix under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the effect of 1-Phenyldecane-1,3-dione on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity and aneugenicity of 1-Phenyldecane-1,3-dione was tested in two independent experiments.

The study procedures described in this report are in compliance with OECD guideline no. 487 and EC guideline no. B.49.

Batch MR92143651 of 1-Phenyldecane-1,3-dione was a reddish solid below 25°C / liquid above 25°C with a purity of 86.4%. The test item was dissolved in dimethyl sulfoxide.

In the first cytogenetic assay, 1-Phenyldecane-1,3-dione was tested up to 75 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence of S9-fraction and up to 80 μg/ml in presence of S9-fraction. Appropriate toxicity was reached at 75 μg/ml in absence of S9-mix. 1-Phenyldecane-1,3-dione precipitated in the culture medium at 80 μg/ml.

In the second cytogenetic assay, 1-Phenyldecane-1,3-dione was tested up to 40 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei.

In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

1-Phenyldecane-1,3-dione did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Finally, it is concluded that this test is valid and that 1-Phenyldecane-1,3-dione is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 January 2016 to 12 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: IN VITRO MAMMALIAN CELL GENE MUTATION TEST
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: MR92143651
- Expiration date of the lot/batch: 01 January 2017
- Purity: 86.4% (No correction was made for purity of the test substance)
- Purity test date: 18 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Yes, maximum temperature: 50°C, maximum duration: unknown (until liquefaction).
- Solubility and stability of the test substance in the solvent/vehicle: unknown
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: unknown

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was heated to 37°C, to obtain a homogeneous liquid sample.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).

MEDIA USED
- Type and identity of media including CO2 concentration:
Exposure medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies) supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). All incubations were carried out in a humid atmosphere (80 - 100%, actual range 42 – 90%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 – 37.3 °C).
- Properly maintained: not specified
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for
1 day in R10 medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Merck) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Test concentrations:
Based on the results of the dose range finding test, 1-Phenyldecane-1,3-dione was tested in two mutation assays. The first experiment was performed in the absence and presence of S9-mix with a 3-hour treatment period. The second mutation experiment was performed in the absence of S9-mix with a 24-hour treatment period. An additional experiment (1A) was performed in the presence of S9-mix with a 3-hour treatment period.
- First mutagenicity test:
Without S9-mix: 0.05, 0.1, 0.5, 1, 5, 7.5, 10, 20, 30, 40 and 50 μg/ml exposure medium.
With S9-mix: 5, 10, 25, 40, 50, 60, 70, 80, 90 and 100 μg/ml exposure medium.
- Second mutagenicity test (without S9-mix): 0.5, 1, 5, 7.5, 10, 12.5, 15, 17.5, 20, 30 and 40 μg/ml exposure medium.
Since in the first mutation experiment in the presence of S9-mix, no dose level with a RTG between 10 and 20% was tested and increases above the positive threshold of MF(controls) + 126 were observed at very toxic dose levels (RTG <10%), an additional experiment was performed. The following dose levels were tested in this additional experiment (1A) in the presence of S9-mix with a 3-hour treatment period: 10, 25, 40, 50, 60, 65, 70, 75, 80, 85, 90 and 100 μg/ml exposure medium.

Justification for top dose:
Since 1-Phenyldecane-1,3-dione was poorly soluble in the exposure medium, the highest tested concentration for the dose range finding test was 164 μg/ml exposure medium. In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained in a dose range finding test by treating cells with a number of test item concentrations increasing by approximately half log steps.
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 5.2 to 164 μg/ml in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO. The final concentration of the solvent in the exposure medium was 1% (v/v).
- Justification for choice of solvent/vehicle: solvent in which the test item was found soluble.
Negative controls:
yes
Remarks:
= Solvent control
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: Per culture 8 x 106 cells (106 cells/ml for 3 hours treatment) or 6 x 106 cells (1.25 x 105 cells/ml for 24 hours treatment) were used.

DURATION
- Preincubation period: non
- Exposure duration: 3 hours or 24 hours
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period.
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT) (Sigma).

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: N/A

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A

NUMBER OF CELLS EVALUATED: For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A

DETERMINATION OF CYTOTOXICITY
- Method: Parameters: Cloning efficiency; rRelative Total Growth; relative survival; Suspension Growth; Relative Suspension Growth.

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER:
Rationale for test conditions:
L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes. The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions and clastogenic effect.
Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT).
TK deficient cells cannot incorporate the analogue into its phosphorylated derivative (nucleotide); the nucleotides needed for cellular metabolism are obtained solely from de novo synthesis. In the presence of TK, TFT is converted into nucleotides, which is lethal to the cells. Thus, cells that are able to proliferate in culture medium containing TFT are mutated, either spontaneously or by the action of the test item, to a TK deficient phenotype. Furthermore, by applying the TFT-selection procedure it is possible to discriminate between two different classes of TFT-resistant mutants (small and large colonies). The large colonies are believed to be the result of mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are believed to be the result of chromosomal damage to the TK and adjacent genes.
A test article, which induces a positive response in this assay, is presumed to be a potential mammalian cell mutagen.
Evaluation criteria:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Statistics:
not performed.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not assessed
- Effects of osmolality: Not assessed
- Evaporation from medium: Not assessed
- Water solubility: Not assessed
- Precipitation: 1-Phenyldecane-1,3-dione precipitated in the exposure medium at concentrations of 164 µg/mL and above. This concentration was used as the highest test item concentration for the dose range finding test.
- Definition of acceptable cells for analysis: Not assessed
- Other confounding effects: N/A

RANGE-FINDING/SCREENING STUDIES:

In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 5.2 to 164 μg/ml in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
With the 3-hour treatment period, in the absence of S9-mix, the relative suspension growth was 35% at the test item concentration of 28 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test item concentrations of 52 μg/ml and above.
With the 3-hour treatment period, in the presence of S9-mix, the relative suspension growth was 10% at the test item concentration of 100 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentration of 164 μg/ml.
With the 24-hour treatment period,in the absence of S9-mix, the relative suspension growth was 18% at the test item concentration of 17 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test item concentrations of 28 μg/ml and above.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 7.6.1/8 below.
- Negative (solvent/vehicle) historical control data: see table 7.6.1/9 below.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: cloning efficiency, relative survival, Relative Total Growth, Suspension Growth, Relative Suspension Growth.
- Other observations when applicable: The growth rate, as an indicator of optimally growing cultures, was calculated for the solvent control cultures.

Table 7.6.1/5: Experiment 1: Cytotoxic and mutagenic response of 1-Phenyldecane-1,3-dione in the mouse lymphoma L5178Y test system

Dose (µg/ml)

RSG (%)

CEday2(%)

RSday2(%)

RTG (%)

Mutation frequency per 106survivors

total

Small / Large

Without metabolic activation – 3 hours treatment

SC1

100

101

100

100

99

50 / 44

SC2

84

100

55 / 41

0.5

91

83

89

81

113

61 / 47

1

92

91

99

91

114

63 / 45

5

81

101

109

89

132

57 / 66

7.5

66

116

126

84

147

71 / 64

10

59

89

96

57

147

77 / 61

20

37

75

81

30

182

101 / 69

30

27

110

119

32

98

21 / 73

40

9

70

76

7

128

30 / 93

MMS

60

62

67

40

747

173 / 505

With metabolic activation – 3 hours treatment

SC1

100

93

100

100

116

61 / 49

SC2

95

101

53 / 43

5

97

108

115

112

71

23 / 45

10

95

81

87

82

129

30 / 94

25

72

88

93

67

144

43 / 93

40

41

93

99

41

186

49 / 123

50

36

99

106

38

175

64 / 97

60

22

105

112

24

196

41 / 139

80

12

69

74

9

361

153 / 163

90

10

79

84

8

248

123 / 101

CP

52

39

41

22

2108

1012 / 685

 

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

Table 7.6.1/6: Experiment 1A: Cytotoxic and mutagenic response of 1-Phenyldecane-1,3-dione in the mouse lymphoma L5178Y test system

Dose (µg/ml)

RSG (%)

CEday2(%)

RSday2(%)

RTG (%)

Mutation frequency per 106survivors

total

Small / Large

With metabolic activation – 3 hours treatment

SC1

100

90

100

100

130

62 / 60

SC2

108

118

62 / 49

10

98

110

111

108

122

73 / 42

25

90

83

83

75

181

100 / 68

40

71

108

109

78

121

64 / 50

50

49

99

100

49

193

99 / 76

60

30

101

102

30

212

116 / 74

65

29

110

111

32

186

108 / 61

75

20

110

111

22

190

63 / 109

80

11

93

93

10

172

51 / 109

CP

70

79

80

55

924

332 / 428

 

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; CP = Cyclophosphamide

 

Table 7.6.1/7: Experiment 2: Cytotoxic and mutagenic response of 1-Phenyldecane-1,3-dione in the mouse lymphoma L5178Y test system

Dose (µg/ml)

RSG (%)

CEday2(%)

RSday2(%)

RTG (%)

Mutation frequency per 106survivors

total

Small / Large

Without metabolic activation – 24 hours treatment

SC1

100

90

100

100

82

33 / 46

SC2

111

70

23 / 44

0.5

86

88

87

75

106

41 / 60

1

90

108

107

97

86

45 / 37

5

82

89

88

72

120

31 / 83

7.5

43

95

95

41

106

35 / 66

10

34

95

95

32

58

25 / 32

12.5

20

88

87

18

68

31 / 36

15

14

95

95

13

90

37 / 49

17.5

11

107

106

12

61

30 / 29

MMS

85

99

99

84

533

302 / 166

  

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate

Table 7.6.1/8 :Historical control data of the mutation frequencies of the positive controls for the mouse lymphoma assay

 

Mutation frequency per 106survivors

 

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

 

Mean

877

745

1718

SD

236

193

762

n

67

63

91

Upper control limit

(95% control limits)

1416

1252

3911

Lower control limit

(95% control limits)

338

239

-475

 

SD = Standard deviation

n = Number of observations

Table 7.6.1/9 :Historical control data of the spontaneous mutation frequencies of the solvent controls for the mouse lymphoma assay

 

Mutation frequency per 106survivors

 

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

 

Mean

80

73

83

SD

21

22

28

n

133

132

193

Upper control limit

(95% control limits)

123

122

138

Lower control limit

(95% control limits)

37

24

28

 

SD = Standard deviation

n = Number of observations

Conclusions:
Under the experimental conditions described in the report, 1-Phenyldecane-1,3-dione was found not mutagenic in the mouse lymphoma L5178Y test system in the absence or presence of a metabolic activation system (S9-mix).
Executive summary:

The objective of this study was to evaluate of the mutagenic activity of 1-Phenyldecane-1,3-dione in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

This report describes the effects of 1-Phenyldecane-1,3-dione on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hours treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). An additional experiment was performed in the presence of S9-mix with a 3 hours treatment period.

The study procedures described in this report were based on the most recent OECD guideline no. 490.

Batch MR92143651 of 1-Phenyldecane-1,3-dione was a reddish solid below 25°C and liquid above 25°C with a purity of 86.4%. The test item was dissolved in dimethyl sulfoxide.

In the first experiment, 1-Phenyldecane-1,3-dione was tested up to concentrations of 40 and 90 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) of these highest concentrations was 7 and 8% in the absence and presence of S9-mix, respectively.

In the absence of S9-mix, none of the tested concentrations reached a mutation frequency of MF(controls) + 126 (226 x 10-6) (Global evaluation factor, GEF).

In the presence of S9-mix, increases above the positive threshold of MF(controls) + 126 (GEF = 235 x 10-6) were observed at the very toxic top doses of 80 and 90 μg/ml. Since the RTG was below 10% in both dose levels, these concentrations were too toxic according to the guidelines and therefore the mutagenic responses were not biologically relevant. Furthermore, the next dose level of 60 μg/ml with a RTG of 24%, did not even show a two-fold increase in the mutation frequency and was not above the GEF.

Since in the first mutation experiment in the presence of S9-mix, no dose level with a RTG between 10 and 20% was tested and increases above the positive threshold of MF(controls) + 126 were observed at very toxic dose levels (RTG <10%), an additional experiment (1A) was performed in the presence of S9-mix. The test item was tested up to a concentrations of 80 μg/ml. The incubation time was 3 hours. The Relative total growth (RTG) of this highest concentration was 10%. None of the tested concentrations reached a mutation frequency of MF(controls) + 126 (GEF = 250 x 10-6).

In the second experiment, the test item was tested up to concentrations of 17.5 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG of this highest concentration was 12%. None of the tested concentrations reached a mutation frequency of MF(controls) + 126 (GEF = 202 x 10-6).

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Since the observed increases in the first experiment in the presence of S9-mix were only observed at concentrations which were too toxic according to the guidelines (below 10% RTG) and no increase was observed in the additional experiment with acceptable RTG values, the mutagenic responses are not biologically relevant and 1-Phenyldecane-1,3-dione is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial gene mutation:

A GLP-compliant Ames test conducted according to OECD TG 471 (of reliability 1 according to Klimisch cotation criteria) is available and was selected as key study (Pharmaco, 1995). There was no evidence of mutagenic activity in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA at any dose level of test item (up to 5000 µg/plate) in the presence and the absence od S9 -mix.

The registered substance 1 -phenyldecane-1,3 -dione is therefore considered not to be mutagenic in this Ames test.

 

Mammalian gene mutation:

One GLP-compliant Mouse Lymphoma Assay conducted according to OECD TG 490 (of reliability 1 according to Klimisch cotation criteria) is available and was selected as key study (WILResearch, 2016). In the absence of S9-mix, 1-Phenyldecane-1,3-dione tested in mouse lymphoma L5178Y cells

did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment. In the presence of S9-mix, increases above the positive threshold were observed at the very toxic top doses of 80 and 90μg/ml in the first experiment. Since the observed increases in the first experiment in the presence of S9-mix were only observed at concentrations which were too toxic according to the guidelines (below 10% Relative Total Growth (RTG)) and no increase was observed in the additional experiment with acceptable RTG values, the mutagenic responses are not biologically relevant and 1-Phenyldecane-1,3-dione is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of this study.

 

Mammalian micronucleus:

One GLP-complant in vitro micronucleus test conducted according to OECD TG 473 (of reliability 1 according to Klimisch cotation criteria) is available and was selected as key study. In the first cytogenetic assay, 1-Phenyldecane-1,3-dione was tested in cultured peripheral human lymphocytes up to 75 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence of S9-fraction and up to 80 μg/ml in presence of S9-fraction. Appropriate toxicity was reached at 75 μg/ml in absence of S9-mix. 1-Phenyldecane-1,3-dione precipitated in the culture medium at 80 μg/ml.

In the second cytogenetic assay, 1-Phenyldecane-1,3-dione was tested up to 40 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. 1-Phenyldecane-1,3-dione did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

1-Phenyldecane-1,3-dione is therefore not clastogenic or aneugenic in human lymphocytes under the experimental conditions of this study.

Justification for classification or non-classification

 Three in vitro genotoxicity tests are available. None of the tests showed evidence of genotoxicity. The substance is therefore not regarded to have genotoxic effects and does not require classification for genetic toxicity according to the CLP and GHS-UN regulations.