Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic or 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:
2017 October 11 - 2018 January 30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Guideline study under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Remarks:
Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire DE72 2GD UK
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0000150418
- Expiration date of the lot/batch: 14 August 2018
- Purity test date: Not reported

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Expected to be stable
- Solubility and stability of the test substance in the solvent/vehicle: Not reported
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS:
Target gene:
histidine for the S. typhimurium strains, and tryptophan for E. coli.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S-9 liver fraction (10% v/v), from male rats induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
The test item was tested using the following method. The maximum concentration was 5000 ug/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
DMSO, FIsher Scientific, batch 1714907, >99% pure
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
concurrent
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (first test) and preincubation (second test)
- Cell density at seeding (if applicable): 0.1 ml of a 10-h bacterial culture (having a density of at least 10E9/mL)

DURATION
- Preincubation period: 0.5 h
- Exposure duration: 48-72 h for plate incorporation; preincubation for 30 m before plating in top agar
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells): cells not fixed

SELECTION AGENT (mutation assays): The Ames assay employs, as an indicator of mutation, observable (and countable) growth (colonies) in agar deficient in histidine. Colonies of bacteria represent mutants which have back-reverted to a histidine auxotroph. An automated colony counter is used.


NUMBER OF REPLICATIONS: 3 (triplicate)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: reduction in bacterial lawn

Rationale for test conditions:
The first plate incorporation test included 7 concentrations, in triplicate. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and phosphate buffer. All plates were incubated at 34 to 39°C for 48-72 hours. After this period, the appearance of the background bacterial lawn were examined and revertant colonies counted using an automated colony counter. Colonies were counted manually if automated counting is not possible (e.g. if dense precipitate is present).
Any toxic effects of the test item were detected as thinning or absence of the background lawn of non-revertant colonies, and/or reduction in revertant colony numbers to ≤ 50% of the concurrent vehicle control count. In the absence of any toxic effects the maximum concentration used in the second test were the same as that used in the first. If toxic effects were observed at more than one concentration, a lower concentration was chosen. A minimum of four non-toxic concentrations were obtained. If this was not achieved then the first test was repeated using a more appropriate concentration range. If a negative or equivocal response was obtained a variation on the above procedure will be used, with a minimum of five concentrations of test item used.
If the required number of non-toxic concentrations was not obtained, the second test was repeated using a more appropriate concentration range. It may also have been repeated to confirm a positive response or to confirm a dose-response.
Evaluation criteria:
For a test material to be classified as toxic, the test item must cause a reduction in the number of spontaneous revertants (below a factor of 0.5 fold under the concurrent solvent control) and/or the bacterial lawn should exhibit evidence of thinning when viewed microscopically.

For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range of the laboratory unless otherwise justified by the Study Director.

The positive control compounds must produce an increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls.

Criteria for a positive result (any, one, or all of the following may be used to determine the overall results of the study, weighing the results in terms of their biological significance. The criteria are listed in order of priority:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase in mean revertant colony numbers at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Fold increase greater than two times thef the concurrent solvent control for any tester strain (especially if accompanied by an out-of historical range response (Cariello and Piegorsch, 1996).
5. Statistical analysis of data as determined by the UKEMS (Mahon et al., 1989)

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, the test data should be evalueated by expert judgment and or further possible investigations.

Fold-increase: (three times in the case of strains TA1535 and TA1537) those of the concurrent vehicle controls.
Statistics:
The automated scoring system (Delta Building Monitoring System, Ames Study Manager and Sorcerer Imaging System) automatically conducts statistical analysis during the scoring process using Dunnetts Regression Analysis. Statistical significance will be confirmed for those values that indicate statistically significant increases in the frequency of revertant colonies compared with the concurrent solvent control (* = p < 0.05). Values that the program concludes are statististically significant but are within the in-house historical vehicle/untreated control range will not be reported.
See (Mahon et al, 1989, Analysis of data from microbial colony assays in: Kirkland, D.J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part III. Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, Cambridge, pp.26-65.).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Representative results:

Table 1            Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

72

 

29

 

24

 

18

 

10

 

98

(93)

25

(28)

18

(19)

18

(17)

9

(9)

108

 

29

 

15

 

14

 

9

 

Table 2            Test Results: Experiment 1 – Without Metabolic Activation(Plate Incorporation)

Test Period

From: 14 November 2017

To: 17 November 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

96

98

76

(90)

12.2#

23

38

21

(27)

9.3

32

24

16

(24)

8.0

21

21

15

(19)

3.5

9

9

12

(10)

1.7

1.5 µg

108

97

95

(100)

7.0

40

24

26

(30)

8.7

29

20

22

(24)

4.7

9

9

13

(10)

2.3

14

8

13

(12)

3.2

5 µg

94

83

90

(89)

5.6

33

24

23

(27)

5.5

19

26

26

(24)

4.0

21

20

19

(20)

1.0

14

13

13

(13)

0.6

15 µg

87

88

82

(86)

3.2

42

33

25

(33)

8.5

29

25

23

(26)

3.1

19

11

12

(14)

4.4

8

13

12

(11)

2.6

50 µg

93

89

91

(91)

2.0

42

33

38

(38)

4.5

23

24

23

(23)

0.6

19

19

18

(19)

0.6

15

12

15

(14)

1.7

150 µg

89

92

98

(93)

4.6

25

33

30

(29)

4.0

33

21

22

(25)

6.7

20

19

16

(18)

2.1

15

11

14

(13)

2.1

500 µg

77

89

89

(85)

6.9

28

31

30

(30)

1.5

25

25

23

(24)

1.2

21

25

24

(23)

2.1

12

13

11

(12)

1.0

1500 µg

80

92

87

(86)

6.0

28

40

25

(31)

7.9

27

24

16

(22)

5.7

19

16

12

(16)

3.5

9

10

13

(11)

2.1

5000 µg

81 F

74 F

79 F

(78)

3.6

32 F

18 F

13 F

(21)

9.8

24 F

22 F

16 F

(21)

4.2

19 F

27 F

10 F

(19)

8.5

15 F

2 F

6 F

(8)

6.7

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

665

538

606

(603)

63.6

465

450

489

(468)

19.7

848

735

822

(802)

59.2

160

139

163

(154)

13.1

540

324

395

(420)

110.1

Table 3            Test Results: Experiment 1 – With Metabolic Activation(Plate Incorporation)

Test Period

From: 14 November 2017

To: 17 November 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

84

114

116

(105)

17.9#

22

36

28

(29)

7.0

27

26

25

(26)

1.0

34

19

35

(29)

9.0

12

18

14

(15)

3.1

1.5 µg

106

93

93

(97)

7.5

30

25

32

(29)

3.6

25

25

31

(27)

3.5

15

19

21

(18)

3.1

19

5

12

(12)

7.0

5 µg

105

98

105

(103)

4.0

25

32

35

(31)

5.1

30

32

26

(29)

3.1

26

30

27

(28)

2.1

13

19

8

(13)

5.5

15 µg

103

91

96

(97)

6.0

27

21

21

(23)

3.5

25

26

28

(26)

1.5

17

21

19

(19)

2.0

14

19

13

(15)

3.2

50 µg

97

98

102

(99)

2.6

30

20

30

(27)

5.8

26

25

28

(26)

1.5

18

15

16

(16)

1.5

16

19

12

(16)

3.5

150 µg

92

106

112

(103)

10.3

40

23

28

(30)

8.7

28

36

25

(30)

5.7

12

23

25

(20)

7.0

12

15

9

(12)

3.0

500 µg

116

94

96

(102)

12.2

25

23

26

(25)

1.5

30

28

30

(29)

1.2

22

22

14

(19)

4.6

10

10

11

(10)

0.6

1500 µg

94

113

90

(99)

12.3

30

22

20

(24)

5.3

28

30

26

(28)

2.0

26

28

23

(26)

2.5

14

8

10

(11)

3.1

5000 µg

103 F

97 F

113 F

(104)

8.1

15 F

17 F

17 F

(16)

1.2

18 F

26 F

18 F

(21)

4.6

19 F

11 F

24 F

(18)

6.6

4 F

12 F

7 F

(8)

4.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

2395

2473

2494

(2454)

52.2

276

298

268

(281)

15.5

181

175

212

(189)

19.9

194

184

202

(193)

9.0

338

346

365

(350)

13.9

ENNG     N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO     4-Nitroquinoline-1-oxide

9AA        9-Aminoacridine

2AA   2 -Aminoanthracene

BP           Benzo(a)pyrene

Conclusions:
The substance was tested in a guideline Ames Assay (OECD 471) and found to be non-mutagenic. The substance does not meet the criteria for classification as a mutagen according to Regulation EC No. 1272/2008.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
March 26th, 2010 - June 08th 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The analogue approach is used for the hazard assessment of toxicological endpoints for the registration of the target substance pentaerythritol ricinoleate (CAS 78-22-8) based on generation of different breakdown/metabolic products, resulting not only in similar physical and biological systems (Scenario 2 of the Read-Across Assessment Framework (RAAF, ECHA, 2015), but also consequently in similar physico-chemical and toxicological properties. The source compounds for read-across are fatty acids, C16-18, esters with pentaerythritol (CAS 85116-93-4) and trimethololpropane ricinoleate (CAS 67025-99-4). It is proposed that the different alcohols resulting from ester hydrolysis of the source compounds and the target substance will not result in significant variation in biological effects.
Neither target nor source compounds are classified for mammalian hazardous effects. The use of reliable experimental data, all evaluated as reliable according to Klimisch scores of 1 or 2, to estimate the toxicity of the registered substance is adequate for the purposes of fulfilling the data requirements of registration and classifying potential hazards. Similar grouping into categories has been accepted by other regulatory agencies (U.S. EPA, 2010; U.S. FDA for food notifications). Thus, this read-across approach is adequate for the purposes of risk assessment and classification and labeling.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
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.

Key result
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: negative all strains/cell types tested

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: negative. These data are relevant to the registered (target) substance to fill the data requirements of REACH.
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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The analogue approach is used for the hazard assessment of toxicological endpoints for the registration of the target substance pentaerythritol ricinoleate (CAS 78-22-8) based on generation of different breakdown/metabolic products, resulting not only in similar physical and biological systems (Scenario 2 of the Read-Across Assessment Framework (RAAF, ECHA, 2015), but also consequently in similar physico-chemical and toxicological properties. The source compounds for read-across are fatty acids, C16-18, esters with pentaerythritol (CAS 85116-93-4) and trimethololpropane ricinoleate (CAS 67025-99-4). It is proposed that the different alcohols resulting from ester hydrolysis of the source compounds and the target substance will not result in significant variation in biological effects.
Neither target nor source compounds are classified for mammalian hazardous effects. The use of reliable experimental data, all evaluated as reliable according to Klimisch scores of 1 or 2, to estimate the toxicity of the registered substance is adequate for the purposes of fulfilling the data requirements of registration and classifying potential hazards. Similar grouping into categories has been accepted by other regulatory agencies (U.S. EPA, 2010; U.S. FDA for food notifications). Thus, this read-across approach is adequate for the purposes of risk assessment and classification and labeling.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
none
Species / strain / cell type:
lymphocytes: lymphocytes: Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation
Remarks:
primary culture
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced male Sprague-Dawley rat liver S9 mix containing MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphatebuffer (100 mM, pH 7.4)
Test concentrations with justification for top dose:
320 μg/mL, maximum solubility. Higher concentrations produced an oily liquid on the surfa
ce of the culture medium.
Vehicle / solvent:
acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 500 μL heparinized whole fresh blood in 4.5 mL complete cult
ure medium
DURATION : See table
ANALYSIS OF METAPHASE CELLS: Evaluation of the cultures is performed using microscopes with
100x oil immersion objectives.
SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.3 μg/ml
STAIN: 5% Gurrs Geimsa
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in
mammalian cells): At least 300
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The cultures were harvested by centrifugation 24 h after beginning of treatment. The supernatant was
discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.4 % KCl). The
cell suspension is incubated at room temperature for 20 min. After removal of the hypotonic solution
by centrifugation the cells are fixed with 3:1 methanol :glacial acetic acid. The fixative was changed at
least three times and the cells stored at 4oC for at least four hours to ensure complete fixation.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index. 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.
- Any supplementary information relevant to cytotoxicity:
OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells is scored
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole
or fragmented chromosomes (if applicable): N/A
Rationale for test conditions:
The OECD Guideline for Testing of Chemicals Section 4, No 473 – “In Vitro Mammalian Chromo
some Aberration Test”, adopted 29 July, 2016 – recommends using a variety of cell lines or primary
cell cultures (e.g. Chinese hamster fibroblasts, human or other mammalian peripheral blood
lymphocytes).
Evaluation criteria:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted,
where there was approximately 50% of cells with aberrations, slide evaluation was terminated, at 50
cells.
All structural chromosomal aberrations lsuch as breaks, fragments, deletions, exchanges and
chromosomal disintegration are recorded. Gaps are recorded as well but not included in the calculat
ion of the aberration rates. The definition of a gap is as follows: an achromatic region (occurring in one
or both chromatids) independent of its width. The remaining visible chromosome regions should not
be dislocated either longitudinally or laterally.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive
if the % cells with aberrations, excluding gaps, markedly exceeds that seen in the concurrent control,
either with or without a clear dose-relationship. For modest increases in aberration frequency a dose
response relationship is generally required and appropriate statistical tests may be applied in order to
record a positive response.
Statistics:
Fisher's Exact test
Key result
Species / strain:
lymphocytes: primary culture
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: primary culture
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
When the solvent selection was performed it was noted that the test material formed a greasy oily pr
ecipitate at and above 312.5 μg/ml, and a cloudy precipitate at 156.25 μg/ml. With the test material
producing a greasy oily precipitate at and above 312.5 μg/ml, it was considered that maximum test
material exposure to the cells would occur at dose levels below this level. Therefore it was consi
dered that the maximum dose level would be limited to 320 μg/ml. This was later increased slightly to
400 μg/ml.
Conclusions:
The test substance was evaluated for potential to cause chromosome aberrations using an in vitro
Chromosome Aberration test according to OECD 473 in human lymphocytes at concentrations up
to the limit of solubility (400 μg/ml). No increase in the incidence of chromosome breaks, gaps or ot
her aberrations were observed. The test substance was determined to be non-clastogenic under the
GLP conditions of this assay. These data are applicable to the target (registered) substance and fill the data requirements of REACH.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable

Additional information

Justification for classification or non-classification

No adverse effects were found in genotoxicity studies on the substance(s). The criteria for classification of Regulation EC No. 1272/2008 are not met.