Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are three key studies on an analogue substance (EC613 -848 -7):
- Ames Test 2013 (OECD 471, GLP, K, rel. 1): non mutagenic up to 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & WP2uvrA-
- Human lymphocyte chromosome aberration test 2013 (OECD 473, GLP, K, rel. 1): non clastogenic up to precipitation limit
- Gene mutation assay in mouse lymphoma L5178Y cells 2013 (OECD 476, GLP, K rel. 1): non mutagenic up to precipitation limit

Read-across to the properties of Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester (EC 613-848-7, CAS 65870-94-2), Fatty acid polyols (Fatty acids, C5-9, esters with pentaerythritol (EC 270-290-3, CAS 68424-30-6) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)) and their analogues is applicable based on the similarity in structure and physico-chemical properties. The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.


Genetic toxicity in vitro
Supp, EC 234-392-1 Exxon, Bailey, 1996, Ames, - not mutagenic
Supp, EC 234-392-1 Exxon, Gudi, 1996, CA, - not clastogenic
Supp, CAS 131459-39-7, Croda, Wright, 1999, ChrAb, human – not clastogenic
Supp, CAS 85186-89-6, Verspeek-Rip, 2010, gene mut in mamm cells – not mutagenic

Key, CAS 68424-31-7 Croda, Griffiths, 1992, CTL/P/3792, Micronuc., - not genotoxic

Key, CAS 68424-31-7 Croda, Griffiths, 1992, CTL/P/3792, Micronuc., - not genotoxic


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-05-23 to 2013-06-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: 40 CFR 799.9510 TSCA bacterial reverse mutation test and the USA, EPA (TSCA) OCSPP harmonized guidelines.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
None
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Non-mammalian study
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Non-mammalian study
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9; S9 fraction was prepared from liver homogenates of male rats induced with phenobarbitone/β-naphthoflavone
Test concentrations with justification for top dose:
Mutation tests (plate incorporation method)
Experiment 1:
1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate for all S.typhimurium strains (TA1535, TA1537, TA98 and TA100) and E. coli strain WP2uvrA, with and without S9-mix

Experiment 2:
50, 150, 500, 1500 and 5000 μg/plate for all S.typhimurium strains (TA1535, TA1537, TA98 and TA100) and E. coli strain WP2uvrA, with and without S9-mix

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
- Preparation of formulation: Test item was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent (Acetone)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene - 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 and 10 µg/plate for WP2uvrA; Benzo(a)pyrene - 5 µg/plate for TA98
Remarks:
With metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent (Acetone)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitro-N-nitrosoguanidine - 2 µg/plate for WP2uvrA, 3 µg/plate for TA100 and 5 µg/plate for TA1535; 9-Aminoacridine - 80 µg/plate for TA1537; 4-Nitroquinoline-1-oxide - 0.2 µg/plate for TA98
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) at multiple dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors).

DURATION
- Exposure duration: Treated plates were incubated at 37 °C ± 3 °C for approximately 48 h and scored for the presence of revertant colonies using an automated colony counting system.

NUMBER OF REPLICATIONS: 3 replicates of each strain at each concentration both in the presence and absence of S9

DETERMINATION OF CYTOTOXICITY
- Method: The treated plates were viewed microscopically for evidence of thinning (toxicity).

OTHER:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response) (Cariello and Piegorsch, 1996)).

- A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
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:
valid
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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (globular in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were comparable with historical negative, solvent and positive control data (2011 and 2012) of laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation in both experiments.

OTHERS:
- The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile.
- The culture density for each bacterial strain used in each experiment was also checked and considered acceptable.

See the attached document for tables of results

Conclusions:
Under the test conditions, the test material is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) with and without metabolic activation
Executive summary:

Test Guidance

OECD Guideline 471

Method and materials

Strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA)were exposed to the test item at the following concentrations using plate incorporation method.

 

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9 mix in all strains

Experiment 2: 50, 150, 500, 1500 and 5000 μg/plate, with and without S9 mix in all strains

 

Metabolic activation system used in this test was10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with phenobarbitone/β-naphthoflavone. Negative, vehicle and positive control groups were also included in mutagenicity tests.

Results

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in any of the experiment. A test item precipitate (globular in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 or 2. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

Conclusion

Under the test conditions, the substance is not considered as mutagenic in bacterial systems.

.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
May 06th, 1996 - August 29th, 1996
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:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Genes involved in Histidine synthesis
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (Spraque Dawley rats, male, Aroclor 1254 induced)
Test concentrations with justification for top dose:
0, 10, 33, 100, 333 and 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see table
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h
- Expression time (cells in growth medium): 48 to 72 h

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial backgroung lawn wit a dissecting microscope
Evaluation criteria:
Revertant colonies were counted and the mean and standard deviation were calculated and compared to the controls.
All Salmonella tester strains must demonstrate the presence of the deep rough mutation and the deletion of the uvrA gene. Cultures of the TA98 and TA100 strains must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion of the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls. Tester strain titers must be above 30.000.000 cells/ml. The mean of each positive control must be at least three-fold increased to the controlls. A minimum of three non-toxic dose levels are recquired to evaluate assay data.
Statistics:
Mean and standard deviation were calculated
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, but tested up to precipitating concentrations
Vehicle controls validity:
other: An unacceptable vehicle control value with the tester strain TA1537, contamination of tester strain TA98, experiments were repeated
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: >100 µg/plate

RANGE-FINDING/SCREENING STUDIES: Yes

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

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

Experiment 1:

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 1535

TA1537

TA98

TA100

 

TA 1538

WP2uvrA

-

Vehicle

12

4

17

111

5

21

 

10

3

4

15

115

6

16

 

33

5

5

19

113

6

11

-

100

9

4

19

98

7

12

-

333

7

5

11

116

4

10

-

1000

8

6

21

120

7

13

Positive

controls

- S9

Name

SA

9AA

2NF

SA

2NF

MMS

Concentrations

(μg/plate)

1.0

75

1.0

1.0

1.0

1000

Number of colonies/plate

241

40

105

377

179

143

+

Vehicle

14

5

18

133

7

11

 

10

9

5

27

114

12

16

+

33

9

3

21

113

8

13

+

100

8

7

26

108

9

13

+

333

9

4

27

115

6

8

+

1000

10

5

17

111

9

13

Positive

controls

+ S9

Name

2AA

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1.0

1.0

1.0

1.0

1.0

10

Number of colonies/plate

72

127

888

904

783

57

SA: Sodium azide

9AA : 9-Aminoacridine

MMS: Methylmethanesulfonate

2-AA: 2-Aminoanthracene

2NF: 2-Nitrofluorene

 

 

Experiment 2/3:

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 1535

TA1537

TA98

TA100

 

TA 1538

WP2uvrA

-

Vehicle

14

10

23

126

11

27

 

10

7

13

16

117

7

27

 

33

9

15

23

124

8

19

-

100

5

13

22

120

6

18

-

333

12

9

17

110

11

16

-

1000

8

14

17

125

5

21

Positive

controls

- S9

Name

SA

9AA

2NF

SA

2NF

MMS

Concentrations

(μg/plate)

1.0

75

1.0

1.0

1.0

1000

Number of colonies/plate

429

757

125

601

221

195

+

Vehicle

8

5

19

147

14

24

 

10

9

7

17

142

16

30

+

33

10

5

19

136

18

25

+

100

10

5

20

132

13

31

+

333

10

4

17

138

12

19

+

1000

10

7

19

125

12

19

Positive

controls

+ S9

Name

2AA

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1.0

1.0

1.0

1.0

1.0

10

Number of colonies/plate

85

97

530

647

1041

88

SA: Sodium azide

9AA : 9-Aminoacridine

MMS: Methylmethanesulfonate

2-AA: 2-Aminoanthracene

2NF: 2-Nitrofluorene

 

 

Conclusions:
Under the tested experimental conditions the test substance did not induce gene mutations in S. typhimurium strains and in an E. coli strain up to the maximum of solubility. Therefore it is not considered to be mutagenic in this bacterial mutagenicity test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 June 2013 to 11 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
N/A
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum,
- Properly maintained: NDA
- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1
0*, 25, 50*, 100*, 200*, 300, 400 µg/ml without S9
0*, 25, 50*, 100*, 200*, 300, 400 µg/ml with S9
* = Dose levels selected for metaphase analysis

Experiment 2
0*, 25*, 50, 100*, 200*, 300, 400 µg/ml without S9
0*, 25*, 50*, 100*, 200, 300, 400 µg/ml with S9
* = Dose levels selected for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test item was insoluble in water and DMSO but soluble in acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.4 and 0.2 ug/ml in Expteriment 1 and 2 respectively
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
5.0 ug/ml
Details on test system and experimental conditions:
- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum,
- Properly maintained: NDA
- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA

Experiment 1
0*, 25, 50*, 100*, 200*, 300, 400 µg/ml without S9
0*, 25, 50*, 100*, 200*, 300, 400 µg/ml with S9
* = Dose levels selected for metaphase analysis

Experiment 2
0*, 25*, 50, 100*, 200*, 300, 400 µg/ml without S9
0*, 25*, 50*, 100*, 200, 300, 400 µg/ml with S9
* = Dose levels selected for metaphase analysis

METHOD OF APPLICATION: in medium
With Metabolic Activtion
Cultures were established approximately 48 hours prior to treatment. Cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing.

Without Metabolic Activation
Cultures were established approximately 48 hours prior to treatment. In Experiment 1, cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing. In Experiment 2, the cultures were incubated in the presence of the substance at 37°c for 24 hours
DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 20 and 0 hours
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): Mitosis was arrested by addition of democolcine two hours prior to the required harvest time and the cells were harvested and fixed

SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): 5% Gurrs Giemsa


NUMBER OF REPLICATIONS: Treatments performed in duplicate.


NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Where possible the first 100 consecutive well-spread metaphases from each culture were counted.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes in comparison to controls
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated
Evaluation criteria:
A test item can be classified as non-genotoxic if:
1 . The number of induced chromosome aberrations in all evaluated dose groups is within the range of laboratory historical control data.
2. No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.
A test item can be classified as genotoxic if:
1 . The number of induced structural chromosome aberrations is not in the range of laboratory historical control data.
And
2. Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.
Biological relevance of the results will be considered first. Statistical methods will be used to analyze the increases in aberration data as recommended in the OECD 473 guideline. However, statistical significance will not be the only determining factor for a positive response.

A toxicologically significant response is recordedwhen the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Statistics:
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.
Species / strain:
other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 9.77 - 2500 µg/ml.

The maximum dose was based on solubility in the vehicle.

* Precipitate was observed at and above 39.06 µg/ml in the 4-hour exposure group in the absence of metabolic activation and the continuous exposure group. In the presence of metabolic activation a precipitate was observed at and above 78.13 ug/ml.

* Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 2500 ug/ml in all three exposure groups.

The selection of the maximum dose level was based on the presence of a precipitate:

Chromosome Aberration Test - Experiment 1

400 ug/ml


Chromosome Aberration Test - Experiment 2

400 ug/ml

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that was the lowest precipitating dose level. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

See attached background material.

Conclusions:
Interpretation of results (migrated information):
negative

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that was the lowest precipitating dose level. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations. The test methods described are designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

• OECD Guidelines for Testing of Chemicals No. 473 " In Vitro Mammalian Chromosome Aberration Test"

• Method BIO of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

• US EPA OPPTS 870.5375 Guideline.

• 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test.

• Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were 25, 50, 100,200, 300, 400 ug/ml for all exposure groups.

 

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that was the lowest precipitating dose level.

 

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
February 01st, 1999 - April 21st, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance CAS 131459-39-7. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No details on the purity of the test substance
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No details on the purity of the test substance
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced S9-Mix from male Spraque-Dawley rats
Test concentrations with justification for top dose:
1. Experiment:
4(20) h without S9: 0; 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/ml
4(20) h with S9: 0; 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/ml

2. Experiment
24 h without S9: 0; 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/ml
4(20) h with S9: 0; 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
in absence of S9-Mix Migrated to IUCLID6: 750 µg/ml
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in presence of S9-Mix Migrated to IUCLID6: 25 µg/ml
Details on test system and experimental conditions:
Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations at up to three dose levels. For each experiment, whole blood was drawn from the peripheral circulation of a volunteer who had previously been screened for suitability. The cell cycle length of approx. 14 h was determined by BrdU incorporation.
METHOD OF APPLICATION: in medium (Eagle´s minimal essential medium with HEPES buffer supplemented with L-glutamine, Pen/Strep, amphotericin B and 15% FCS

DURATION
- Exposure duration: Experiment I: 4 h with or without S9 mix and 20 h expression time.
Experiment II: Either 4 h with S9 mix and 20 h expression time or 24 h without S9 mix.
- Expression time (cells in growth medium): 20 h after 4 h exposure to the test substance


SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)
STAIN (for cytogenetic assays): 5% Gurrs Giemsa for 5 min

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER: Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations at up to three dose levels. For each experiment, whole blood was drawn from the peripheral circulation of a volunteer who had previously been screened for suitability. The cell cycle length of approx. 14 h was determined by BrdU incorporation.
Evaluation criteria:
All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.
Statistics:
The frequency of cells with aberrations and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher´s Exact test.
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at and above 1250 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes:
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at and above 1250 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No detectable effects
- Effects of osmolality: No detectable effects
- Precipitation: Medium was "cloudy" from 156.25 µg/ml and a precipitate was seen from 1250 µg/ml without effects on the toxicity responsefects:

COMPARISON WITH HISTORICAL CONTROL DATA: Yes
Remarks on result:
other: strain/cell type: lymphocytes
Remarks:
Migrated from field 'Test system'.

Experiment 1: 4 h treatment without metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

5

0

1

0

1

0

7

2

7

2

1250

200

5

0

0

3

0

0

8

3

8

3

2500

200

2

2

0

0

0

0

4

2

4

2

5000

200

5

4

0

0

1

0

10

5

9

5

EMS 750

200

24

9

5

5

0

0

43

19

35

17

Experiment 1: 4 h treatment with metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

4

1

0

0

0

0

5

1

4

1

1250

200

6

4

0

0

0

0

10

4

9

4

2500

200

4

2

0

0

0

0

6

2

5

2

5000

200

2

4

0

1

0

0

7

5

7

5

CP 25

200

13

12

3

2

0

0

30

17

24

15

Experiment 2: 20 h treatment without metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

2

2

1

0

0

0

5

3

5

3

1250

200

7

0

0

1

0

0

8

1

8

1

2500

200

0

1

0

0

0

0

1

1

1

1

5000

200

6

0

0

0

0

0

6

0

6

0

EMS 500

200

53

41

13

2

0

0

109

56

74

48

Experiment 2: 4 h treatment with metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

0

0

0

2

0

0

2

2

2

2

1250

200

4

2

2

3

0

0

9

5

7

3

2500

200

5

2

0

1

0

0

8

3

8

3

5000

200

3

2

0

2

0

0

7

4

6

3

CP 25

200

10

10

2

0

0

0

22

12

15

11

The test substance was non-clastogenic to human lymphocytes in vitro.

Conclusions:
Interpretation of results (migrated information):
negative non-clastogenic to human lymphocytes in vitro
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
May 22nd, 1996 - October 28th, 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study Basic data on test substance given
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Only basic data on test substance given
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy´s 5A medium supplemented with 10% FBS, 100 units penicillin and 100 µg streptomycin/ml and 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9-mix (Spraque-Dawley)
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 0.0 (Vehicle control), 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/ml
Chromosomal aberration assay: 0.0 (Vehicle control), (157, 313), 625, 1250, 2500, 5000 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:
mitomycin C
Remarks:
8 and 15 µg/mL Migrated to IUCLID6: in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
1000 µg/mL Migrated to IUCLID6: in water
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 h without S9 activation and 4 h with activation.

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF CELLS EVALUATED: A minimum of 200 metaphase spreads (100 per duplicate flask) at each concentration used.
Evaluation criteria:
Criteria for cytotoxicity of the test substance: (1) cell growth inhibition relative to the solvent control
Criteria for chromosomal damage: (1) Number and types of aberrations found, the percentage of structurally and numerically damaged cells in the total population were counted.
Statistics:
The frequency of structural aberrations per cell was calculated. The statistical analysis of the percent aberrant cells was performed with Fisher´s Exact Test. It was used to compare pair wise the percent aberrant cells of each treatment group with that of the solvent control. In the event of positive control, the Cochran-Armitage test was used to measure dose- responsiveness.
The dose response was estimated by linear regression curves. Difference in the sensitivity of mutagenic compounds was established by comparing the slopes of corresponding dose-response regression curves. For a comparison of mean values, Student's t test was used.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
48% in the highest dose for treatment of 4 h without metabolic activation, 33% with metabolic activation and 3% for 20 h exposure without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 8 in the highest dose
- Effects of osmolality: 285 in the highest dose

RANGE-FINDING/SCREENING STUDIES: Yes, cell growth inhibition of 83% without and 3% with metabolic activation at the highest dose tested (5000 µg/ml).


COMPARISON WITH HISTORICAL CONTROL DATA: Yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Chromosomal aberration – Summary

 

Treatment

[µg/ml]

S9 activation

Treatment/

harvest time [h]

Mitotic index

Cells scored

Aberrations/cell (mean±SD)

Cells with aberrations [%]

numerical

structural

Vehicle (Ethanol)

-

4/20

6.9

200

0.005±0.071

2.5

0.5

625

-

4/20

5.9

200

0.000±0.000

2.0

0.0

1250

-

4/20

5.9

200

0.025±0.186

4.0

2.0

2500

-

4/20

6.5

200

0.025±0.186

2.5

2.0

5000

-

4/20

4.4

200

0.000±0.000

0.5

0.0

MMC (0.08)

-

4/20

6.1

200

0.150±0.788

3.5

9.0*

Vehicle (Ethanol)

+

4/20

7.8

200

0.025±0.157

2.0

2.5

625

+

4/20

7.0

200

0.020±0.140

1.0

2.0

1250

+

4/20

6.9

200

0.035±0.184

3.0

3.5

2500

+

4/20

7.4

200

0.030±0.222

2.0

2.0

5000

+

4/20

7.7

200

0.010±0.100

2.5

1.0

CP (10)

+

4/20

2.3

200

0.950±1.591

2.5

45.5*

Vehicle (Ethanol)

-

20/20

7.6

200

0.020±0.140

1.0

2.0

625

-

20/20

6.4

200

0.015±0.158

1.5

1.0

1250

-

20/20

5.5

200

0.025±0.186

2.0

2.0

2500

-

20/20

6.0

200

0.025±0.157

2.5

2.5

5000

-

20/20

6.2

200

0.020±0.172

2.0

1.5

MMC (0.08)

-

20/20

5.8

200

0.220±0.513

1.0

18.0*

MMC = Mitomycine C

CP = Cyclophosphamide

* = p≤0.01; Fisher´s exact test

The test substance did not cause chromosome aberrations unter the conditions tested.

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 June 2013 to 31 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
The thymidine kinase, TK +1-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ug/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ug/ml) and 10% donor horse serum (giving R10 media) at 37 oC with 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared in-house from the livers of male Wistar Han™ rats weighing -200g. These had each received, orally, three consecutive daily doses of phenobarbitall~-naphthoflavone(80/100 mg per kg per day) prior to S9 preparation on the fourth day.
Test concentrations with justification for top dose:
Experiment 1 (ug/ml) without S9: 0, 9.75, 19.5, 39, 78, 156, 312 ug/ml
Experiment 1 (ug/ml) with S9: 0, 9.75, 19.5, 39, 78, 156, 312 ug/ml
Experiment 2 (ug/ml) without S9: 0, 9.75, 19.5, 39, 78, 156, 312 ug/ml
Experiment 2 (ug/ml) with S9: 0, 9.75, 19.5, 39, 78, 156, 312 ug/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Insoluble in water and DMSO but soluble in acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
400 ug/ml in 4 hour exposure, 150 ug/ml in 24 hour exposure
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
2 ug/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ~g/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ~g/ml) and 10% donor horse serum (giving R10 media). Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The 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 cells were exposed to doses of the test material, vehicle and positive control, both with and without metabolic activation. Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air.
The treatment regimes were as follows:
EXPERIMENT 1:
a). Without metabolic activation: 4-hour exposure, dose levels 0, 9.75, 19.5, 39, 78, 156, 312 ug/ml
b). With metabolic activation (2% S9): 4-hour exposure groups, dose levels 0, 9.75, 19.5, 39, 78, 156, 312 ug/ml
EXPERIMENT 2:
a). Without metabolic activation: 24-hour exposure, dose levels 0, 9.75, 19.5, 39, 78, 156, 312 ug/ml
b). With metabolic activation (1% S9):4-hour exposure, dose levels 0, 9.75, 19.5, 39, 78, 156, 312 ug/ml

DURATION
- Preincubation period: Not applicable.
- Exposure duration: 4 h (Experiment 1 and 2), or 24 h (Experiment 2).
- Expression time (cells in growth medium): 2 days (viability test)
- Selection time (if incubation with a selection agent): 10~14 days (plate scoring for colony formation)
- Fixation time (start of exposure up to fixation or harvest of cells): ~ 2 h


SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): Not applicable


NUMBER OF REPLICATIONS: Duplicate


NUMBER OF CELLS EVALUATED: seeded 2000 cells/well for mutant frequency; 2 cells/well for viability.


DETERMINATION OF CYTOTOXICITY
- Method: other: Suspension Growth values (SG)


OTHER EXAMINATIONS:
- Determination of polyploidy: No.
- Determination of endoreplication: No.
- Other:


OTHER: Calculation of Day 2 Viability (%V), Calculation of Relative Total Growth (RTG), Calculation of Mutation Frequency (MF) were performed, and the experimental data was analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.
Evaluation criteria:
The normal range for mutant frequency per survivor i s 50-170 x 10*6 for the TK+/- locus in L5178Y cells at this 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 normallyacceptable and will be repeated.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
The Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10*6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that i s greater than the corresponding vehicle control by the GEF of 126 x 10*6 and demonstrates a positive linear trend will be considered positive.
Statistics:
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Overall, precipitate of the test item was observed at and above 39.06 ug/nil. A greasy / oily precipitate was also observed at and above
312.5 ug/ml in the 24-hour exposure group in the absence of metabolic activation. With no evidence of any significant test item-induced
toxicity, the maximum dose level in the subsequent Mutagenicity Test was limited by the onset of greasy / oily precipitate.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. 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 at any of the dose levels in either the absence or presence of metabolic activation. Overall, precipitate of the test item was observed at and above 39 pg/ml.

Conclusions:
Interpretation of results (migrated information):
negative

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

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locu s of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9 final concentration). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9 final concentration) and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item was selected following the results of a preliminary toxicity test and for Experiments 1 and 2 was 9.75 to 312 ug/ml in both the absence and presence of metabolic activation. With no evidence of significant toxicity, the maximum dose level used in the Mutagenicity Test was limited by the onset of greasy / oily precipitate. Overall, precipitate of the test item was observed at and above 39 ug/ml in the Mutagenicity Test. The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any toxicoiogically significant dose-related increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or second experiment. The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
March 26th, 2010 - June 08th 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance CAS 85186-89-6. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
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
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
mutation at the autosomal thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:RPMI 1640 supplemented with 5% (v/v) heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
First experiment: 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/ml (with and without metabolic activation (8%, v/v))
Second experiment: 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/ml (with and without metabolic activation (12%, v/v))
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in the presence of S9-mix Migrated to IUCLID6: 15 and 5 µg/ml for 3 and 24 h treatment period
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
in the absence of S9-mix Migrated to IUCLID6: 7.5 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: No
- Exposure duration: cells were exposed to the test material for 3 h and 24 h in the presence and absence of S9-mix, respectively.
- Expression time (cells in growth medium): Cells in the final suspension after treatment were counted with the coulter particle counter. For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. Cells were plated for the determination of the cloning efficiency and mutation frequency. For the determination of the mutation frequency cells were plated and incubated for 11-12 d. After that, cells were stained for 2 h by adding 0.5 mg/ml MTT (Sigma) to each well. The plates were scored for cloning efficiency and mutation frequency with the naked eye or with the microscope.

SELECTION AGENT (mutation assays): RPMI 1640 supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/ml trifluorothymidine (TFT).

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
Measurement of cytotoxicity by determining the relative cloning efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period.
There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency.
Statistics:
The cloning efficiency (CE) was determined as follows:
P(0)= Number of empty wells divided by the total number of wells
CE= P(0)/number of cells plated per well

Relative survival rate (RS): RS= [CE(test)/CE(control)] x 100
Relative total growth (RTG): RTG= RSG x RSday2 / 100
Suspension growth (SG): [Day 0 cell count/1.25x10exp5] x [Day 1 cell count/1.25x10exp5] x [Day 2 cell count]
Relative suspension growth (RSG): SG(test)/SG(control) x 100

RSday2= CEday2(test) / CEday2(control) x 100

The growth rate (GR) was calculated for the solvent control cultures:
- 3 h treatment: [Day 1 cell count/1.25x105] x [Day 2 cell count/1.25x10exp5]
- 24 h treatment: [Day 0 cell count/1.25x105] x [Day 1 cell count/1.25x1exp05] x [Day 2 cell count/1.25x10exp5]


The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:

MF= {-ln P(0)/number of cells plated per well)/CE day2 x 10exp-6

Small and large colony mutation frequencies were calculated in an identical manner.

Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at and above 333 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 333 µg/mL

RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity data was obtained by treating cells for 3 h and 24 h respectively with a number of increasing test substance concentrations. The highest concentration tested was 750 µg/ml due to poor solubility of the test substance. No toxicity was observed with and without metabolic activation up to and at the maximum dose level tested with 3 h or 24 h incubation.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all controls were in the range of the historical controls
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10-6

 

 

 

 

 

total

Without metabolic activation, 3 h treatment

SC1

100

104

100

100

74

SC2

85

97

0.3

99

98

104

102

74

1

101

102

108

109

71

3

100

101

107

107

94

10

93

98

104

97

67

33

120

94

100

120

63

100

113

101

107

121

61

333*

104

113

120

124

64

750*

405

101

107

112

74

MMS

71

68

72

51

835

With 8% (v/v) metabolic activation, 3 h treatment

SC1

100

70

100

100

65

SC2

69

64

0.3

96

60

86

83

74

1

115

68

98

113

60

3

109

40

57

62

84

10

127

72

104

132

52

33

114

46

66

75

84

100

122

76

108

133

63

333*

115

62

89

102

72

750*

104

58

84

87

53

CP

50

32

45

22

1617

 

 

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10-6

 

 

 

 

 

total

Without metabolic activation, 24 h treatment

SC1

100

66

100

100

90

SC2

79

75

0.3

112

77

106

119

88

1

116

80

110

128

82

3

117

72

100

117

79

10

120

85

117

140

66

33

114

74

101

116

83

100

121

69

95

115

83

333*

116

70

97

112

70

750*

116

66

91

106

71

MMS

101

49

67

68

1502

With 12% (v/v) metabolic activation, 3 h treatment

SC1

100

93

100

100

80

SC2

93

76

0.3

103

84

90

93

74

1

113

83

89

101

81

3

107

97

104

112

60

10

105

94

101

107

80

33

103

93

100

103

67

100

102

105

114

116

57

333*

106

91

99

104

74

750*

103

93

100

103

73

CP

72

75

81

58

1082

 

RSG: Relative Suspension Growth; CE: Cloning efficiency; RS: Relative Survival; RTG: Relative Total Growth; SC: Solvent Control (DMSO); MMS: Methylmethansulfonate; CP: Cyclophosphamide

*: Precipitation of test substance

It is concluded that Pentaerythritol tetravalerate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

Conclusions:
Interpretation of results (migrated information):
negative
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)
Rationale for reliability incl. deficiencies:
other: analogue substance
Justification for type of information:
Bacterial gene mutation in vitro does not need to be investigated because available data indicate that structural variation does not influence test results or adverse effect profile (see read-across and category justifications 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:
valid
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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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)
Rationale for reliability incl. deficiencies:
other: analogue substance
Justification for type of information:
Chromosome aberration in vitro does not need to be investigated because available data indicate that structural variation does not influence test results or adverse effect profile (see read-across and category justifications attached in Section 13).
Reason / purpose:
read-across source
Species / strain:
other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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)
Rationale for reliability incl. deficiencies:
other: analogue substance
Justification for type of information:
An in vitro mouse lymphoma assay does not need to be conducted because available data indicate that structural variation does not influence test results or adverse effect profile (see read-across and category justifications 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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read-across to the properties of Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester (EC 613-848-7, CAS 65870-94-2), Fatty acid polyols (Fatty acids, C5-9, esters with pentaerythritol (EC 270-290-3, CAS 68424-30-6) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)) and their analogues is applicable based on the similarity in structure and physico-chemical properties. The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Supp, CAS 68424-31-7 Croda, Griffiths, 1992, CTL/P/3792, Micronuc., - not genotoxic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
May 13th, 1992 - July 08th, 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance CAS 68424-31-7. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
: Few details on test compound
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding , Margate, Kent, UKLaboratories
- Age at study initiation: 5-9 weeks for phase I and 7-9 weeks for phase II of the study
- Assigned to test groups randomly: Yes
- Housing:5 per cage in mobile mouse cage racks
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): 25
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no. (if required): Y00790/004
Details on exposure:
Route = intraperitoneal

Dosing solutions of the test material were prepared in corn oil, the cyclophosphamide was prepared in physiological saline. The vehicle control, the test substance or cyclophosphamide was administered as a single intraperitoneal injection.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
24 h and 48 h
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 65 mg/kg in physiological saline
Tissues and cell types examined:
Monochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
No deaths or severe adverse effects occurred in Phase I of the study with doses up to 5000 mg/kg. This dose was selected as MTD.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 h and 48 h after dosing


DETAILS OF SLIDE PREPARATION: Bone Marrow smears were stained with polychrome methylene blue and eosin


METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were evaluated for micronuclei per slide. In addition, 1000erythrocytes were counted to determine the percentage of polychromatic erythrocytes in the total erythrocyte population.


OTHER:
Evaluation criteria:
Increase in the incidence of micronucleated polychromatic erythrocytes in any sex or at any time point.
Percentage of polychromatic erythrocytes.
Statistics:
The incidence of micronucleated polychromatic erythrocytes and percentage of polychromatic erythrocytes in the erythrocyte sample were considered by analysis of variance regarding each combination of sampling time, dose level and sex as a separate group. Results were examined to determine weather any differences between vehicle control and test substance treated groups were consistent between sexes and across sampling times.
Each group mean was compared with the vehicle control group mean at the corresponding sampling time using a one-sided Student´s t-test based on the error mean square in the analysis.
Sex:
male/female
Genotoxicity:
negative
Remarks:
except for a small but significant decrease of polychromatic erythrocytes in male mice at 5000 mg/kg
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Mean incidence of micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes ±SD at two sampling times. n=5

 

Males

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

0.8 ± 0.8

1.0 ± 1.2

12

Cyclophosphamide

65 mg/kg

24.4 ± 6.0

 

13

Test substance

5000 mg/kg

0.6 ± 0.6

0.4 ± 0.6

 

Female

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

0.2 ± 0.5

1.4 ± 1.1

12

Cyclophosphamide

65 mg/kg

18.4 ± 7.3

 

13

 Test substance

5000 mg/kg

0.4 ± 0.9

0.4 ± 0.9

 

 

Mean percentage of polychromatic erythrocytes ±SD at two sampling times. n=5

 

Males

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

48.0 ± 5.6

44.3 ± 7.5

12

Cyclophosphamide

65 mg/kg

41.4 ± 4.4

 

13

 Test substance

5000 mg/kg

42.2 ± 7.0

43.3 ± 1.9

 

Female

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

41.9 ± 4.8

41.9 ± 1.7

12

Cyclophosphamide

65 mg/kg

45.9 ± 3.49

 

13

 Test substance

5000 mg/kg

46.5 ± 5.8

48.0 ± 5.2

Under the conditions tested, the test substance is not clastogenic in the mouse marrow micronucleus test.

Conclusions:
Interpretation of results (migrated information): negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

There are three key in vitro studies on an analogue test item:

Gene Mutation Assays

A Bacterial Reverse mutation Assay (Ames test) was performed according to the OECD 471 test guideline with an analogue substance (EC 613 -848 -7). The study (2013) was selected as a key study. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains in both experiments, with any dose of the test material, either with or without metabolic activation. The study results indicate that the substance does not induce gene mutations in bacteria, whereas all positive control chemicals (with and without metabolic activation) induced a significant increase in the number of colonies. The substance is therefore considered as non-mutagenic in the Ames test.

 

A mammalian cell gene mutation assay in L5178Y cells was performed according to the OECD 476 test guideline with an analogue substance (EC 613 -848 -7). The study (2013) was selected as a key study. No significant increase in the frequency of mutant colonies was recorded, either in the absence or presence of metabolic activation. The substance does not induce gene mutations in L5178Y cells under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of mutations.The substance is therefore considered as negative for inducing gene mutations in L5178Y cells under the activation and non-activation conditions used in this assay.

 

Chromosomal aberration

The clastogenic potential of the substance was determined using ananalogue substance (EC 613 -848 -7) and an in vitro chromosome aberration test in Human Lymphocyte cells, which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured cells. None of the test substance dose levels, up to the precipitation limit, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The substance does not induce structural aberrations in the chromosomes of human lymphocyte cells under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.The substance is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under the activation and non-activation conditions used in this assay.

 

Read-across to the toxicological properties of fatty acid polyols (Fatty acids, C5-9, esters with pentaerythritol (EC 270-290-3, CAS 68424-30-6) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)) and their analogues is applicable based on the similarity in structure and physico-chemical properties. The substances with the CAS No. 131459-39-7, 85186-89 -6 and 68424 -31-7 are structural analogues of the read-across substances and can therefore be used for read-across. The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

 

Genetic toxicity in vitro

Data from supporting studies using structural analogues, conducted according to OECD Guideline 471 (Bacterial Reverse Mutation Test), OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test), and OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) were also used for read-across.

 

In an Ames test conducted with Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6), Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E.coli WP2 uvr A were treated according to OECD Guideline 471 (Bailey, 1996, Ames). The test substance was diluted in ethanol and test substance concentrations of 0, 10, 33, 100, 333 and 1000 µg/plate were tested in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). Precipitation of the test substance was observed at and above 100 µg/plate. The test material caused no cytotoxicity up to the highest, precipitating dose. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

An in vitro mammalian chromosome aberration test was performed in chinese hamster ovary (CHO) cells with Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6) (Gudi, 1996, CA). The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0 (Vehicle control), 157, 313, 625, 1250, 2500, 5000 µg/mL diluted with ethanol. Cytotoxicity of 48% was observed in the highest dose for treatment of 4 h without metabolic activation, 33% with metabolic activation and 3% for 20 h exposure without metabolic activation. The test substance did not induce an increase in the number of metaphases with aberrations at any preparation time and dose level. Positive control substances significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. In conclusion, the test substance did not induce chromosome aberrations in CHO cells, neither in the presence nor in the absence of a metabolic activation system, under experimental conditions.

 

An in vitro mammalian chromosome aberration test was performed with CAS No. 131459-39-7 (3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid) in human lymphocytes (Wright, 1999, CA). The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0, 312.5, 625, 1250, 2500, 5000 µg/mL diluted with acetone. The test substance did not induce a significant increase in the number of metaphases with aberrations at any preparation time and dose level. No relevant cytotoxic effects were reported. Precipitation of the test substance was observed at and above 1250 µg/ml. Positive controls significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. In conclusion, the test substance did not induce chromosome aberrations in human lymphocytes, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.

 

An in vitro Mammalian Cell Gene Mutation Test was conducted with the structural analogue substance CAS No. 85186-89-6 (Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane) according to OECD Guideline 476. Mouse lymphoma L5178Y cells were dosed with 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/ml with and without metabolic activation. No signs of toxicity were observed, DMSO was used as vehicle, precipitation was observed at and above 100 µg/ml. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, indicated by the total number of colonies per plate (Verspeek-Rip, 2010, gene mut in mamm cells).

 

Genetic toxicity in vivo

Negative results were obtained in key studies using a close analogue of the registered substance (EC 613-848-7) during investigation of in vitro genetic toxicity. The Ames test, chromosome aberration test and mouse lymphoma assay were all negative. 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.

In a supporting study, Fatty acids, C5-10, esters with pentaerythritol (CAS No. 68424-31-7) were found to be not genotoxic in the micronucleus assay in vivo after intraperitoneal application. A single intraperitoneal injection was given to groups of 5 male and 5 female mice at a dose level of 5000 mg/kg bw. Bone marrow samples were taken 24 and 48 hours after dosing. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times.

Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is, however, considered not to be biologically significant compared to the concurrent control values.

The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen (Griffiths, 1992, CTL/P/3792, Micronuc.).

 

Considering the clearly negative results of the in vitro and in vivo experiments used as read-across and the proven physiological metabolism of fatty acids and excretion of polyols, it can be assumed that the registered substance (EC 939 -415 -5) is not genotoxic, in vitro or in vivo.


Justification for classification or non-classification

According to CLP criteria for classification and labelling of substances the analogue test item (EC 613 -848 -7) used in key studies is not classified as mutagenic.

Harmonized classification:

The analogue substance used in key studies has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification is proposed for the registered substance (EC 939 -415 -5) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).