Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: An analogue test item (EC 265-205-1) was determined to be non-mutagentic in the Ames test (OECD 471, EU Method B.13/14 and relevant Japanese guidelines).

 

Chromosome aberration: An analogue test material (EC 265-205-1) was considered to be non-mutagenic (OECD 473 and EU Method B.10).

 

Mouse lymphoma assay: An analogue substance(EC 265-205-1) was considered to be non-mutagenic to L5178Y cells under the conditions of the test (OECD 476, EU Method B.17, EPA OPPTS 870.5300 and relevant Japanese guidelines).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Species / strain:
S. typhimurium TA 1535
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
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 98
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
Species / strain:
S. typhimurium 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
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
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:
13 August 2003 to 15 September 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
MHLW and MAFF
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine auxotrophs of S. typhimurium and tryptophan auxotrophs of E. coil
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: uvrB, rfa
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (10% liver S9 in co-factors)
Test concentrations with justification for top dose:
Preliminary Study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Main Study: 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Remarks:
see table 1 in the field "any other information on material and methods inc tables"
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
see table 1 in the field "any other information on material and methods inc tables"
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation method

PRELIMINARY STUDY
- The dose range was determined through a preliminary toxicity assay
- Test strains, TA 100 and WP2uvrA
- 0.1 mL of the culture was added to 2 mL of molten top agar (either histidine or tryptophan supplemented), 0.1 mL of the test material and 0.5 mL of S9 mix or phosphate buffer. This was overlaid onto sterile agar plates of Vogel-Bonner Minimal agar (30 mL /plate).
- The solvent control and sterility controls were performed.
- The plates were incubated for 48 hours at 37 ºC.
- Revertant colonies were counted using a Domino colony counter.

MAIN TEST
- The main test was carried out twice on different days, following the same procedure, with fresh bacterial cultures.
- The procedure is the same as in the preliminary test; all plates were produced in triplicate.
- The colonies had to be counted by hand in the 2nd test as there was interference from the agar plates.
Evaluation criteria:
he test was considered positive if the following criteria was met:
The test induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

The mutation assay was considered to be valid if the following criteria were met:
All culture strains in the vehicle and untreated controls exhibit a characteristic number of spontaneous revertants per plate.
Strain characteristics have been confirmed i.e. rfa and pKM101 plasmid R-factor.
All strain cultures meet the approximate range of 1 to 9.9 x 10⁹ bacteria per mL.
Positive controls should be at least two times the respective vehicle control values.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.
Statistics:
Dunnett's method of linear regression.
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
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.

MUTATION TEST:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments was shown to be sterile.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 pg/plate. A light, particulate precipitate was observed at 5000 pg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested

Table 2. Revertant Colony Counts from Experiment 1 and 2 Including Controls

Experiment No.

With or Without S9-mix

Concentration of Test Material

Mean Number of Revertants per Plate (standard deviation)

Strain

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

1

-

0

66 (13.1)

11 (1.0)

22 (6.1)

20 (3.5)

7 (1.2)

50

70 (6.0)

12 (3.1)

22 (6.1)

19 (3.5)

9 (2.1)

150

87 (13.2)

13 (3.5)

21 (4.6)

21 (7.5)

8 (0.6)

500

71 (13.0)

14 (10.1)

15 (1.2)

16 (7.1)

6 (2.6)

1500

71 (4.4)

13 (1.5)

21 (3.5)

17 (3.2)

8 (0.6)

5000

72 (13.9)

7 (2.3)

15 (3.5)

20 (5.0)

9 (2.5)

ENNG

514 (10.4)

275 (16.0)

797 (22.5)

 

4NQO

240 (13.1)

 

9AA

2316 (390.7)

+

0

71 (5.6)

13 (5.6)

24 (6.4)

27 (3.6)

15 (5.5)

50

71 (4.7)

10 (3.8)

18 (3.5)

22 (0.0)

16 (5.0)

150

68 (5.7)

8 (0.6)

22 (6.6)

23 (3.5)

15 (2.5)

500

69 (1.2)

11 (3.6)

18 (6.8)

25 (9.9

17 (3.5)

1500

67 (5.8)

9 (2.3)

20 (0.6)

21 (5.5)

17 (6.7)

5000

69 (4.7)

14 (4.0)

21 (2.1)

25 (7.8)

10 (3.5)

2AA

1440 (32.1)

156 (1.5)

791 (16.4)

502 (53.5)

BP

205 (15.1)

 

2

-

0

130 (9.5)

35 (3.5)

20 (2.0)

24 (3.8)

12 (3.5)

50

115 (2.1)

37 (3.5)

23 (5.2)

21 (2.3)

9 (3.2)

150

129 (14.4)

39 (0.0)

17 (2.3)

23 (5.0)

15 (2.5)

500

115 (15.7)

31 (6.1)

17 (3.5)

16 (2.6)

13 (1.5)

1500

99 (13.0)

27 (12.3)

19 (1.5)

22 (1.5)

11 (3.0)

5000

88 (4.6)

15 (9.5)

18 (4.6)

18 (1.7)

6 (2.1)

ENNG

381 (11.8)

137 (25.0)

817 (39.1)

 

4NQO

210 (9.9)

 

9AA

827 (65.5)

+

0

103 (17.2)

13 (4.4)

29 (2.3)

35 (5.5)

19 (2.1)

50

115 (9.1)

14 (1.7)

20 (0.0)

34 (4.0)

20 (4.0)

150

101 (9.8)

15 (2.6)

25 (4.5)

30 (2.1)

18 (2.6)

500

90 (3.1)

14 (1.5)

22 (2.1)

26 (3.0)

19 (6.7)

1500

77 (6.7)

13 (1.2)

17 (2.5)

20 (6.6)

15 (2.8)

5000

89 (11.0)

17 (1.7)

23 (1.5)

13 (0.6)

19 (1.2)

2AA

881 (50.6)

229 (59.7)

613 (64.6)

 

316 (40.8)

BP

 

 

 

260 (20.2)

 

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of the test, the test material was determined to be non mutagenic in several strains of S. typhimurium and E. coli confirmed in an Ames Test.
Executive summary:

In a GLP compliant study performed to standardised guidelines OECD 471 and EU Method B.13/14, the mutagenic potential of the test material was assayed in an Ames Test. S. typhimurium, TA 1535, TA 1537, TA 100, TA 98 and E. coli WP2 uvr A were exposed to the test material in varying concentrations with and without metabolic activation. Under the conditions of the test it was determined that the test material is non-mutagenic as there was no significant increase in revertant colony counts.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 September 2003 to 25 November 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: UK department of Health Committee on Mutagenicity Guidelines for the Mutagenicity Testing of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Average generation time: ~ 17 hours for this donor, determined under typical experimental conditions.
- Medium: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 15 % foetal calf serum, at 37 ºC with 5 % CO2 in air.
- Cells were stimulated to divide by the addition of phytohaemagglutinin (PHA) at 90 µg/mL final concentration.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment 1
4 hour exposure with and without S9 mix: 0, 156.25, 312.5, 625, 1250, 1875, 2500 µg/mL

Experiment 2
4 hour exposure with S9 mix: 0, 39.06, 78.13, 156.25, 312.5, 625 and 1250 µg/mL
24 hour exposure without S9 mix: 0, 39.06, 78.13, 156.25, 312.5, 468.75 and 625 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.

PRELIMINARY STUDY:
A dose range finding study was performed using concentrations of the test material between 19.5 and 5000 µg/mL.

MAIN TEST: The study was carried out in two experiments, utilising four different exposure conditions.

Experiment 1
- The cells were exposed for 4 hours without S9-mix followed by 20 hour culture in media free from the test material, prior to cell harvest.
- The cells were exposed for 4 hours with S9-mix followed by 20 hour culture in media free from the test material, prior to cell harvest.

Experiment 2
- Continuous exposure to the test material for 24 hours prior to cell harvesting.
- The cells were exposed to the test material for 4 hours with S9-mix followed by 20 hour culture in test material free media, prior to cell harvest.

TEST CONDITIONS:
- Preincubation period: 48 hours at 37 ºC, 5% CO2 in humidified air.
- Volume of test material, vehicle or positive control: 0.1 mL
- Total nominal volume: 10 mL
- Volume and concentration of S9 mix: 1 mL of 20 % S9-mix in experiment 1 and 1 mL of 10 % S9-mix in experiment 2.
- Expression time (cells in growth medium): 4 hours with the test material and 20 hours without the test material.
- In the case of the continuous exposure the expression time was 24 hours, with the test material.

SPINDLE INHIBITOR (cytogenetic assays): demecolcine, Colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): 5% Gurrs Giemsa.

NUMBER OF REPLICATIONS: Tests performed in duplicate.

NUMBER OF CELLS EVALUATED: 2000 lymphocytes cell nuclei were counted and the number of cells in methaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Evaluation criteria:
Scoring:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976).

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test. Samples which showed a statistical increase in aberrations were considered to have given a positive result.
Statistics:
Fisher’s Exact test.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Experiment 1:

-The test material did not induce a statistically significant increase in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

- The maximum dose level with scoreable metaphases present was 1250 µg/mL in the presence and absence of metabolic activation.

- The mitotic data confirms the qualitative assessment that the dose-related inhibition of mitotic index was observed and that 33 % mitotic inhibition was achieved at 1250 µg/mL in the absence of S9 and 35 % in the presence.

- Complete inhibition was observed at 1875 and 2500 µg/mL in both exposure groups.

Experiment 2:

-The test material did not induce a statistically significant increase in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

- Qualitative assessment of the slides determined that the maximum dose level with scorable metaphases was 1250 µg/mL with S9 mix and 468.75 µg/mL without S9 mix in experiment 2.

- The mitotic index data confirms the qualitative observations that a dose-related inhibition of mitotic index was observed and that 67 % mitotic inhibition as achieved at 468.75 µg/mL in the absence of S9. In the presence of S9 50 % mitotic inhibition was achieved at 1250 µg/mL.

Table 1. Results of Chromosome Aberrations

Experiment No.

With or Without S9 mix

Treatment Group

Dose Level (µg/ml)

Total No. of Aberrations

Frequency of Aberrant Cells (%)

(+ Gaps)

(- Gaps)

(+ Gaps)

(- Gaps)

1

Without

Vehicle Control

 

2

1

1

1

Test Material

312.5

1

1

1

1

625

3

2

3

2

1250

2

1

2

1

1875

TOXIC

Positive Control MMC

0.4

39

29

28

22***

1

With

Vehicle Control

 

7

5

3

1

Test Material

312.5

2

2

2

2

625

1

0

1

0

1250

5

3

5

3

1875

TOXIC

Positive Control CP

10

89

50

65

39***

2

Without

Vehicle Control

 

3

3

3

3

Test Material

156.25

1

0

1

0

312.5

1

0

1

0

468.75

1

1

1

1

625

TOXIC

Positive Control MMC

0.2

72

53

50

39***

2

With

Vehicle Control

 

2

1

2

1

Test Material

312.5

1

0

1

0

625

2

2

2

2

1250

8

3

8

3

Positive Control CP

0.2

96

73

52

42***

MMC = Mitomycin C

CP = Cyclophosphamide

*** = P < 0.001

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of the test, the test material was determined to be non-clastogenic to human lymphocytes. There was no statistically significant increase in the frequency of cell aberrations in the absence or presence of metabolic activation.
Executive summary:

In a GLP compliant chromosome aberration study performed according to the standardised guidelines; OECD 473, EU Method B.10 and UK department of Health Committee on Mutagenicity Guidelines for the Mutagenicity Testing of Chemicals, the test material was determined to be non-clastogenic. Under the conditions of the test the human lymphocyte did not produce a statistically significant increase in the frequency of cell aberrations in the absence or presence of metabolic activation. Therefore the test material is considered to be non-mutagenic.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 April 2012 to 2 July 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
METI/MHLW
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-, locus in L5178Y mouse lymphoma cells. Mutant form is TK-/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
-Master stocks of cells were tested and found to be free of mycoplasma.
-Cell Cleansing: TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Cells were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/ml), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL).
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from the livers of rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
4 hour exposure without S9 mix: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 234.38, 312.5, 468.75 µg/mL
4 hour exposure with S9 mix: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 468.75, 625 µg/mL

Experiment 2
24 hour exposure without S9 mix: 0, 6.25, 12.5, 25, 50, 100, 150, 200, 250, 300, 350 µg/mL
4 hour exposure with S9 mix: 0, 50, 100, 200, 300, 350, 400, 450, 500 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
CELL CULTURE
-The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air.
-The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.

PREPARATION OF TEST AND CONTROL ITEMS
-The test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared. Due to formulation difficulties, the maximum dose level suitable for dosing was set at 2500 µg/mL.
-Acetone is toxic to L5178Y at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 250 mg/mL and dosed at 0.5 % to give a maximum achievable dose level of 1250 µg/mL.
-The test item was a complex mixture and, therefore, purity was not accounted for when formulating the dosing solutions.

MICROSOMAL ENZYME FRACTIONS
-PB/βNF S9 was prepared in-house from the livers of male Sprague-Dawley rats weighing approximately 250 g. These had each received, orally, three consecutive daily doses of phenobarbital/β-naphthoflavone (80/100 mg/kg/day) prior to S9 preparation on the fourth day. The S9 was stored at approximately -196 °C in a liquid nitrogen freezer.
-S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.
-20% S9 mix (i.e. 2 % final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1. In Experiment 2, 10 % S9 mix (i.e. 1 % final concentration of S9), was added.

PRELIMINARY TOXICITY TEST
-A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 4.88 to 1250 µg/mL for all three of the exposure groups.
-Following the exposure period the cells were washed twice with R10, re-suspended in R20 medium, counted using a coulter counter and then serially diluted to 2 x 10^5 cells/mL.
-The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth (%RSG) value.
-Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments.

MUTAGENICITY TEST
Experiment 1
-An exponentially growing stock culture of cells was set up. Cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9 mix). To each universal was added 2 mL of S9 mix if required, 0.1 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL.
-The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2
-As in experiment 1, cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL cultures in R10 medium for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3 x 10^6 cells/mL in 10 ml cultures were established in 25 cm² tissue culture flasks.
-The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix). To each universal was added 2 mL of S9 mix if required, 0.1 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to give a final volume of 20 mL (R10 is used for the 24-hour exposure group).
-The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
-At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then re-suspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and subcultured every 24 hours for the expression period of two days by counting and diluting to 2 x 10^5 cells/mL.
-On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
-The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

PLATE SCORING
Microtitre plates were scored using a magnifying mirror box after 10 to 14 days’ incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded.
-Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately 0.25 to 0.75 of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25 % of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours.

CALCULATION OF PERCENTAGE RELATIVE SUSPENSION GROWTH (%RSG)
-The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the %RSG.
-Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
-Day 0 Factor = (dose 0-hour cell count)/(vehicle control 0-hour cell count)
-%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

CALCULATION OF DAY 2 VIABILITY (%V)
-Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.
-P(0) = number of negative wells/total wells plated
-%V = - (lnP(0) x 100)/(number of cells/well)

CALCULATION OF RELATIVE TOTAL GROWTH (RTG)
-For each culture, the relative cloning efficiency, RCE, was calculated:
RCE = (%V/Mean Solvent Control %V) x 100%
-Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100%

CALCULATION OF MUTATION FREQUENCY (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
Evaluation criteria:
INTERPRETATION OF RESULTS
-The normal range for mutant frequency per survivor is 50-170 x 10^-6 for the TK+/- locus in L5178Y cells at the test laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10^-6 mutant frequency per survivor are not normally acceptable and require repetition.
-Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
Statistics:
-The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al, 1989).
-For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
-There was evidence of marked dose-related reductions in the %RSG of cells treated with the test item when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult.
-A greasy/oily precipitate of the test item was present at higher concentrations. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity.


MUTAGENICITY TEST
The results are summarised in Tables 1 and 2.

Experiment 1
-There was once again evidence of marked dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation.
- There was also evidence of a modest reduction in %V in the presence of metabolic activation, therefore indicating that residual toxicity had occurred.
-Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the presence of metabolic activation. Optimum levels of toxicity were not achieved in the absence of metabolic activation. This was considered to be due to a combination of the steep toxicity curve of the test item, and the presence of greasy/oily precipitate giving variable exposure of the test item to the cells. However, with no evidence of any toxicologically significant increases in mutant frequency in the presence of metabolic activation in Experiment 1, or in the absence of metabolic activation in Experiment 2 where optimum levels of toxicity were achieved, the test item was considered to have been overall adequately tested.
-The excessive toxicity observed at 468.75 µg/mL in the absence of metabolic activation, and at 625 µg/mL in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.
-Neither of the vehicle control mutant frequency values were outside the acceptable range. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
-The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in the absence of metabolic activation.
-A very modest statistically significant dose related (linear-trend) increase was observed in the presence of metabolic activation. However, there was no evidence of any marked increases in mutant frequency, the GEF was not exceeded at any of the dose levels, and there was no evidence of any increases in absolute number of mutant colonies. Therefore, the response was considered artefactual and of no toxicological significance.

Experiment 2
-As was seen previously, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values.
-There was no evidence of any significant reductions in %V in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred.
-Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the absence of metabolic activation. On this occasion optimum levels of toxicity were not achieved in the presence of metabolic activation. This was considered to be due to a combination of the steep toxicity curve of the test item, the presence of greasy/oily precipitate giving variable exposure of the test item to the cells, and the lowering of the S9 concentration resulting in lower levels of toxicity than seen previously. However, with no evidence of any toxicologically significant increases in mutant frequency in the absence of metabolic activation in Experiment 2, or in the presence of metabolic activation in Experiment 1 where optimum levels of toxicity were achieved, the test item was considered to have been overall adequately tested.
-The excessive toxicity observed at 350 µg/mL in the absence of metabolic activation resulted in this dose level not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.
-The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item. It should be noted that the lowering of the S9 concentration to 1% in this second experiment resulted in lower levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2 % metabolic activation in the Preliminary Toxicity Test and Experiment 1.
-Neither of the vehicle control mutant frequency values were outside the acceptable range. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
-The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in either the absence or presence of metabolic activation.
Remarks on result:
other: all strains/cell types tested

Table 1 Summary of Results from Experiment 1

Treatment (µg/mL)

4-Hours -S9

Treatment (µg/ml)

4-Hours +S9

%RSG

RTG

MF*

%RSG

RTG

MF*

0

9.77

19.53

39.06

78.13

156.25

234.38

312.5

468.75

100

100

93

95

89

81

76

57

1

1.00

 

1.03

1.05

0.91

0.85

0.77

0.58

115.33

 

98.86

84.26

78.54

91.48

85.07

82.63

0

9.77

19.53

39.06

78.13

156.25

312.5

468.75

625

100

97

99

92

95

89

67

14

1

1.00

 

1.08

0.93

0.87

0.84

0.59

0.09

102.83

 

88.77

119.03

111.70

105.31

138.15

139.82

 

Linear trend: Not significant                                  

Linear trend: p<0.05

Positive Control

EMS 400

 

69

 

 0.48

 

802.31

Positive Control

CP 2

 

59

 

0.30

 

1320.32

*5-TFT resistant mutants/10^6 viable cells 2 days after treatment

†Not plated for viability or TFT resistance

EMS = Ethylmethanesulphonate

CP = Cyclophosphamide

 

Table 2 Summary of Results from Experiment 2

Treatment (µg/mL)

24-Hours -S9

Treatment (µg/ml)

4-Hours +S9

%RSG

RTG

MF*

%RSG

RTG

MF*

0

6.25

12.5

25

50

100

150

200

250

300

350

100

109

83

99

93

57

49

37

21

9

5

1.00

 

 

 

0.93

0.64

0.62

0.49

0.35

0.18

 

176.95

 

 

 

186.78

169.79

159.56

167.58

159.79

97.47

 

0

50

100

200

300

350

400

450

500

100

98

87

81

75

70

64

59

40

1.00

 

 

0.90

0.90

0.84

0.80

0.62

0.43

172.76

 

 

145.89

159.43

146.53

134.78

171.61

166.75

 

Linear trend: Not significant                                          

Linear trend: Not significant

Positive Control

EMS 150

 

71

 

0.49

 

1836.31

Positive Control

CP 2

 

66

  

0.46

  

1355.21

*5-TFT resistant mutants/10^6 viable cells 2 days after treatment

†Not plated for viability or TFT resistance

EMS = Ethylmethanesulphonate

CP = Cyclophosphamide

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Executive summary:

A GLP compliant study was performed according to the standardised guidelines OECD 476, Method B.17 of Commission Regulation (EC) No. 440/2008, US EPA OPPTS 870.5300, and would be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

The study was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line, both in the absence and presence of metabolic activation (S9 mix).

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, with or without metabolic activation, in either the first or the second experiment.

Therefore the test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
9 October 2000 to 26 October 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
other: ASTM Standard Method (E 1687-98)
Deviations:
yes
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with
Metabolic activation system:
S9 mixture
Test concentrations with justification for top dose:
0, 6, 12, 18, 24 and 30 µL/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material is not soluble in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: reference oil
Details on test system and experimental conditions:
PRELIMINARY STUDY:
A preliminary dose range finding study was performed to confirm the concentration range for the main test

NUMBER OF REPLICATIONS:
- Tested in triplicate with the exception of the vehicle control which was only performed once

Preparation of Test Material and Controls:
The test material was prepared at 1:5 v/v with DMSO, in a glass tube at 70 ºC. The mixture was then vortexed for 30 seconds every five minutes for 30 minutes. The phases were separated by centrifugation and the extract removed with a pipette and stored until testing. The test material was diluted 1:2 with DMSO before preparing the dose concentrations.

The reference oil (positive control) was diluted 1:4 with DMSO before preparing the dosing solutions at 0, 3, 6, 9, 12 and 15 µL/plate.

Tester Strain:
The strain was developed by B.N. Ames.
An aliquot from the initial inoculums of frozen stock was incubated for 16 hours in nutrient broth at 37 ºC. The resulting culture was then diluted and incubated for a further 3 hours to ensure that the bacteria were in long-phase growth.
 
Microsomal Enzyme Fraction:
The S9 fraction was derived from Syrian Golden Hamster liver. The fraction was mixed with the following salts and co-factors:
S9 fraction: 0.8 mL
0.104 M NADP: 0.075 mL
1.0 M G-6-P: 0.005 mL
0.65 M KCl or 0.4 M MgCl₂: 0.02 mL
1M Phosphate buffer (pH 7.4): 0.1 mL
Evaluation criteria:
Substances with MI > 1.0 are likely to carcinogenic in the mouse skin-painting bioassay.
Substances with MI < 1.0 are not carcinogenic.
Substances with MI > 50 induce tumours in virtually all mice in skin-painting assays.
Statistics:
The slope of the dose-response curve was determined by linear regression analysis.
The MI, Mutagenicity Index, was also calculated.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Revertant Colony Counts and Mutagenic Index

Dose µl/plate

Revertant Colonies (per plate)

Mean

S.D

MI

Fold Increase

Mutagenic Potency Index

Test A

Test B

TestC

0

60

57

52

54

5

0.16

1.1

1.34

54

45

54

6

53

57

59

58

4

65

62

53

12

77

50

58

62

10

63

70

52

18

66

58

50

58

5

54

56

61

24

62**

52*

61*

59*

4

63**

55*

61*

30

55**

57**

58**

61**

7

62**

56**

76**

* = light precipitate present

** = moderate precipitate present

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation

Under the conditions of the test, the MI was calculated to be 0.16, which according to the criteria set out in the study means that the test material would not be carcinogenic in a mouse skin-painting assay and thus would be non-mutagenic.
Executive summary:

In a non-GLP compliant study performed in part according to ASTM Standard Method (E 1687-98), the Mutagenic Index (MI) was determined in a modified Ames Test. S. typhimurium TA 98 was exposed to the test material at varying concentrations, diluted in DMSO. Under the conditions of the test, the MI was calculated to be 0.16 which, according to the criteria set out in the study, means that the test material would not be carcinogenic in a mouse skin-painting assay and thus would be classed as non-mutagenic.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro

 

Key Ames test:In a GLP compliant study performed to standardised guidelines OECD 471 and EU Method B.13/14, the mutagenic potential of an analogue test material (EC 265-205-1) was assayed in an Ames Test. S. typhimurium, TA 1535, TA 1537, TA 100, TA 98 and E. coli WP2 uvr A were exposed to the test material in varying concentrations with and without metabolic activation. Under the conditions of the test it was determined that the analogue test material is non-mutagenic as there was no significant increase in revertant colony counts.

 

Supporting Ames test:In a non-GLP compliant study performed in part according to ASTM Standard Method (E 1687-98), the Mutagenic Index (MI) was determined in a modified Ames Test. S. typhimurium TA 98 was exposed to an analogue test material (EC 265-205-1) at varying concentrations, diluted in DMSO. Under the conditions of the test, the MI was calculated to be 0.16 which, according to the criteria set out in the study, means that the analogue test material would not be carcinogenic in a mouse skin-painting assay and thus would be classed as non-mutagenic.

 

Key chromosome aberration test:In a GLP compliant chromosome aberration study performed according to the standardised guidelines; OECD 473, EU Method B.10 and UK department of Health Committee on Mutagenicity Guidelines for the Mutagenicity Testing of Chemicals, an analogue test material (EC 265-205-1) was determined to be non-clastogenic. Under the conditions of the test the human lymphocyte did not produce a statistically significant increase in the frequency of cell aberrations in the absence or presence of metabolic activation. Therefore the analogue test material is considered to be non-mutagenic.

 

Key mouse lymphomas assay:A GLP compliant study was performed on an analogue test item (EC 265-201-1) according to the standardised guidelines OECD 476, Method B.17 of Commission Regulation (EC) No. 440/2008, US EPA OPPTS 870.5300, and would be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances. The study was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line, both in the absence and presence of metabolic activation (S9 mix). The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, with or without metabolic activation, in either the first or the second experiment. Therefore the analogue test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

In vivo

 

Negative results were obtained during investigation of an analogue substance (EC 265-201-1) using in vitro gene mutation in bacteria (Ames test), in vitro cytogenicity in mammalian cells (chromosome aberration study) and in vitro gene mutation in mammalian cells (mouse lymphoma assay). As a result, and in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 5.0; December 2016), the analogue substance is not considered to be genotoxic and no further testing is required.

Justification for classification or non-classification

In vitro tests demonstrated that the analogue test material (EC 265-201-1) was non-mutagenic (Ames test) and non-clastogenic (chromosome aberration test in vitro). An in vivo study (mouse micronucleus test) also concluded that the analogue test item was non-genotoxic. Classification in accordance with Regulation (EC) No 1272/2008 is therefore not required.