Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471, GLP): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 100 and TA 98, and E. coli WP2 uvrA

Read-across from structural analogue source substances Dipentaerythritol hexaesters with fatty acids, C5 and C9iso (CAS No. 647028-25-9), Fatty acids, C16-18 (even numbered), esters with pentaerythritol (CAS No. 85116-93-4) and Fatty acids, C8-10 mixed esters with dipenaterythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8)

Chromosome aberration (OECD 473, GLP): negative in cultured peripheral human lymphocytes with and without metabolic activation

Read-across from structural analogue source substances Dipentaerythritol hexaesters with fatty acids, C5 and C9iso (CAS No. 647028-25-9) and Fatty acids, C8-10 mixed esters with dipenaterythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8)

Gene mutation in mammalian cells (OECD 476, GLP): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Read-across from structural analogue source substance Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane (CAS No. 85186-89-6)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 Feb - 15 Apr 1999
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)
Version / remarks:
adopted in 1997
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media:
Eagle’s MEM medium supplemented with:
-sodium bicarbonate
-HEPES buffer
-L-glutamine
-penicillin/streptomycin
-amphotericin B
-15% foetal calf serum (FCS)

Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
First experiment:
4(20) h without and with S9: 39.06; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 µg/mL

Second experiment:
20 (20) h without S9: 156.25; 312.5; 625; 1250; 2500 and 5000 µg/mL
4(20) h with S9: 156.25; 312.5; 625; 1250; 2500 and 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:
other: cyclophosphamide, 25 µg/mL in phopsphate buffered saline, +S9; ethyl methanesulphonate , 750 µg/mL and 500 µg/mL (Experiment 1 and 2 respectively), -S9 in dimethyl sulphoxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (experiment 1, with and without S9); 4 and 20 h (experiment 2, with and without S9, respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 20h

SPINDLE INHIBITOR (cytogenetic assays): colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): Gurrs Giemsa R66 5%

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded 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:
The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test or Chi-squared test.
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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: there was no observable change in pH
- Effects of osmolality: the osmolality did not increase by more than 50 mOSM
- Precipitation: a cloudy appearance of the test material was noted at all dose levels in both treatment groups, after four hours exposure

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: in Experiment 1 and 2 no dose-related toxicity was observed, a 50% mitotic inhibition was not achieved and the precipitate of the test item had no effect on the toxicity response curve. Thus, 1250, 2500 and 5000 µg/mL dose levels were selected for chromosome analysis.

Table 1: Test results of experiment 1

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

Exposure period 4 h, fixation time 20 h, without S9 mix

Acetone

100

3.0

2.5

EMS

750

50

20

12.5

Test substance

1250

78

3.0

2.0

2500

83

5.5

1.0

5000

70

2.0

1.5

Exposure period 4 h, fixation time 20 h, with S9 mix

Acetone

100

3.5

2.0

CP

25

29

14.5

9.0

Test substance

1250

87

3.0

3.0

2500

125

3.5

0.5

5000

117

1.5

1.0

EMS: ethyl methanesulphonate;

CP: Cyclophosphamide (positive controls)

 

Table 2: Test results of experiment 2

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

Exposure period 20 h, fixation time 20 h, without S9 mix

Acetone

0

100

1.5

0.5

EMS

750

58

36.7

20.0

Test substance

1250

123

2.0

0.5

2500

110

0.5

0.0

5000

99

2.0

0.0

Exposure period 4 h, fixation time 20 h, with S9 mix

Acetone

0

100

2.0

0.5

CP

25

25

19.0

11.5

Test substance

1250

89

1.5

0.0

2500

89

1.0

0.0

5000

112

1.0

0.0

EMS: ethyl methanesulphonate;

CP: Cyclophosphamide (positive controls)

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 Dec 1998 - 19 January 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EU MEthod B.14 of Commission Directive 92/69/EEC
Qualifier:
according to
Guideline:
other: USA, EPA (TSCA) OPPTS harmonised guidelines
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Type of assay:
bacterial reverse mutation assay
Target gene:
"his operon" (for S. typhimurium strains) and "trp operon" (for E.coli strains)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers treated with Aroclor 1254
Test concentrations with justification for top dose:
Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
First and second experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
yes
Remarks:
untreated control plates
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Other: -S9: N-ethyl-N´-nitro-N-nitrosoguanidine (3, 5 and 2 µg/plate respectively) for TA100, TA1535 and WP2uvrA; 9-Aminoacridine (80 µg/plate) TA1537; 4-Nitroquinolone-1oxide (0.2 µg/plate) for TA98
Untreated negative controls:
yes
Remarks:
untreated control plates
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: +S9: 2 aminoanthracene 1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537, and 10 µg/plate for WP2uvrA; Benzo(a)pyrene (5 µg/plate) for TA98.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn


OTHER: a preliminary toxicity test was carried out in the strains TA100 and WP2uvrA.
Evaluation criteria:
The test material may be considered positive in this test if the following criteria are met: the test item to be considered mutagenic should have induced, a reproducibile, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Mean values and standard deviation were calculated. Dunnett’s method of linear regression was used.
Species / strain:
S. typhimurium, other: 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
Remarks:
oily precipitate was observed at 5000 µg/plate
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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
oily precipitate was observed at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: an oily precipitate was observed at 5000 µg/plate; however this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: The dose range for the main test was determined in a preliminary toxicity assay where the test material was tested at the following doses: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test material was non-toxic to the strains of bacteria used (TA 100 and WP2uvrA-).

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Furthermore positive control values were at least two times the respective vehicle control value for each strain. The historical control ranges are presented in Table 1.

Table 1: Historical control ranges

History profile-Vehicle control values

 

-S9

TA 100

TA 98

TA 1535

TA 1538

TA 1537

WP2uvrA

TA 102

Extreme values

61-196

13-56

10-39

6-41

4-17

12-39

216-339

Mean

117± 24.7

28± 6.7

24± 5.2

21± 6.8

10± 2.0

20± 4.6

264 29.8

                       History profile-Vehicle control values

+S9

TA 100

TA 98

TA 1535

TA 1538

TA 1537

WP2uvrA

TA 102

Extreme values

63-177

14-52

11-39

11-50

5-20

11-40

205-343

Mean

120± 22.2

33± 7.1

18± 4.0

25± 6.2

11± 2.3

21± 4.8

283± 32.7

                       History profile-Positive control values

-S9

TA 100

TA 98

TA 1535

TA 1538

TA 1537

WP2uvrA

TA 102

Extreme values

277-1126

127-698

163-1005

190-799

161-1149

342-1209

540-1188

Mean

613±175.7

203±57.2

466±208.4

484±146.0

812±201.3

834±197.2

785±172.1

+S9

TA 100

TA 98

TA 1535

TA 1538

TA 1537

WP2uvrA

TA 102

Extreme values

412-1315

198-757

139-516

212-915

123-718

225-1089

511-1090

Mean

949± 189.7

420± 112.5

291± 63.6

454± 131.0

266± 90.2

734± 210.5

705± 126.6

Table 2: Test Results of Experiment 1 (plate incorporation)

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 100

TA1535

WP2uvr A-

TA98

TA1537

0

81 ± 11.0

16 ± 1.5

25 ± 3.5

19 ± 2.5

7 ± 3.8

50

77 ± 2.1

14 ± 0.6

22 ± 0.6

24 ± 0.6

9 ± 3.6

150

80 ± 12.2

14 ± 1.0

22 ±0.6

20 ± 6.7

10 ± 3.8

500

83 ± 10.8

13 ± 2.3

23 ± 2.9

21 ± 6.0

7 ± 2.6

1500

76 ± 1.5

18 ± 5.2

18 ± 2.6

22 ± 1.2

7 ± 3.2

5000

90 ± 11.8 P

18 ± 8.2 P

24 ± 4.0 P

28 ± 4.9 P

5 ± 0.6 P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3)

323 ± 11.7

283 ± 10.0

503 ± 24.2

166 ± 5.3

1027 ± 358.2

 

 

TA 100

TA1535

WP2uvr A-

TA98

TA1537

+

0

105 ± 7.8

11 ± 1.2

28 ± 3.8

30 ± 3.1

20 ± 3.6

+

50

101 ± 7.0

12 ± 0.6

27 ± 3.8

29 ± 6.6

20 ± 3.2

+

150

102 ± 5.5

16 ± 4.0

32 ± 1.5

27 ± 4.0

23 ± 2.0

+

500

93 ± 16.6

14 ± 4.0

28 ± 6.6

30 ± 6.7

20 ± 1.2

+

1500

86 ± 3.6

15 ± 3.5

26 ± 4.0

31 ± 3.1

17 ± 6.5

+

5000

93 ± 7.8 P

12 ± 1.2 P

24 ± 1.5 P

33 ± 5.7 P

15 ± 2.6 P

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3)

920 ± 153.0

198 ± 9.3

555 ± 27.8

551 ± 132.0

196 ± 6.5

Table 3: Test Results of Experiment 2 (plate incorporation)

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates  

± SD)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvr A-

TA98

TA1537

0

96 ± 4.5

18 ± 4.2

26 ± 3.8

18 ± 5.3

11 ± 6.5

50

97 ± 18.6

17 ± 4.0

29 ± 6.5

16 ± 3.6

12 ± 0.6

150

100 ± 9.5

20 ± 9.0

28 ±0.0

16 ± 3.8

11 ± 1.7

500

101 ± 8.4

18 ± 6.2

33 ± 6.0

19± 4.4

12 ± 1.2

1500

87 ± 6.4

17 ± 7.1

29 ± 7.1

22 ± 0.0

12 ± 1.2

5000

93 ± 1.0 P

19 ± 3.1 P

33 ± 5.1 P

17 ± 3.2 P

15 ± 6.0 P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3)

880 ± 31.5

257 ± 34.4

1191 ± 59.3

166 ± 4.6

1084 ± 263.4

 

 

TA 100

TA1535

WP2uvr A-

TA98

TA1537

+

0

85 ± 4.0

14 ± 3.5

35± 1.2

31 ± 5.0

21 ± 1.5

+

50

91 ± 7.5

19 ± 0.7

35 ± 10.1

30 ± 8.0

24 ± 1.5

+

150

84 ± 9.5

14 ± 1.0

34 ± 6.1

29 ± 10.7

22 ± 3.5

+

500

101 ± 14.3

10 ± 0.6

42 ± 0.6

28 ± 4.5

22 ± 2.1

+

1500

94 ± 8.7

14 ± 0.6

36 ± 5.3

31 ± 4.7

21 ± 1.0

+

5000

101 ± 6.7 P

14 ± 3.2 P

34 ± 7.0 P

30 ± 5.5 P

22 ± 1.2 P

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3)

865 ± 43.2

246 ± 7.5

1051 ± 90.0

611 ± 51.5

182 ± 7.0

ENNG = N-Ethyl-N´-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

BP = Benzo(a)pyrene

2AA = 2-Aminoanthracence

P = precipitate

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Mar - 08 June 2010
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)
Version / remarks:
adopted in 1997
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:
TK 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:
co-factor supplemented post-mitochondrial fraction (S9-mix), prepared from rats pretreated with 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
Remarks:
+ S9: cyclophosphamide, 15 and 5 µg/mL for 3 and 24 h treatment, respectively; - S9: methylmethanesulfonate, 7.5 µg/mL
Positive control substance:
cyclophosphamide
methylmethanesulfonate
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): For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. For determination of the mutation frequency cells were plated and incubated for 11-12 days. 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:
Several criteria including a concentration-related, or a reproducible increase in mutation frequencies determined a positive 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

Table 1: Results of experiment 1

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10E6

 

 

 

 

 

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

 

Table 2: Results of experiment 2

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10E6

 

 

 

 

 

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

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23 Aug - 1 Sep 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restrictions (no E.coli strain or equivalent S. typhimurium strain was tested; lack of data on test substance, methodological deficiencies, no bacteria were added to the plates for tester strain TA1538 in the repeat experiment)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(no E.coli strain or equivalent S. typhimurium strain was tested; lack of data on test substance, methodological deficiencies, no bacteria were added to the plates for tester strain TA1538 in the repeat experiment
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of Sprague-Dawley rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
First experiment (Initial assay): 0.5, 5, 50, 500, 5000 µg/plate with and without metabolic activation
Second experiment (Repeat assay): 50, 100, 500, 1000, 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone for the test substance, DMSO for the positive control substances
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone, DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: 9-Aminoacridine (9AA; 100 µg/plate) for TA1537; N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG; 10 µg/plate) for TA100 and TA1535; 2-nitrofluorene (2NF; 5 µg/plate) for TA98 and TA1538; +S9: 2-Aminoanthracene (2AA; 2.5 µg/plate) for all strains
Remarks:
test substance was dissolved in acetone; positive control substances were dissolved in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Triplicates each in two independent experiments with one exception: in the repeat assay, inadvertently no TA 1538 bacteria were added to the plates at 50, 100 and 500 µg/plate without S9-mix.

DETERMINATION OF CYTOTOXICITY
- Method: notable reduction of the bacterial background lawn and/or a greater than 50% reduction in the mean number of revertants in comparison to the vehicle control
Evaluation criteria:
The test substance may be considered positive in this test system if the following criteria are met: For the test item to be considered mutagenic, three-fold (or more) increases in mean revertant numbers must be observed in comparison with vehicle control plates.
In addition, there must be evidence of a dose-related increase in the number of revertants over at least three concentrations of the test substance. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
A lack of response in the positive controls or spontaneous revertant frequencies which were not in keeping with historical laboratory values would render that portion of the test invalid.
Statistics:
Mean values and standard deviation were calculated.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was observed in the initial assay and the repeat assay.
- Other confounding effects: Beading of the test substance was observed in the initial assay and the repeat assay at 500 µg/plate and higher with and without metabolic activation in all strains. The only exception was the strain TA 100 in the repeat assay without metabolic activation, where a beading of the test substance was observed at 1000 and 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES: A range-finding test was conducted to determine the doses used for the main studies. Therefore, concentrations of the test substance of 0.5, 1, 5, 10, 50, 100, 500, 1000, 2000 and 5000 µg/plate were tested. Toxicity was not observed at any concentration tested. Precipitation of the test substance was observed at 1000 and 2000 µg/plate with metabolic activation and between 1 and 500 µg/plate without metabolic activation. Beading of the test substance was observed at 500 µg/plate and higher with metabolic activation and at 100 µg/plate and higher without metabolic activation.

Table 1: Test results of the initial assay

Bacterial Reverse Mutation Assay, mean revertants colonies/plate (mutation factor)

INITIAL ASSAY (Plate Incorporation Test)

S9-Mix

Without

 

Test item (µg/plate)

TA98

TA 100

TA 1535

TA 1537

TA 1538

Negative control

17.7

105.7

10.0

3.0

5.3

Acetone

17.3

97.0

6.3

3.3

8.0

0.5

14.7

98.7

11.7

5.0

5.7

5

10.0

109.0

12.3

6.7

8.0

50

14.3

101.3

7.7

4.3

7.7

500

18.7 B

122.3 B

12.3 B

3.3 B

4.0 B

5000

16.0 B

114.3 B

11.3 B

5.3 B

4.7 B

DMSO

13.0

112.0

9.3

4.0

3.3

9AA

---

---

---

674.0*

---

MNNG

---

945.0*

1089.7*

---

---

2NF

595.0*

---

---

---

826.7*

S9-Mix

 

With

Test item (µg/plate)

TA98

TA 100

TA 1535

TA 1537

TA 1538

Negative control

22.3

111.7

9.3

4.0

12.3

Acetone

20.3

119.7

11.7

6.3

7.0

0.5

18.3

126.0

12.3

4.7

9.3

5

17.0

124.3

7.3

6.0

13.0

50

15.7

129.0

11.0

5.0

5.3

500

25.7 B

121.7 B

10.7 B

4.3 B

9.7 B

5000

18.0 B

116.3 B

8.7 B

5.3 B

14.7 B

DMSO

13.7

105.0

12.0

5.3

13.7

2AA

338.3*

911.3*

113.7*

31.7*

471.3*

9AA: 9-aminoacridine (100 µg/plate)

MNNG: N-methyl-N-nitro-N-nitrosoguanidine (10 µg/plate)

2NF: 2-nitrofluorene (5 µg/plate)

2AA: 2 -aminoanthracene (2.5 µg/plate)

*, p<0.05; **, p<0.01

B: test substance beaded on the plate

Table 2: Test results of the repeat assay

Bacterial Reverse Mutation Assay, mean revertants colonies/plate (mutation factor)

REPEAT ASSAY (Plate Incorporation Test)

S9-Mix

Without

 

Test item (µg/plate)

TA98

TA 100

TA 1535

TA 1537

TA 1538

Negative control

14.0

120.0

9.3

4.7

7.0

Acetone

16.7

102.7

10.7

5.3

3.7

50

16.7

117.7

13.7

5.7

0#

100

15.7

108.3

10.0

7.3

0#

500

21.7 B

99.0

12.0 B

5.7 B

0#

1000

16.7 B

125.3 B

7.7 B

4.0 B

8.3 B

5000

15.0 B

112.3 B

9.0 B

3.3 B

4.3 B

DMSO

16.3

101.7

10.7

3.7

6.3

9AA

---

---

---

544.3*

---

MNNG

---

1644.3*

1341.3*

---

---

2NF

708.3*

---

---

---

1019.3*

S9-Mix

 

With

Test item (µg/plate)

TA98

TA 100

TA 1535

TA 1537

TA 1538

Negative control

24.0

108.0

7.3

6.0

16.0

Acetone

21.0

123.7

7.0

4.7

14.7

50

25.3

109.3

12.7

5.0

10.0

100

30.3

110.7

7.0

6.3

17.3

500

27.7 B

104.3 B

15.3 B

5.7 B

16.7 B

1000

24.7 B

110.3 B

9.7 B

11.0 B

19.3 B

5000

22.3 B

121.7 B

11.3 B

4.7 B

13.3 B

DMSO

17.3

110.0

9.0

7.0

14.0

2AA

270.3*

738.3*

70.0*

36.7*

241.3*

9AA: 9 -aminoacridine (100 µg/plate)

MNNG: N-methyl-N-nitro-N-nitrosoguanidine (10 µg/plate)

2NF: 2-nitrofluorene (5 µg/plate)

2AA: 2 -aminoanthracene (2.5 µg/plate)

*, p<0.05; **, p<0.01

B: test substance beaded on the plate

#: bacteria were inadvertently not added

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (shorter exposure period. Lack of data on test substance, no positive controls for 40 h time point)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 1997
Deviations:
yes
Remarks:
(in both experiments, cultures without metabolic activation were exposed to the test substance for about 16 h, no positive control for the 40 h time point)
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 containing 10% (v/v) fetal bovine serum and 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
other: WBL clone
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague-Dawley rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
40, 80 and 160 µg/mL with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on results of a solubility test, acetone was selected as the vehicle. The test substance was not soluble in water or dimethyl sulfoxide at any of the concentrations (10, 25, 50% (v/v)) tested. The test substance was soluble as a 50% mixture in acetone.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 0.6 µg/mL (v/v) in acetone; + S9: 7,12-Dimethylbenz[a]anthracene (DMBA), 10 µg/mL (v/v) in acetone
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
+S9: ca. 3 h (± 0.5 h)
- S9: ca. 16 h (± 0.5 h)
- Fixation time (start of exposure up to fixation or harvest of cells): ca. 16 h (± 0.5 h); second experiment - ca. 16 and 40 h (± 0.5 h)

SPINDLE INHIBITOR (cytogenetic assays): 0.2 mL Colcemid® (10 mg/mL (v/v) in cell culture medium)
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2 replications (16 h) and 1 replication (40 h), respectively

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
Evaluation criteria:
A test substance was considered positive in the chromosome aberration test if:
1. A statistically significant dose-related increase in the percentage of aberrant cells and in at least one of the treatment groups, the percentage of aberrant cells exceeds 5%. OR
2. A reproducible and statistically significant response for at least one of the treatment groups is observed. In addition, the mean percentage of aberrant cells exceeds 5%.
A positive result indicates that under the test conditions the test substance induces chromosomal aberrations in cultured mammalian somatic cells.
If neither of the above conditions exist, the test substance is considered nonmutagenic or negative for inducing chromosomal aberrations in this system.
Species / strain:
Chinese hamster Ovary (CHO)
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance is not soluble in water, therefore it was dissolved in acetone.
- Precipitation: Final concentrations of the test substance in medium of 10, 20, 39, 78, 156, 313, 625, 1250 and 2500 µg/mL were tested by visual and microscopic methods for precipitation immediately, 30 minutes and 3 h after dosing. Traces of the test substance were observed microscopically at all test concentrations equal to or greater than 78 µg/mL. Therefore, the upper limit of the culture medium solubility of the test substance was considered to be between 39 and 78 µg/mL. Based on these results, the study director selected the following concentrations for the toxicity pretest: 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL.
In the main experiments, slight precipitation was observed in the second experiment after 16 h at 160 µg/mL without metabolic activation. Precipitation was not noted at any other harvest of a 160 µg/mL culture.

RANGE-FINDING/SCREENING STUDIES: To determine a concentration selection for the aberration assay, a toxicity pretest was conducted with concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL of the test substance with and without metabolic activation. The concentrations tested were based on the results of a culture medium solubility test. The cultures with metabolic activation were treated for 3 h (± 0.5 h). The cultures without metabolic activation were treated until 2-3 h prior to harvest. All cultures were harvested about 16 h from the beginning of treatment. After harvest, the number of cells that survived treatment were counted using a hemacytometer to evaluate cytotoxicity and the mitotic indices (number of mitotic cells per 1000 total cells) were determined to evaluate cell cycle suppression. The selected concentrations for the aberration assay were based on the results of the cell count data and mitotic index data. The highest reduction in cell survival was observed at 160 µg/mL without metabolic acvtivation, where reduction in viability of 37% was noted. Other less notable reductions in cell survival were noted (see table 3), but were not indicative of a concentration-related trend. Based on these results, the concentrations selected for the aberration assay were 40, 80 and 160 µg/mL.

Table 1. Test results of experiment 1

Test item

Concentration

Mitotic Index

Aberrant cells

Aberration frequency

 

 in µg/mL

in %

in %

in %

Exposure period 16 h, fixation time 16 h, without S9 mix

vehicle

0.5% (v/v)

6.8

0.5

0.5

MNNG

0.6

6.2

22.5**

24.5

Test substance

40

5.6

0.5

0.5

80

6.5

0.5

0.5

160

7.2

1.0

1.0

Exposure period 3 h, fixation time 16 h, with S9 mix

Acetone

0.5% (v/v)

5.5

1.0

1.0

DMBA

10

2.6

33.5**

42.0

Test substance

40

5.3

1.5

1.5

80

6.3

0.5

0.5

160

4.6

2.0

2.0

 

**statistically significantly higher than vehicle control (p<0.001)

MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)

 

Table 2. Test results of experiment 2

Test item

Concentration

Mitotic Index

Aberrant cells

Aberration frequency

 

 in µg/mL

in %

in %

in %

Exposure period 16 h, fixation time 16 h, without S9 mix

vehicle

0.5% (v/v)

7.2

1.0

1.0

MNNG

0.6

5.4

17.5**

17.5

Test substance

40

7.1

1.0

1.0

80

5.4

0.5

0.5

160

7.1

2.5

2.5

Exposure period 3 h, fixation time 16 h, with S9 mix

Acetone

0.5% (v/v)

2.2

0.0

0.0

DMBA

10

4.5

33.0**

43.0

Test substance

40

2.2

1.0

1.0

80

2.4

2.0

2.0

160

2.0

1.5

1.5

Exposure period 16 h, fixation time 40 h, without S9 mix

Acetone

0.5% (v/v)

3.4

2.5

2.0

MNNG #

0.6

---

---

---

Test substance

40

3.0

4.0

4.5

80

2.2

3.0

3.0

160

3.4

2.0

2.0

Exposure period 3 h, fixation time 40 h, with S9 mix

Acetone

0.5% (v/v)

4.8

2.5

2.5

DMBA #

10

---

---

---

Test substance

40

5.4

2.0

2.0

80

4.8

2.0

2.0

160

5.0

0.5

0.5

 

**statistically significantly higher than vehicle control (p<0.001)

MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)

# According to the study report, positive controls were not required for the 40 h harvest.

Table 3. Toxicity pretest results

Treatment Group
in µg/mL

Cell Survival in %*

+ S9

- S9

non-treated

107

102

vehicle

100

100

0.625

122

114

1.25

95

72

2.5

121

88

5

108

68

10

111

108

20

89

102

40

99

93

80

78

93

160

120

63

  * % cell survival as compared to vehicle

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
16 July - 27 July 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (Strain S. typhimurium TA102 or E.coli WP2 were not tested).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Strain S. typhimurium TA102 or E.coli WP2 not tested.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His-operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa-, uvR-, Strains 98 and 100 also R+
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: rfa-, uvR-
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
8, 40, 200, 1000 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tween 80
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
2 µg/plate; TA100 and TA1535 (-S9)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate; TA1537 (-S9)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
40 µg/plate; TA98 and TA1538 (- S9)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
all strains with metabolic activation; 2.5 µg/plate: TA1535, TA1537; 5 µg/plate: TA100, TA1538, TA98
Species / strain:
S. typhimurium, other: 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
Remarks:
Precipitate was seen at concentrations of 1000 µg/plate and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: Mutagenicity of the test item on bacteria - experiment I

 

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 100

TA1535

TA1538

TA98

TA1537

-

Buffer

116

9

10

22

7

 

Vehicle

112

11

12

34

10

-

8

114

10

7

29

10

-

40

111

10

8

34

7

-

200

114

8

9

29

8

-

1000

120

12

7

25

6

-

5000

132

6

9

29

6

Positive

controls

- S9

Name

SA

SA

4NP

4NP

9AA

Concentrations

(μg/plate)

2.0

2.0

40

40

80

Number of colonies/plate

818

621

1792

1571

1025

+

Buffer

116

11

15

38

6

+

Vehicle

103

12

15

35

10

+

8

115

12

16

34

8

+

40

120

9

11

39

8

+

200

113

12

13

38

10

+

1000

126

11

14

39

7

+

5000

130

13

12

3934

7

Positive

controls

+ S9

Name

2AA

2AA

 2AA

 2AA

 2AA

Concentrations

(μg/plate)

5.0

2.5

5.0

5.0

2.5

Number of colonies/plate

1594

210

1769

1572

66

 

4NP= 4-nitro-o-phenylendiamine

SA = sodium azide

9AA = 9 -aminoacridine

2AA = 2 -aminoanthracene

 

 

 

Table 2: Mutagenicity of the test item on bacteria - experiment II

 

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 100

TA1535

TA1538

TA98

TA1537

-

Buffer

122

11

13

34

6

 

Vehicle

127

13

9

39

13

-

8

116

12

11

30

10

-

40

110

14

12

31

8

-

200

121

13

14

39

5

-

1000

111

17

18

29

7

-

5000

122

16

13

32

10

Positive

controls

- S9

Name

SA

SA

4NP

4NP

9AA

Concentrations

(μg/plate)

2.0

2.0

40

40

80

Number of colonies/plate

1018

824

1929

1643

1017

+

Buffer

127

15

19

40

9

+

Vehicle

116

21

20

43

10

+

8

128

11

19

46

10

+

40

126

17

19

49

6

+

200

115

13

19

45

9

+

1000

121

19

16

49

10

+

5000

127

21

14

40

7

Positive

controls

+ S9

Name

2AA

2AA

 2AA

2AA

2AA

Concentrations

(μg/plate)

5.0

2.5

5.0

5.0

2.5

Number of colonies/plate

1535

327

1384

1515

64

 

4NP= 4-nitro-o-phenylendiamine

SA = sodium azide

9AA = 9 -aminoacridine

2AA = 2 -aminoanthracene

 

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

Genetic toxicity in vivo

Description of key information

Mammalian erythrocyte micronucleus formation in vivo (OECD 474, GLP): negative after intraperitoneal and oral application

Read-across from structural analogue source substances Pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS No. 68424-31-7) and Fatty acids, C8-10 mixed esters with dipenaterythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (lack of data on test substance)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
(lack of data on test substance)
GLP compliance:
yes
Type of assay:
other: in vivo micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, St. Constant, Quebec, Canada
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: 32.8 - 40.1 g (males); 27.1 - 31.8 g (females)
- Assigned to test groups randomly: yes, by a computer-generated body weight sorting program
- Housing: the animals were housed individually in suspended stainless steel and wire mesh cages with absorbent paper below the cages.
- Diet: Certified Rodent Diet # 5002 (pellets) (PMI Feeds Inc.), ad libitum
- Water: tap water, ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 18 Sep 1995
To: 21 Sep 1995
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: peanut oil
- Justification for choice of solvent/vehicle: The test substance was soluble in peanut oil at the concentrations required for this study.
- Amount of vehicle: ≤ 1 mL/100 g bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was thoroughly mixed with the vehicle.
Duration of treatment / exposure:
three applications
Frequency of treatment:
approximately every 24 h
Post exposure period:
24 h after the last treatment
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral by gavage
- Dose: 20 mg/kg bw
Tissues and cell types examined:
Tissue: bone marrow of the femur
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: range finding study performed to find the maximum tolerated dose

DETAILS OF SLIDE PREPARATION: 2 slides per animal were prepared. The slides were stained with acridine orange and wet mounted.

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCEs) from each animal were examined for the presence of micronuclei.
Evaluation criteria:
The test substance may be considered positive in this test system if at least one of the following criteria is met:
- The mean number of the micronucleated PCEs of at least one dose point is statistically different from the mean number of the micronucleated PCEs of the vehicle control. This value also must be outside the normal range of the micronucleated PCEs of the vehicle control. Additionally, a dose related statistical increase in the mean number of micronucleated PCEs must be observed.
- The mean number of the micronucleated PCEs of at least two dose points is statistically different from the mean number of the micronucleated PCEs of the vehicle control. This values also must be outside the normal range of the micronucleated PCEs of the vehicle control.
Statistics:
The statistical analysis included means and standard deviations of the micronuclei data and a test of equality of group means performed by a standard one-way analysis of variance (ANOVA).
Residuals from the ANOVA were analyzed for normality by either Wilk's Criterion or the Kolomogorov-Smirnov Statistic. If the residuals were not normally distributed (at 0.01 level of significance) in more than 25% of the analyses then nonparametric analyses were performed. The nonparametric analyses included the Kruskal-Wallis one-way analysis of variance followed by Dunn's Summed Rank Test if differences were indicated. Dose response was evaluated by Jonkheere's test of Ordered Response.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No toxicity was observed up to the highest dose tested.
- Harvest times: 24 h

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: Treatment of the animals with the test substance did not lead to a dose-dependent and statistically significant increase in micronuclei formation.
- Cytotoxicity: No cytotoxicity was observed, since there were no statistically significant decreases in the percentage of PCEs of the treated animals in comparison with the vehicle control.

Table 1: Results of the in-vivo micronucleus assay in male animals

 

%PCE

at sampling time

Total micronuclei per 1000 PCEs at sampling time

Exp group

Number

of animals

Dose [mg/kg]

24 h

24 h

Vehicle control

(peanut oil)

5

0

54.14 ± 6.00

0.6 ± 0.7

Positive control

(cyclophosphamide)

5

20

40.24 ± 11.18**

18.7 ± 7.5**

Test substance

5

500

54.76 ± 4.81

0.9 ± 0.4

Test substance

5

1000

51.80 ± 7.46

1.2 ± 0.8

Test substance

5

2000

51.84 ± 8.85

0.8 ± 0.6

**statistically significant (p<0.01)

Table 2: Results of the in-vivo micronucleus assay in female animals

 

%PCE

at sampling time

Total micronuclei per 1000 PCEs at sampling time

Exp group

Number

of animals

Dose [mg/kg]

24 h

24 h

Vehicle control

(peanut oil)

5

0

55.30 ± 4.72

1.5 ± 0.4

Positive control

(cyclophosphamide)

5

20

44.54 ± 5.17**

10.5 ± 3.4**

Test substance

5

500

56.26 ± 5.89

0.6 ± 0.4*

Test substance

5

1000

60.68 ± 3.63

0.8 ± 0.7*

Test substance

5

2000

59.90 ± 2.7

0.4 ± 0.4*

*statistically significant (p<0.05);**statistically significant (p<0.01)

Conclusions:
Interpretation of results: negative
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 May - 08 July 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted in 1983
Deviations:
no
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 Laboratories (UK Limited), Margate, Kent, UK
- Age at study initiation: 5-9 weeks for phase I (determination of the maximum tolerated dose) and 7-9 weeks for phase II (Micronucleus test) of the study
- Assigned to test groups randomly: Yes
- Housing: 5 per cage in mobile mouse cage racks, housed per sex
- Diet: Porton Combined Diet, ad libitum
- Water: filtered tap 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
Details on exposure:
The study consisted in two phases: in phase I the maximum tolerated dose (MTD) was determined, on the basis of lethalities or severe toxicity observed over a four-day observation period following a single intraperitoneal injection.
In phase II, male and female animals were weighed and given a single intraperitoneal injection of corn oil (vehicle control), cyclophosphamide (positive control) or test substance prepared in corn oil.

Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
24 h and 48 h
Dose / conc.:
5 000 mg/kg bw/day (nominal)
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 bw 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 bw. This dose was selected as MTD.

TREATMENT AND SAMPLING TIMES: 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, 1000 erythrocytes were counted to determine the percentage of polychromatic erythrocytes in the total erythrocyte population.

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 wether 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
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
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 statistically significant compared to the concurrent control values.
The positive control induced stastistically 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.

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

 

Table 1: 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

 

Table 2: Females

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 ± Standard Deviation at two sampling times. n=5

 

Table 3: 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

 

Table 4: 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

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

Additional information

Justification for grouping of substances and read-across

The read-across from analogue source substances approach comprises aliphatic esters of poly-functional alcohols containing two to six reactive hydroxyl groups and one to six fatty acid chains. The analogue approach contains mono constituent, multi-constituent and UVCB substances with fatty acid carbon chain lengths ranging from C5 - C22, which are mainly saturated but also mono unsaturated C16 and C18, polyunsaturated C18, branched C5 and C9, branched C14 - C22 building mono-, di-, tri-, tetra- and hexa esters with an alcohol (i.e. the polyol).

The available data allows for an accurate hazard and risk assessment of the target substance and the read-across concept is applied for the assessment of environmental fate and environmental and human health hazards. Thus, where applicable, environmental and human health effects are predicted from adequate and reliable data for source substances within the group by interpolation to the target substance applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No. 1907/2006. In particular, for each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 7.1 and 13).

In vitro gene mutation in bacteria

There is a reliable and adequate key study performed with a structural analogue source substance available investigating the potential to induce gene mutation in bacteria and using all relevant bacterial strains. This study is further supported by additional tests of structural analogue substances.

The potential to induce gene mutation in bacteria has been investigated using Dipentaerythritol hexaesters with fatty acids, C5 and C9iso (CAS No. 647028-25-9). This key study was conducted according to OECD Guideline 471 and under GLP conditions (Key, RA-A, 647028-25-9, 1999c). The bacterial strains S. typhimurium TA 1535, TA 1537, TA 98, and TA 100 and E. coli WP2 uvr A were exposed to solutions of the test item in acetone in a plate incorporation test design. Concentrations applied in the first and second experiment were 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation. Metabolic activation was achieved by means of a cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers treated with Aroclor 1254. An oily precipitate was observed at 5000 µg/plate. Vehicle and appropriate positive controls were included into the study design. All positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolising system. Investigation of the number of revertant colonies after exposure to the test item revealed no increase either in the presence nor in the absence of metabolic activation and hence the test substance was established not to induce gene mutations in the bacterial strains tested under the experimental conditions applied.

In a first supporting study, the mutagenic potential of Fatty acids, C16-18 (even numbered), esters with pentaerythritol (CAS No. 85116-93-4) was tested in a reverse mutation assay comparable to OECD Guideline 471 and under GLP conditions (BASF, 1991c). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 were used. Tester strains were incubated with test material dissolved in Tween 80 at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (no toxicity but tested up to precipitating concentrations) with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9 mix). Vehicle, negative and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent controls was observed in all strains treated with the test substance, neither in the presence nor in the absence of metabolic activation. Thus, the test substance did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

In another supporting study, the mutagenic potential of Fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8) was tested in a reverse mutation assay comparable to OECD Guideline 471 and under GLP conditions (Exxon, 1995e). The following Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 were used. Tester strains were incubated with the test material dissolved in acetone at concentrations of 0.5, 5, 50, 500, 5000 µg/plate in the first experiment and 50, 100, 500, 1000 and 5000 µg/plate in the repeat experiment with and without the addition of a metabolic activation system (Arochlor 1254 induced rat liver S9 mix). Vehicle, negative and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. No cytotoxicity was observed but beading of the test substance occurred in the initial assay and repeat assay at 500 µg/plate and above with and without metabolic activation in all strains. Thus, the test substance did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

In vitro cytogenicity / chromosome aberration in mammalian cells

A relevant key study investigating the potential to induce chromosome aberration in mammalian cells performed with a suitable structural analogue source substance is available. The result of this study is further supported by another test with a structural analogue.

An in vitro mammalian chromosome aberration test was performed with Dipentaerythritol hexaesters with fatty acids, C5 and C9iso (CAS No. 647028-25-9) in cultured peripheral human lymphocytes according to OECD Guideline 473 and under GLP conditions (Key, RA-A, 647028-25-9, 2000b). Duplicate cultures of peripheral human lymphocytes cells were evaluated for chromosome aberrations in the presence as well as the absence of metabolic activation. Metabolic activation was achieved by a cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with Aroclor 1254. In the first experiment, cells were exposed to the test substance for 4 h followed by 20 h expression time with and without metabolic activation. The test substance was dissolved in acetone and used at concentrations of 39.06; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 µg/mL. In the second experiment cells were exposed for 20 h (without metabolic activation) and for 4 h (with metabolic activation) followed by 20 h expression time. Concentrations applied without metabolic activation were 156.25; 312.5; 625; 1250; 2500 and 5000 µg/mL and those in the experiment with metabolic activation 156.25; 312.5; 625; 1250; 2500 and 5000 µg/mL. A cloudy appearance of the test material was noted at all dose levels in both treatment groups after 4 h of exposure. In experiment 1 and 2 no dose-related toxicity was observed, a 50% mitotic inhibition was not achieved and the precipitate of the test item had no effect on the toxicity response curve. Thus, 1250, 2500 and 5000 µg/mL dose levels were selected for chromosome analysis. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. Cyclophosphamide was used as positive control material inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolising system. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations did not reveal an increase in the frequency of chromosome aberrations and polyploid cells at any dose level tested in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to cultured peripheral human lymphocytes cells in vitro under the conditions of this test.

A supporting study investigating the in vitro mammalian chromosome aberration was performed with Fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8) in Chinese hamster ovary cells (CHO cells) comparable to OECD Guideline 473 and under GLP conditions (Exxon, 1995f). Duplicate cultures of CHO cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In the first experiment, cells were exposed to the test substance for 3 h and for 16 h followed by 16 h expression time with and without metabolic activation, respectively. The test substance was dissolved in acetone and used at concentrations of 40, 80 and 160 µg/mL. In the second experiment cells were again exposed for 3 h and for 16 h followed by 16 h expression time with and without metabolic activation, respectively. Additionally, cells were exposed for 3 and 16 h followed by 40 h expression time with and without metabolic activation, respectively. The same substance concentrations as in first experiment were used. The test substance did not induce cytotoxicity but a precipitate was visible in the second experiment at 160 µg/mL after 16 h incubation without metabolic activation. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. N-Methyl-N-Nitro-N-Nitrosoguanidine and 7,12-Dimethylbenz[a]anthracene were used as positive control materials inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level tested in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to CHO cells in vitro.

In vitro gene mutation in mammalian cells

There is a relevant key study investigating the potential to induce gene mutation in mammalian cells available. The study has been performed with a suitable structural analogue source substance.

An in vitro mammalian cell gene mutation assay according to OECD Guideline 476 and GLP was performed with Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane (CAS No. 85186-89-6) in mouse lymphoma L5178Y cells (Key, RA-A, 85186-89-6, 2010). The cells were treated for 3 and 24 hours with 8% (v/v) and without S9-mix in the first experiment, respectively, and with 12% (v/v) with and without S9-mix in the second experiment, respectively. In the first experiment the test substance was tested at 0.3, 1, 3, 10, 33, 100, 333 and 750 μg/mL up to precipitation with 8% (v/v) and without S9-mix for 3 hours. In the second experiment the test substance was tested at 0.3, 1, 3, 10, 33, 100, 333 and 750 μg/mL up to precipitation with 12% (v/v) for 3 hours and without S9-mix for 24 hours. Cyclophosphamide and Methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. Positive and negative controls were valid and in range of historical control data. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. It was concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

In vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

There are two relevant studies with structural analogue source substances available investigating the potential to induce micronuclei in erythrocytes in vivo. Both investigations are taken into account in a Weight-of-Evidence approach.

Fatty acids, C5-10, esters with pentraerythritol (CAS No. 68424-31-7) was found to be not genotoxic in the micronucleus assay in vivo after intraperitoneal application (WoE, RA-A, 68424-31-7, 1992). 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 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.

Fatty acids, C8-10 mixed esters with dipenaterythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8) was tested for its clastogenic potential in an in vivo micronucleus assay (Exxon, 1995g). Male and female CD-1 mice (5 per sex and dose) were administered 500, 1000, and 2000 mg/kg bw by oral gavage for three times. The period between the oral applications was approx. 24 h. Cyclophosphamide (20 mg/kg bw) as positive control substance and concurrent vehicle controls were also part of the study design. No toxicity was observed up to the highest dose tested. Bone marrow samples were taken 24 h after the last application of the test substance. No cytotoxicity was observed, since there were no statistically significant decreases in the percentage of polychromatic erythrocytes of the treated animals in comparison with the vehicle control. Treatment of the animals with the test substance did not lead to a dose-dependent and statistically significant increase in micronuclei formation. The sensitivity of the test system and the validity of the assay were demonstrated by a statistically significant increase in micronucleated polychromatic erythrocytes induced by the positive control, when compared to the vehicle control. In summary, the test substance was found not to be genotoxic in the in vivo micronucleus assay.

Conclusion on genetic toxicity

Several reliable studies performed with analogue source substances are available investigating the genotoxic potential. Genotoxic effects considered include gene mutation in bacteria and mammalian cells as well as clastogenicity both in vitro and in vivo. The available data demonstrate the lack of genotoxic effects since all tests performed were negative. Thus, no hazard regarding genotoxicity is identified for the target substance Octadecanoic acid, 1,1'-[2-[[3-[(1-oxooctadecyl)oxy]-2,2-bis[[(1-oxooctadecyl)oxy]methyl]propoxy]methyl]-2-[[(1-oxooctadecyl)oxy]methyl]-1,3-propanediyl] ester (CAS No. 70967-57-2).

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 information on intrinsic properties of substances may be generated by means other than tests, e.g. using information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the read-across concept is applied to the target substance Octadecanoic acid, 1,1'-[2-[[3-[(1-oxooctadecyl)oxy]-2,2-bis[[(1-oxooctadecyl)oxy]methyl]propoxy]methyl]-2-[[(1-oxooctadecyl)oxy]methyl]-1,3-propanediyl] ester (CAS No. 70967-57-2), data gaps can be filled by interpolation from representative structural analogue source substances to avoid unnecessary animal testing.

The read-across concept is also used to derive the classification of the target substance taking the properties of the source substances into account. Based on the read-across concept, all available data on genotoxicity both in vitro and in vivo do not meet the classification criteria according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.