Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A key Ames bacterial mutagenicity test demonstrated that no increases in mutations were observed in 5 different Salmonella typhimurium strains with and without metabolic activation up to cytotoxic concentration of 5000 µg/plate. In a key mammalian gene mutation test in mouse lymphoma L5178Y cells, the test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation. In a key in vitro mammalian micronucleus assay in Chinese Hamster V79 Cells under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage.

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:
2012 - 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): DE07_2012_001_PLSW
- Substance type: Alkylsulfate/alkylethersulfate
- Chemical name: Alcohols, C16-18 (even numbered, C18-unsatd.), ethoxylated, and alcohols C20-22 (even numbered), sulfates, ammonium salts
- CAS: n/a
- Physical state: ivory paste-like solid at 20 °C
- Batch No.: PU20740016, PU20740019
- Purity: 100 % (UVCB)
- Storage condition of test material: Room temperature, protected from light
- Stability: stable under test conditions
Target gene:
his- / his+ reversions
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver, induced with phenobarbital and 5,6-benzoflavone)
Test concentrations with justification for top dose:
Dose-finding study:
3.16, 10.0, 31.6, 100, 316, 2500, 5000 μg/plate (TA 98, TA 100)
Doses: 3.16, 10.0, 31.6, 100, 316, 2500, 5000 μg/plate

Main study:
-S9 mix: 0, 31.6, 100, 316, 1000, 2500, 5000 μg/plate (all strains)
+S9 mix: 0, 31.6, 100, 316, 1000, 2500, 5000 μg/plate (all strains)

The test item was tested in a pre-experiment eith the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

The test item concentration to be applied in the main experiments were choosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Three independent experiments were performed with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
dist. Water
Untreated negative controls:
yes
Remarks:
dist. water
Negative solvent / vehicle controls:
yes
Remarks:
dist. water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine, 2-aminoanthracene
Details on test system and experimental conditions:
Procedures:
Pre-incubation method
Plate incorporation method

For each strain and dose level, including controls, three plates were used.

Number of replicates: 3

Positive controls:
-S9 mix; Sodium azide (TA 100, TA 1535), 4-nitro-o-phenylene-diamine (TA 98, TA 1537), methylmethanesulfonate (TA 102)
+S9 mix; 2-Aminoanthracene (for all strains)
Evaluation criteria:
To be considered positive if:
- a clear and dose related increase in the number of revertants accours and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows;

- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
Statistics:
no data
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
a slight toxic effect oberved in tester strain TA 1535 at a concentration of 5000 µg/plate (+ S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No biological relevant increases in revertant colony numbers of any of the five strains were observed in the test with either the non-activation method (-S9 mix) or the activation method (+S9 mix).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 Revertants per plate (mean of 3 plates, data of experiment 1 of 3 shown)

Concentration µg/plate

TA 98

TA100

TA1535

TA1537

TA102

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Negative control

25

32

106

128

5

6

7

9

309

177

31.6

19

33

108

113

9

11

9

12

239

220

100.0

14

30

116

119

8

6

14

14

256

191

316

22

30

125

122

8

5

7

11

252

182

1000

20

24

127

125

8

5

9

7

297

158

2500

27

28

115

117

12

5

11

13

268

207

5000

22

22

114

105

17

4

17

11

341

193

4-NOPD/NaN3 (10µg)/MMS ()* 382  - 711 387 30  - 1784
2-AA (2.5 µg)**  -  2500  -  2004  -  66  -  210  - 379 

* positive control with 4-Nitro-o-phenylene-diamine (4-NOPD, 10 µg (TA 98) and 40 µg (TA 1537)), sodium azide (NaN3, 10 µg (TA 100, TA 1535)) or Methylmethansulfonate (MMS, 1µL (TA 102))

** positive control with 2-Aminoanthracene (2-AA, 2.5 µg (TA 98, TA 100, TA 1535, TA 1537) and 10 µg (TA 102))

Conclusions:
Interpretation of results (migrated information): negative
The test material was considered to be non-mutagenic to Salmonella typhimurium under the conditions of the test.
Executive summary:

The test item did not increase the reverse mutation rate in histidine dependent bacterial strains of Salmonella typhimurium in the presence or absence of metabolic activation at concentrations up to and including 5000 µg/plate. It is concluded that the test substance is negative for mutagenicity in bacteria under the conditions of the test.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012 - 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
- Name of test material (as cited in study report): DE07_2012_001_PLSW
- Substance type: Alkylsulfate/alkylethersulfate
- Chemical name: Alcohols, C16-18 (even numbered, C18-unsatd.), ethoxylated, and alcohols C20-22 (even numbered), sulfates, ammonium salts
- CAS: n/a
- Physical state: ivory paste-like solid at 20 °C
- Batch No.: PU20740016, PU20740019
- Purity: 100 % (UVCB)
- Storage condition of test material: Room temperature, protected from light
- Stability: stable under test conditions
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cells (ATCC, CCL-93) are stored over liquid nitrogen (vapour phase). Routine checking of mycoplasma infections
were carried out before freezing. For the experiments thawed cultures are set up in 75 cm 2 cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 10 E05 cells per flask are seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (foetal bovine serum) and subcultures are made every 3-4 days.
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 I: with metabolic activation: 10.00, 20.00, 30.00, 40.00, 45.00, 50.00, 55.00, 60.00, 65.00,
70.00 and 80.00 µg/mL
without metabolic activation: 5.00, 15.00, 20.00, 25.00, 30.00, 35.00, 40.00, 45.00,
50.00, 60.00, and 70.00 µg/mL
Experiment II: without metabolic activation: 0.16, 0.31, 0.63, 1.25, 2.50, 5.00, 10.00, 15.00, 20.00,
25.00 and 30.00 µg/mL
The following concentrations were selected for the microscopic analyses for micronucleated frequencies:
Experiment I with short exposure (4 h):
with metabolic activation: and 20.00, 50.00, 60.00 and 65.00 µg/mL
without metabolic activation: 5.00, 15.00 and 20.00 µg/mL
Experiment II with extended exposure (24 h):
without metabolic activation: 0.31, 5.00 and 10.00 µg/mL
Untreated negative controls:
yes
Remarks:
cell culture medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Remarks:
with and without metabolic activation
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
other: Colcemide
Evaluation criteria:
All slides, including those of positive and negative controls were independently coded before microscopic analysis. For each experimental point, at least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei according to the criteria of Countryman and Heddle, i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered. No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated.
Statistics:
Statistical significance at the 5% level (p < 0.05) was evaluated by the non-parametric χ² test. The number of micronucleated cells found in the groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control.

Experiment I without metabolic activation:
In experiment I without metabolic activation the micronucleated cell frequencies of the negative control (1.00%) were within the range of the historical control data of the negative control (0.45% – 1.60%). The number of micronucleated cells found in the groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control.

Experiment I with metabolic activation:
In experiment I with metabolic activation the micronucleated cell frequency of the negative control (1.20%) was within the historical control data of the negative control (1.20%). The number of micronucleated cells found in the groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control.

Experiment II without metabolic activation:
The micronucleated cell frequency of the negative control (1.05 %) was within the historical control data of the negative control (0.45% – 1.60%). The number of micronucleated cells found in the groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control.

Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Exposure Concentrations selected for the microscopic analyses:
In experiment I 20.00 µg/mL without metabolic activation and 65.00 µg/mL with metabolic activation were selected as the highest dose groups for the microscopic analysis of micronuclei.
In experiment II without metabolic activation 10 µg/mL was selected as highest dose group for the microscopic analysis of micronuclei.
The cells were prepared 24 h after start of treatment with the test item. The treatment intervals were 4 h with and without metabolic activation (experiment I) and 24 h without metabolic activation (experiment II). Two parallel cultures were set up and 1000 binucleated cells per culture were scored for micronuclei. Duplicate cultures were performed at each dose level.
The following concentrations were evaluated for micronucleated frequencies:

Experiment I with short exposure (4 h):
with metabolic activation: and 20.00, 50.00, 60.00 and 65.00 µg/mL
without metabolic activation: 5.00, 15.00 and 20.00 µg/mL

Experiment II with extended exposure (24 h):
without metabolic activation: 0.31, 5.00 and 10.00 µg/mL

Precipitation:
The test item was prepared in culture medium (MEM medium). No precipitate of the test item was noted in all dose groups evaluated in experiment I and II.

Toxicity:
As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined.
According the OECD Guideline 487 the maximum of cytotoxicity should not exceed the limit of 55% ± 5%. Higher levels of cytotoxicity induce chromosome damage as a secondary effect of cytotoxicity.According to laboratory experience a culture showing reduced cell viability (less than 70% rel. CBPI) of the negative control displays cytotoxicity. Due to this the acceptable limit of cytotoxicity is ≥ 30%. This corresponds
to ≤ 70% of rel. CBPI. In experiment I without metabolic activation no decrease of the relative CBPI below 70% was noted at a concentration of 5.00 µg/mL. At a concentration of 15.00 µg/mL a relative CBPI of 52%, at a concentration of 20.00 µg/mL a relative CBPI of 43% and at a concentration of 25.00 µg/mL a relative CBPI of 29% were noted. In experiment I with metabolic activation no decrease of the relative CBPI below 70% was noted at aconcentration of 20.00 µg/mL. At a concentration of 50.00 µg/mL a relative CBPI of 64%, at a concentration of 60.00 µg/mL a relative CBPI of 55% and at a concentration of 65.00 µg/mL a relative CBPI of 35% were noted. In experiment II without metabolic activation no decrease of the relative CBPI below 70% was noted at a concentration of 0.31 µg/mL. At a concentration of 5.00 µg/mL a relative CBPI of 65%, at a concentration of 10.00 µg/mL a relative CBPI of 50% and at a concentration of 30.00 µg/mL a relative CBPI of 31% were noted.

Positive controls:
EMS (600 and 900 µg/mL) and CPA (2.5 µg/mL) were used as clastogenic controls and Colcemide as aneugenic controls (0.08 and 0.80 µg/mL). They induced distinct and biologically relevant increases of micronucleus frequency. This demonstrates the validity of the assay.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Summary of Micronuclei Effects: Experiment I with and without metabolic activation

Dose Group

Concentration [µg/mL]

Treatment time

Fixation interval

Micronucleated Cells Frequency

without metabolic activation

C

0

4 h

24 h

1.00

1

5

4 h

24 h

1.10

2

15

4 h

24 h

0.70

3

20

4 h

24 h

1.05

EMS

900

4 h

24 h

4.70

Colcemide

0.8

4 h

24 h

2.30

with metabolic activation

4 h

24 h

C

0

4 h

24 h

1.20

2

20

4 h

24 h

1.25

6

50

4 h

24 h

0.80

8

60

4 h

24 h

0.95

9

65

4 h

24 h

0.85

CPA

2.5

4 h

24 h

2.30

2000 binucleated cells evaluated for each concentration

C:  Negative Control (Culture medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate) 

Colcemide: Positive Control (without metabolic activation: Colcemide) 

CPA: Positive Control (with metabolic activation: Cyclophosphamide)

Table 2: Summary of Micronuclei Effects: Experiment II with and without metabolic activation

Dose Group

Concentration [µg/mL]

Treatment time

Fixation interval

Micronucleated Cells Frequency

without metabolic activation

C

0

24 h

24 h

1.05

2

0.31

24 h

24 h

1.15

6

5

24 h

24 h

1.20

7

10

24 h

24 h

1.30

EMS

600

24 h

24 h

3.60

Colcemide

0.08

24 h

24 h

4.30

C:  Negative Control (Culture medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

Colcemide: Positive Control (without metabolic activation: Colcemide)

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

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.
Executive summary:

Introduction:

The in vitro micronucleus assay is a genotoxicity test system for the detection of chemicals which induce the formation of small membrane bound DNA fragments i.e. micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from acentric fragments (chromosome fragments lacking a centromer) or whole chromosomes which are unable to migrate with the rest of the chromosomes during the anaphase of the cell division. The in vitro micronucleus assay detects potential clastogenic and aneugenic effects of chemicals. The in vitro micronucleus test is the only in vitro test which is able to detect both numerical and chromosomal aberrations, two mechanisms involved in genetic and carcinogenic risk. Clastogenic or aneugenic incidents are critical lesions for the cell that could cause alterations in protooncogens and tumor suppressor genes. These alterations could result in oncogenesis.

Method:

The test item was investigated for a possible potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation with S9. The selection of the concentrations used in experiment I and II was based on data from

the pre-experiment according to the guidelines. Cytotoxicity was determined with and without metabolic activation by determination of cytokinesis block proliferation index (CBPI). Statistical significance at the 5% level (p < 0.05) was evaluated by the non-parametric

χ² test. In experiment I 20.00 µg/mL without metabolic activation and 65.00 µg/mL with metabolic activation were selected as the highest dose groups for the microscopic analysis of micronuclei.

In experiment II without metabolic activation 10 µg/mL was selected as highest dose group for the microscopic analysis of micronuclei.

The treatment intervals were 4 h with and without metabolic activation (experiment I) and 24 h without metabolic activation (experiment II). Two parallel cultures were set up and 1000 binucleated cells per culture were scored for micronuclei.

Conclusion:

It can be stated that during the study described and under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012 - 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): DE07_2012_001_PLSW
- Substance type: Alkylsulfate/alkylethersulfate
- Chemical name: Alcohols, C16-18 (even numbered, C18-unsatd.), ethoxylated, and alcohols C20-22 (even numbered), sulfates, ammonium salts
- CAS: n/a
- Physical state: ivory paste-like solid at 20 °C
- Batch No.: PU20740016, PU20740019
- Purity: 100 % (UVCB)
- Storage condition of test material: Room temperature, protected from light
- Stability: stable under test conditions
Target gene:
thymidine kinase, TK +/-, locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- RPMI 1640 culture medium supplemented with
9.0 µg/mL hypoxanthine
15.0 µg/mL thymidine
22.5 µg/mL glycine
0.1 µg/mL methotrexate

- Properly maintained: yes
- Routinely checked for Mycoplasma contamination: yes
- Routinely checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction prepared from male Witstar rats induced for 3 days with phenobarbital and naphthoflavone. Cofactors: MgCl2 8 mM, KCl 33 mM, glucose-6-phosphate 5 mM, NADP 5 mM, phosphate buffer (pH 7.4) 100 mM.
Test concentrations with justification for top dose:
1.1 preexperiment I with metabolic activation (µg/ml): 60, 80, 100, 120, 130, 145
1.2 preexperiment I without metabolic activation (µg/ml): 0.05, 0.50, 5.00, 15.00, 25.00, 40.00

2. preexperiment II without metabolic activation (24 h long term exposure) (µg/ml): 0.1, 1.0, 5.0, 10.0, 40.0, 100.0

3.1 main experiment I with metabolic activation (4 h short term exposure) (µg/ml): 5.0, 10.0, 25.0, 50.0, 100.0, 110.0, 120.0, 130.0
3.2 main experiment I without metabolic activation (4 h short term exposure) (µg/ml): 5.0, 10.0, 20.0, 40.0, 46.0, 50.0, 70.0, 80.0

4.1 main experiment II with metabolic activation (24 h long term exposure) (µg/ml): 1.5, 3.5, 7.0, 20.0, 30.0, 60.0, 105.0, 115.0
4.2 main experiment II without metabolic activation (24 h long term exposure) (µg/ml): 1.2, 2.5, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0
Vehicle / solvent:
RPMI cell culture medium with 5% horse serum.
Based on the results of the soluibility tests (DMSO, EtOH, RPMI) the best suited vehicle was RPMI cell culure medium with 5% horse serum.
The solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation, 24 hours without metabolic activation
- Expression time (cells in growth medium): two days

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EXPOSED:
short term: 1 x 10^7 cells/mL
long term: 5 x 10^6 cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: measuring of colony forming ability and the growth rate of cultures

Evaluation criteria:
Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive.
Statistics:
Statisitcal significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Key result
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
Remarks:
Migrated from field 'Test system'.

Table 1. Summary of Results: Experiment I

Treatment (µg/ml)

4-Hours with S-9

Treatment (µg/ml)

4-Hours without S-9

 

% RSG

RCE

MF

 

% RSG

RCE

MF

0

100.0

100.0

77.9/87.2

0

100.0

100.0

112.6/94.9

5.0

93.2

108.1

71.0

5.0

85.2

117.8

83.3

10.0

98.6

87.0

115.9

10.0

101.4

100.4

89.0

25.0

96.0

99.7

97.8

20.0

96.2

130.8

80.4

50.0

89.7

121.8

64.2

40.0

63.8

91.8

93.7

100.0

42.8

98.2

94.6

46.0

53.8

105.2

96.2

110.0

22.9

145.2

71.0

50.0

58.5

114.0

105.6

120.0

12.6

102.9

96.6

70.0

21.6

108.6

117.9

130.0

15.5

108.1

102.5

80.0

15.2

102.0

123.5

Linear Trend = Not Significant

Linear Trend = Not Significant

Benzo[a]pyrene

 

 

 

EMS/MMS

 

 

2.5

37.0

64.4

763.5

200.0/10.0

77.0/74.2

94.5/75.0

546.7/548.9

Table 2. Summary of Results: Experiment II

Treatment (µg/ml)

4-Hours with S-9

Treatment (µg/ml)

24-Hours without S-9

 

% RSG

RCE

MF

 

% RSG

RCE

MF

0

100.0

100.0

72.1/111.5

0

100

100

53.7/57.3

1.5

83.2

97.7

67.9

1.2

100.3

93.3

65.3

3.5

88.1

94.7

92.0

2.5

110.2

93.3

47.1

7.0

97.6

90.5

98.3

5.0

103.4

91.9

49.4

20.0

91.3

109.1

66.6

10.0

98.9

105.5

43.2

30.0

91.4

102.3

92.9

20.0

73.4

114.5

49.4

60.0

93.3

112.8

73.1

30.0

64.9

93.3

62.0

105.0

24.4

72.8

183.3

40.0

35.7

85.3

65.8

115.0

17.0

86.6

195.4

50.0

13.8

62.3

105.4

Linear Trend = Not Significant

Linear Trend = Not Significant

Benzo[a]pyrene

 

 

 

EMS/MMS

 

 

2.5

67.1

78.2

642.7

200.0/10.0

77.0/74.2

56.4/47.3

808.8/943.4

RSG: Relative suspension growth

RCE: Relative cloning efficiency

MF: Mutation factor

Conclusions:
Interpretation of results (migrated information): negative
The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.
Executive summary:

Study Summary

Introduction:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B.17 of the Regulation (EC) No 440/2008.

 

Results:

The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. A precipitate of test material was observed at and above 46 μg/ml in the absence of metabolic activation, and at and above 50 μg/ml in the presence of metabolic activation in the first experiment. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

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

 

Conclusion:

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

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

Additional information

Additional information from genetic toxicity in vitro:

Bacterial mutation:

A Klimisch 1 key Ames bacterial mutagenicity test demonstrated that no increases in mutations were observed in 5 different Salmonella typhimurium strains (S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102) up to cytotoxic concentration of 5000 µg/plate. Three independent experiments were carried out each without and with metabolic activation.

Mammalian mutagenicity:

In a Klimisch 1 key mammalian gene mutation test in mouse lymphoma L5178Y cells, the test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation. Two main experiments were carried out (4h short term exposure with and without metabolic activation and 24 h long term exposure with and without metabolic activation). The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. A precipitate of test material was observed at and above 46 μg/ml in the absence of metabolic activation, and at and above 50 μg/ml in the presence of metabolic activation in the first experiment. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Chromosomal damage:

In a Klimisch 1 key in vitro mammalian micronucleus assay in Chinese Hamster V79 Cells under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage. The test item was investigated for a possible potential to induce micronuclei in Chinese hamster V79 cells in vitro. Cytotoxicity was determined with and without metabolic activation by determination of cytokinesis block proliferation index (CBPI). In experiment I 20.00 µg/mL without metabolic activation and 65.00 µg/mL with metabolic activation were selected as the highest dose groups for the microscopic analysis of micronuclei. In experiment II without metabolic activation 10 µg/mL was selected as highest dose group for the microscopic analysis of micronuclei. The treatment intervals were 4 h with and without metabolic activation (experiment I) and 24 h without metabolic activation (experiment II).

Supporting studies:

Several supporting studies on genetic toxicity of the supporting substances behenyl alcohol and 2-(2-hexyloxyethoxy)ethanol (read across data for structural analogue or surrogate) are available. In a bacterial reverse mutation assay (S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a mammalian gene mutation test (Chinese hamster Ovary (CHO)) for 2-(2 -hexyloxyethoxy)ethanol no increased mutations or induction of any toxicologically significant dose-related increases in the mutant frequency were oberved. In an in vitro mammalian chromosome aberration test (Chinese hamster lung fibroblasts (V79)), as well as an in vitro mammalian cell gene mutation test (Chinese hamster lung fibroblasts (V79)) and in a bacterial reverse mutation assay (S. typhimurium TA1535, TA1537, TA1538, TA98, and TA100) for behenyl alcohol no increases in mutations, no induction of structural and/or numerical chromosomal damage and no toxicologically significant dose-related increases in the mutant frequency were observed.

In addition three in vivo genetic toxicity studies for the supporting substances behenyl alcohol and 2-(2-hexyloxyethoxy)ethanol are available. In a mammalian erythrocyte micronucleus test behenyl alcohol did not increase the incidence of micronuclei in mouse bone marrow cells after a single oral gavage dose of up to 500 mg/kg bw.

In a mammalian erythrocyte micronucleus test in Swiss Webster mice 2-(2-hexyloxyethoxy)ethanol did not increase the incidence of micronuclei in mouse bone marrow cells after a single oral gavage dose of up to 640 mg/kg bw.

In a mammalian bone marrow chromosome aberration test in Sprague-Dawley rats 2-(2-hexyloxyethoxy)ethanol induced no increase of total aberrant cells after a single oral gavage dose of up to 1500 mg/kg bw.

Conclusion:

Standard information requirements according to REACH Guidance Part 3 R7a were fulfilled for genotoxicity testing, including bacterial and mammalian mutagenicity and chromosomal aberration. Based on the available results, there were no indications of mutagenicity or genotoxicity, and no further testing is needed. The substance can be considered to have no mutagenic or genotoxic potential.

The justification to use read-across data from supporting substances is provided in Section 13 of the IUCLID dossier.


Justification for selection of genetic toxicity endpoint
Although the Ames test was indicated as key study, the other endpoints concerning genetic toxicity for the test item are considered equally important.

Justification for classification or non-classification

Based on the available results, there were no indications of mutagenicity or genotoxicity, i.e. no classification is required. The substance can be considered to have no mutagenic or genotoxic potential.