Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Oct - 18 Apr 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance glycerides, castor-oil, mono, hydrogenated, acetates (CAS 736150-63-3). According to the ECHA guidance document ‘Practical guide 6: How to report read-across and categories' (ECHA, 2012), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
primary culture, other: human lymphocytes
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 (500 mg/kg bw)
Test concentrations with justification for top dose:
Preliminary toxicity test:
with and without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL

First experiment (and repeat tests):
without metabolic activation: 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
without metabolic activation (repeat): 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Only slides from cultures of the following dose groups were selected for metaphase analyses:

First experiment:
without metabolic activation: 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL

Second experiment:
without metabolic activation (repeat): 40, 80 and 160 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL



Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycin (0.015 µg/mL, -S9), cyclophosphamide (6 µg/mL, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Preliminary test and first main test:
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

Second main test:
- Exposure duration: +S9: 3 h, -S9: 20 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (0.1 µg/mL)
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: at least 100 metaphases (when possible) and 1000 cells for the determination of mitotic index

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (calculated as percentage of cells in metaphases)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, defined as metaphases with multiples of the haploid chromosome number other than diploid (e.g. 3n, 4n etc.) and determined in 200 metaphases
- Determination of endoreplication: yes, defined by the presence of chromosomes with 4, 8 chromatids and determined in 200 metaphases
Evaluation criteria:
EVALUATION OF RESULTS
The study was considered as valid when:
- the negative control cultures showed a low frequency of metaphases with chromosome aberrations, normally 0 - 3% (excluding gaps)
- the positive control cultures showed a clear increase in the frequency of metaphases with chromosome aberrations

For the evaluation of the results, the number of metaphases with chromosome aberrations of each test condition were compared to the concurrent negative control. Gaps were recorded but excluded from the analyses.

The test material was considered clastogenic in this test system if all of the following criteria were met:
1. increases in the frequency of chromosome aberrations were determined at one or more test concentrations
2. reproducible increases in aberrant chromosomes between replicates
3. statistically significance in the increases of chromosome aberrations
4. increases exceed the historical negative control range
5. increases were not associated with large changes in pH or osmolarity
The evidence of a dose-response relation ship was considered to support the conclusion.

The test material was considered non-clastogenic in this test system when the increase in chromosomal aberrations was not statistically significanct and/or no reproducibility was observed.

Results which failed to meet the above mentioned criteria were considered as equivocal.

Statistics:
When appropriate, Fischer´s Exact Test was performed to evaluate statistical significance.
Key result
Species / strain:
primary culture, other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dose-related toxicity which reduced the mitotic index in the high-dose group (5000 µg/mL) to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation (table 1)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
First main test
As the frequencies of metaphases with chromosomal aberrations were in general unacceptably high (4% for the duplicates without metabolic activation and 7 or 3% for the duplicates with metabolic activation), the repetition of the first experiment was conducted with the identical experimental design as the initial test. The values for chromosome aberrations within the test samples were between 1 and 9%, but they were not reproducible between the replicates nor did they show any dose-related effect (the data from the initial experiment are not included in the study report).
Scoring of slides prepared from the repeat of the initial experiment revealed no appropriate increases in chromosomal aberrations within the positive control samples without metabolic activation. Thus, this part of the test was considered as invalid and therefore repeated.

Second main test
As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups (data not shown), this part of the test was repeated with lower dosages in the second test (table 3).

Polyploid and endoreduplicated metaphases
Single polyploid metaphases were observed at few test points without showing a dose-relation ship. Therefore, this effect is considered as incidental and not treatment-related. In contrast, no endoreduplicated metaphases were observed.

In each test group despite the two positive controls treated with cyclophosphamide, 100 metaphases were counted. In the cyclophosphamide treated samples only 59 and 30 scorable metaphases were detected.

Test validity
The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values (table 4). The positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations in the valid parts of the tests, thereby demonstrating the sensitivity of the test and the efficacy of the S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Test results of the preliminary toxicity test

 

Treatment (µg/mL)

Mitotic index (MI)

Without S9

With S9

Individual values

Relative mean MI (%)

Individual values

Relative mean MI (%)

0

4.8

5.3

100

3.8

2.9

100

313

4.8

4.3

90

3.1

3.5

99

625

4.3

4.1

83

2.9

3.5

96

1250

3.4

3.7

70

3.4

2.8

93

2500

3.7

3.3

69

2.1

1.8

58

5000

3.6

2.9

64

0.4

0.3

10

Mitotic index: percentage of cells at metaphase

Individual values: values for each of the duplicate     

Relative mean MI: relative mean mitotic index for the duplicates

 

 

Table 2. Test results of the first main test (-S9: second repeat test, +S9: repeat test)

Treatment (µg/mL)

S9 mix

Relative mean MI (%)

No. aberrant metaphases

Number and types of aberrations

Number of polyploid metaphases

Gaps

Breaks

Exchanges

 

0

-

100

1 / 0

2 / 1

1 / 0

 

1 / 0

1250

-

110

1 / 1

0 / 2

2 / 1

 

 

2500

-

75

1 / 4

1 / 3

1 / 4

 

 

5000

-

70

2 / 3

2 / 1

2 / 4

 

 

Daunomycin (0.015 µg/mL)

-

91

17 / 14 **

9 / 5

20 / 14

2 / 1

 

0

2%

100

0 / 0

1 / 2

0 / 0

 

 

625

2%

67

0 / 1

0 / 0

0 / 1

 

 

1250

2%

51

0 / 0

2 / 1

0 / 0

 

 

2500

2%

42

0 / 1

1 / 1

0 / 1

 

 

Cyclophosphamide (6 µg/mL)

2%

20

33a /19b **

16 / 3

36 / 20

15 / 9

 

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 3. Test results of the second main test (repeat test)

Treatment (µg/mL)

S9 mix

Relative mean MI (%)

No. aberrant metaphases

Number and types of aberrations

Number of polyploid metaphases

Gaps

Breaks

Exchanges

 

0

-

100

0 / 3

1 / 5

0 / 3

 

 

40

-

84

1 / 4

3 / 6

1 / 4

 

1 / 0

80

-

59

1 / 2

0 / 2

1 / 2

 

 

160

-

49

0 / 2

3 / 1

0 / 2

 

0 / 1

Daunomycin (0.015 µg/mL)

-

102

13 / 14 **

6 / 9

13 / 13

1 / 1

 

0

4%

100

2 / 1

3 / 2

1 / 1

1 / 0

 

625

4%

66

2 / 4

3 / 1

5 / 1

 

0 / 1

1250

4%

61

1 / 1

3 / 4

1 / 1

 

 

2500

4%

33

5 / 1

3 / 0

3 / 4

 

 

Cyclophosphamide (6 µg/mL)

4%

77

36 / 33 **

8 / 8

37 / 33

11 / 11

 

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 4. Historical Data (n = 9 previous study)

Treatment (µg/mL)

S9 mix

Frequency of metaphases with aberrant chromosomes excluding gaps (%)

Number of cultures

Mean

SD

Mimimum

Maximum

 

Negative control

-

0.8

0.8

0

3

32

Daunomycin (0.015 µg/mL)

-

15.1

7.4

7

34

32

Negative control

+

0.7

0.9

0

3

32

Cyclophosphamide (6 µg/mL)

+

43.0

13.6

23

70

32

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Apr - 24 Apr 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions. Due to growth inhibition in Salmonella, but not in E.coli strains, only low test concentrations used.
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
low test substance concentrations in Salmonella strains used, cytotoxic effects (growth inhibition) not further specified
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
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 the livers of rats treated with Phenobarbital (PB) and 5,6-benzoflavone (BF)
Test concentrations with justification for top dose:
0.61, 1.2, 2.4, 4.9, 10, 20, 39, and 78 µg/plate without metablic activation: TA 100, TA1535, TA98 and TA1537
10, 20, 39, 78, 156, and 313 µg/plate with metabolic activation: TA 100, TA1535, TA98 and TA1537
313, 625, 1250, 2500, and 5000 µg/plate with and without metabolic activation: E. coli WP2 uvr A
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test substance was insoluble in water, a solubility test was performed with DMSO and acetone. The test substance was insoluble at 50 mg/mL in DMSO, and was dissolved at 100 mg/mL in acetone, and neither exothermic reaction nor generation of gas was observed. Therefore acetone was used as solvent for preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: +S9: Benzo(a)pyrene (5.0 µg/plate for TA100, TA98 and TA1537); 2-Aminoanthracene (2.0 and 10.0 µg/plate for TA1535 and WP2 uvrA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 and 0.1 µg/plate for TA100, WP2 uvrA and TA98, respectively); 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (1.0 µg/plate for TA1537); sodium azide (0.5 µg/plate for TA1535)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation);

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Two replications each in 3 independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition




Evaluation criteria:
If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates ( based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged postive.
Statistics:
The results at each concentration were demonstrated with the mean and the standard deviation.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation:Yes, at 1250 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary test, the growth inhibition by the test substance was observed at 20 µg/plate and in S. typhimurim TA98, TA1537 without metabolic activation, and at 78 µg/plate and in S. typhimurim TA100, TA1535 without metabolic activation, and at 313 µg/plate and in S. typhimurim TA strains with metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 20 µg/plate dose was selected for S. typhimurim TA98, TA1537 without metabolic activation, and the 78 µg/ plate was selected for S. typhimurim TA100, TA1535 without metabolic activation, and the 313 µg/plate dose was selected for S. typhimurim TA strains with metabolic activation, and these highest dose was diluted 5 times (using a ratio of 2) to provide a total of 6 dose levels. And the 5000 µg/plate dose was selected for E.coli WP2uvrA both with and without metabolic activation, and this highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.

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

1st experiment

number of revertants: mean value of negative control

number of revertants: mean value of positive control

max. number of revertants: mean value of test material [µg/plate]

                without S9-mix

 

 

 

TA 1535

14 ±2

399 ±25

 12±2.5 [39]

TA 100

103±5.0

825 ± 34.6

113 ± 9.7 [4.9]

TA 1537

11±1.5

 1989± 52.0

 13±0.6 [10]

TA 98

19 ±1.7

561 ± 19

18 ±4.4 [1.2]

WP2uvrA

25 ±4.0

243 ±24.7

30 ±2.6 [5000]

with S9-mix

 

 

 

TA 1535

 11± 0.6

399 ±25

12 ±2.5 [20]

TA 100

111 ± 6.1

825 ± 34.6

126 ±7.5 [39]

TA 1537

17 ±4.7

 1989± 52.0

 20±3.8 [20]

TA 98

29±1.5

561 ± 19

28 ±5.0 [39]

WP2uvrA

28 ±0.6

243 ±24.7

 33±7.0 [5000]

2nd experiment

number of revertants: mean value of negative control

number of revertants: mean value of positive control

max. number of revertants: mean value of test material [µg/plate]

          without S9-mix

 

 

 

TA 1535

 10±2.3

 388± 16.4

8 ±0.6 [10]

TA 100

121 ± 6.0

780 ±7.1

114 ±7.8 [20]

TA 1537

11 ±1.0

 2057±200.9

15 ±0 [4.9]

TA 98

17 ±1.2

577 ±32.8

20±1.5  [1.2]

WP2uvrA

20 ±2.0

219 ±28:1

27 ±4.6 [1250]

with S9-mix

 

 

 

TA 1535

9 ±2.1

388 ±16.4

11 ±3.2 [78]

TA 100

114 ±12.7

780 ±7.1

 117±9.3 [10]

TA 1537

23 ±4.7

2057 ±200:9

 21±2.3 [10]

TA 98

25±3.8

577 ±32.8

29 ±2.6 [39]

WP2uvrA

 25± 3.2

219 ±28:1

35 ±2.1 [1250]

 

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 May 2010 - 12 Jun 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052-13-0). According to the ECHA guidance document ‘Practical guide 6: How to report read-across and categories' (ECHA, 2012), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
other: CHL/IU
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
6h treatment with and without S9-mix: 0.02, 0.39, 0.078 and 0.156 mg/mL
24 and 48h treatment without S9-mix: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor, the test substance was insoluble in water and physiological saline. And the solubility test was performed with DMSO and acetone. The test substance was insoluble at 500.0 mg/mL in DMSO, and was dissolved at 500.0 mg/mL in acetone, and neither generation of gas nor exothermic reaction was observed. Therefore acetone was selected as solvent in this study.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix Migrated to IUCLID6: 0.1 µg/mL ( 6 h treatment), 0.05 µg/mL (24 and 48 h treatment)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9-mix Migrated to IUCLID6: 7 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6, 24 and 48 h
- Expression time (cells in growth medium): 18 h after 6 h treatment

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid
STAIN (for cytogenetic assays): 0.1% crystal violet solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells carrying greater than 38 chromosomes including triploid were recorded as polyploidy


Evaluation criteria:
The following criteria were set to evaluate the frequency of aberrant cells in each dose group:
A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps.
Negative (-) less than 5%
Equivocal (+-) 5% or more, less than 10%
Positive (+) 10% or more
Key result
Species / strain:
other: CHU/IU
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 0.078 µg/mL and higher

RANGE-FINDING/SCREENING STUDIES: In the results of the cell growth inhibition test, the dose of 50% cell growth inhibition was 5.0 mg/mL and
more all of without S9 mix, with S9 mix, 24 h and 48 h exposure. In addition, the cell growth inhibition was observed, though the cell growth rate was more than 50% at the 0.156~1.25 mg/mL doses of the 24 h exposure and the 0,313~1.25 mg/mL doses of 48 h exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Table 1. Effects of the test substance on viability and chromosome aberrations.

Test item

Concentration

Cell viability

Aberrant cells in %

 

in µg/mL

in %

Numerical

Structural including gaps

Exposure period 6h without S9 mix

Untreated

--

--

0.0

0.0

Solvent

--

100

0.0

1.5

MMC

0.010

105.5

0.0

39.5

Test substance

0.020

101.0

0.5

0.0

0.039

98.0

1.0

1.5

0.078

88.0

0.0

0.0

0.156

82.5

0.5

1.0

Exposure period 6h with S9 mix

Untreated

--

--

0.0

2.0

Solvent

--

100

0.0

0.5

B(a)P

7.000

96.5

0.0

28.5

Test substance

0.020

96.0

0.0

2.0

0.039

92.0

0.0

0.0

0.078

86.0

1.0

0.5

0.156

84.5

0.5

1.0

Exposure period 24h without S9 mix

Untreated

--

--

1.0

0.5

Solvent

--

100

0.5

0.0

MMC

0.050

102.0

0.0

41.5

Test substance

0.078

90.5

0.0

0.5

0.156

82.5

0.0

1.0

0.313

76.0

1.0

1.0

0.625

66.0

0.0

0.5

1.250

66.0

0.5

2.0

2.500

84.5

0.5

0.0

5.000

92.0

0.0

1.0

Exposure period 48h without S9 mix

Untreated

--

--

0.0

0.0

Solvent

--

100

0.5

0.0

MMC

0.050

94.0

0.0

58.5

Test substance

0.078

99.0

0.0

1.0

0.156

86.5

0.0

0.5

0.313

74.5

1.0

0.0

0.625

70.0

0.0

0.0

1.250

79.5

0.5

0.0

2.500

87.5

0.5

0.0

5.000

92.5

0.5

1.0

MMC: Mitomycin

B(a)P: Benz(a)pyrene

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 - 23 Mar 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study with acceptable restrictions. Incomplete strain selection and only 2-AA used as positive control
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1983
Deviations:
yes
Remarks:
incomplete strain selection; only 2-AA used as positive control.
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
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 (500 mg/kg bw)
Test concentrations with justification for top dose:
First mutation assay (plate incorporation method) = 50, 160, 500, 1600 and 5000 µg/plate in the absence and presence of S9-mix in tester strains TA 1535, TA 1537, TA100 and TA 98.
Second mutation assay (plate incorporation method) = 50, 160, 500, 1600 and 5000 µg/plate in the absence of S9-mix in tester strain TA1537.
Third mutation assay (plate incorporation method) = 800, 1600, 2400, 3200 and 4000 µg/plate in the absence and presence of S9-mix in tester strain TA1537.
Fourth mutation assay (pre-incubation method) = 50, 160, 500, 1600 and 5000 µg/plate in the absence and presence of S9-mix in tester strains TA 1535, TA 1537, TA100 and TA 98.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofurane (THF)
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and Tetrahydrofurane (THF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix: 2-Nitroflurene (2.5 µg/plate) for TA98, Sodium azide (5.0 + 2.5 µg/plate) for TA100 and TA 1535, respectively and 9-Amino-acridine (25 µg/plate) for TA1537. +S9 mix: 2 Amino-anthracene (2.5 µg/plate) for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation


DURATION
- Preincubation period: 30 min
- Exposure duration: 96 h

NUMBER OF REPLICATIONS: 3 replications in 4 independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined

OTHER EXAMINATIONS:
- Other: Determination of the frequency of induced or spontaneous reversion to histidine independence with negative controls (H2O), solvent controls (DMSO), test substance concentrations and positive controls; determination of the titers of overnight cultures
Evaluation criteria:
For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.
Statistics:
Mean values and standard deviation were calculated
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes. at 5000 µg/plate with and without metabolic activation in all strains.

RANGE-FINDING/SCREENING STUDIES: not performed.

ADDITIONAL INFORMATION ON CYTOTOXICTY: None

ADDITIONAL INFORMATION ON MUTAGENICITY: In the first plate incorporation test with TA 13537 (-S9 mix), all plates treated with either the test compound or controls revealed a total absence of the bacterial background lawn. This test was therefore not accepted by the study director and was repeated. In this repeat (second) experiment, a small increase in the revertant frequency was observed at the two top dose levels of 1600 and 5000 µg/plate. At the top dose, this effect however was accompanied by a reduced bacterial background lawn and precipitation of the test compound. For clarification, another repeat (third) experiment with adjusted dose levels was therefore performed. Finally, in the fourth pre-incubation experiment, a precipitation of the test compound was observed at the top dose levels with all bacterial tester strains. This was however not accompanied by any overt signs of toxicity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. 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

TA98

TA1537

0

146±16

8±3

16±3

0±0A

50

166±1

6±1

14±2

0±0A

160

136±16

7±1

18±1

0±0A

500

182±13

10±3

15±7

0±0A

1600

175±11

7±1

18±7

0±0A

5000

166±18P

7±2P

21±4P

0±0A,P

Positive controls, –S9

Name

SA

SA

2NF

9-AC

Concentrations

(μg/plate)

5.0

2.5

2.5

25

Mean No. of colonies/plate

(average of 3)

499±38

242±34

134±25

0A

+

0

168±10

12±3

48±10

13±3

+

50

163±8

9±2

39±8

16±5

+

160

168±3

15±4

50±5

15±8

+

500

172±9

12±2

 49±8

15±4

+

1600

163±15

12±5

59±9

21±2

+

5000

179±8P

18±4P

55±4P

20±4

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

2.5

2.5

2.5

Mean No. of colonies/plate

(average of 3)

2028±39

215±14

1561±88

154±13

SA = Sodium azide

9NF = 2-Nitro-fluorene

2AA = 2-Aminoanthracene

9-AC = 9-Amino-acridine

P = Precipitate

A = Absence of background lawn

Table 2. Test results of experiment 2 (plate incorporation)

Without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Frameshift type

TA1537

0

7±1

50

10±3

160

11±2

500

11±1

1600

15±3

5000

13±1B,P

Positive controls, –S9

Name

9-AC

Concentrations

(μg/plate)

25

Mean No. of colonies/plate

(average of 3)

49±14

9-AC = 9-Amino-acridine

P = Precipitate

B = Background lawn reduced 

Table 3. Test results of experiment 3 (plate incorporation)

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Frameshift type

TA1537

0

15±3

800

12±4

1600

16±6

2400

12±4

3200

11±3

4000

16±2P

Positive controls, –S9

Name

9-AC

Concentrations

(μg/plate)

25

Mean No. of colonies/plate

(average of 3)

40±10

+

0

14±5

+

800

11±5

+

1600

13±3

+

2400

12±4

+

3200

15±4

+

4000

23±4P

Positive controls, +S9

Name

2AA

Concentrations

(μg/plate)

2.5

Mean No. of colonies/plate

(average of 3)

233±35

 

9-AC = 9-Amino-acridine

2-AA = 2-Amino-anthracene

P = Precipitate

Table 4. Test results of experiment 4 (pre-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

TA98

TA1537

0

81±8

9±2

21±5

8±2

50

101±16

7±3

24±4

7±2

160

88±10

6±3

19±4

6±2

500

66±2

4±2

24±4

10±3

1600

82±4

7±3

24±3

7±1

5000

80±10P

10±6P

23±9P

6±4P

Positive controls, –S9

Name

SA

SA

2NF

9-AC

Concentrations

(μg/plate)

5.0

2.5

2.5

25

Mean No. of colonies/plate

(average of 3)

539±17

327±46

103±17

55±22

+

0

84±9

6±3

22±6

11±4

+

50

77±11

9±2

23±6

9±3

+

160

81±11

8±2

17±6

11±3

+

500

82±8

9±2

 20±3

13±1

+

1600

93±15

9±3

17±4

14±2

+

5000

104±20P

11±1P

19±3P

11±3

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

2.5

2.5

2.5

Mean No. of colonies/plate

(average of 3)

1033±163

220±49

1059±330

143±35

SA = Sodium azide

9NF = 2-Nitro-fluorene

2AA = 2-Aminoanthracene

9-AC = 9-Amino-acridine

P = Precipitate

 

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-06 until 2012-09-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts (CAS 91744-38-6). According to the ECHA guidance document ‘Practical guide 6: How to report read-across and categories' (ECHA, 2012), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to category justification report provided in IUCLID section 13
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:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
Experiment II:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
The cultures at the lowest concentration of 4.7 µg/mL in the presence and absence of metabolic activation (experiment I and II) were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Solvent used: Tetrahydrofuran (THF) (99.9%)
- Justification for choice of solvent/vehicle: Solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
- Effects of pH: Not effected pH 7.32 in the solvent control versus pH 7.31 at 1200 µg test item/mL
- Effects of osmolality: Not increased (357 mOsm in the solvent control versus 342 mOsm at 1200 µg test item/mL
- Evaporation from medium: Not examined
- Water solubility: Not indicated
- Precipitation:
In the first experiment precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 1200 µg/mL limited by the solubility of the test item in THF and aqueous medium. Test item concentrations between 9.4 µg/mL and 1200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 1200 µg/mL following 24 hours treatment without metabolic activation. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 75.0 µg/mL and above in the presence (4 hours treatment) and absence (4 and 24 hours treatment) of metabolic activation. .


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Summary Table
  relative relative relative mutant   relative relative relative mutant  
conc. P S9 cloning cell cloning colonies/ induction cloning cell cloning colonies/ induction
µg/mL mix efficiency I density efficiency II 106cells factor efficiency I density efficiency II 106cells factor
        % % %     % % %    
Column 1 2 3 4 5 6 7 8 9 10 11 12 13
Experiment I / 4 h treatment       culture I          culture II
Solvent control with THF - 100.0 100.0 100.0 12.3 1.0 100.0 100.0 100.0 13.8 1.0
Positive control (EMS) 150.0 - 84.1 132.6 113.8 70.9 5.8 80.4 76.7 63.8 114.8 8.3
Test item 4.7 - 97.9 culture was not continued# 87.8 culture was not continued#
Test item 9.4 - 84.6 104.9 118.8 13.4 1.1 79.6 106.1 55.7 24.3 1.8
Test item 18.8 - 88.6 142.2 100.7 9.8 0.8 98.8 105.7 31.0 47.0 3.4
Test item 37.5 - 80.4 155.1 139.6 12.3 1.0 83.9 76.0 39.0 25.3 1.8
Test item 75.0 P - 68.5 70.8 113.2 8.3 0.7 82.0 96.4 42.7 25.1 1.8
Test item 150.0 P - 80.7 90.3 126.2 7.4 0.6 69.8 80.6 35.6 84.9 6.1
Experiment I / 4 h treatment       culture I          culture II
Solvent control with THF + 100.0 100.0 100.0 11.9 1.0 100.0 100.0 100.0 10.7 1.0
Positive control (DMBA) 1.1 + 58.3 70.4 70.2 802.4 67.3 85.0 48.2 119.1 274.5 25.5
Test item 4.7 + 81.4 culture was not continued# 113.4 culture was not continued#
Test item 9.4 + 77.8 120.4 92.1 28.2 2.4 96.0 60.9 109.9 13.3 1.2
Test item 18.8 + 80.5 105.1 100.7 10.3 0.9 119.3 87.5 92.3 17.5 1.6
Test item 37.5 + 78.5 130.0 100.3 11.5 1.0 119.0 97.9 105.2 8.5 0.8
Test item 75.0 P + 72.4 100.9 119.0 9.5 0.8 128.4 77.6 109.0 7.4 0.7
Test item 150.0 P + 72.6 98.2 107.7 6.7 0.6 131.9 76.2 121.7 15.1 1.4
Experiment II / 24 h treatment       culture I          culture II
Solvent control    - 100.0 100.0 100.0 20.4 1.0 100.0 100.0 100.0 9.7 1.0
Positive control (EMS) 150.0 - 108.1 79.6 86.7 381.1 18.7 105.1 126.4 103.5 422.4 43.7
Test item 4.7 - 109.1 culture was not continued# 106.8 culture was not continued#
Test item 9.4 - 98.6 108.0 84.6 26.2 1.3 105.6 95.6 117.2 23.1 2.4
Test item 18.8 - 98.8 92.3 92.4 34.1 1.7 109.3 75.7 98.4 20.2 2.1
Test item 37.5 P - 102.2 93.8 85.5 14.7 0.7 106.1 93.8 99.1 9.9 1.0
Test item 75.0 P - 97.2 104.0 84.7 24.0 1.2 105.8 144.5 113.3 13.2 1.4
Test item 150.0 P - 99.0 115.4 79.0 30.3 1.5 103.9 94.1 95.9 2.6 0.3
Experiment II / 4 h treatment          
Solvent control with THF   + 100.0 100.0 100.0 35.2 1.0 100.0 100.0 100.0 21.0 1.0
Positive control (DMBA) 1.1 + 103.0 120.7 67.8 732.9 20.8 100.0 105.0 93.9 348.7 16.6
Test item 4.7 + 104.4 culture was not continued# 101.6 culture was not continued#
Test item 9.4 + 123.9 109.0 98.6 14.8 0.4 108.7 107.6 95.2 4.5 0.2
Test item 18.8 + 133.2 89.6 93.2 29.6 0.8 112.2 109.0 98.7 10.8 0.5
Test item 37.5 + 94.0 127.6 79.8 26.9 0.8 100.9 93.4 101.3 16.4 0.8
Test item 75.0 P + 97.8 95.3 74.5 23.4 0.7 105.7 123.3 107.4 15.7 0.7
Test item 150.0 P + 106.3 104.3 74.7 15.7 0.4 105.3 99.5 94.7 14.2 0.7

#   culture was not continued since a minimum of only four analysable concentrations is required

P  precipitation observed at the end of treatment

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The test item (Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts) was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration was limited by the solubility of the test item in THF and aqueous medium. Precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL in the first experiment with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control and the range of the historical solvent control data in the second culture of the first experiment without metabolic activation at 18.8 and 150.0 µg/mL. However, the increase at 18.8 µg/mL was marginal (induction factor of 3.4) and was not reproduced in the parallel culture under identical conditions. The increase at 150 µg/mL was substantial (induction factor of 6.1) but again, not reproduced in the parallel culture under identical experimental conditions. Furthermore, the concentration of 150 µg/mL was the second precipitating concentration so, the irreproducible increase of the mutation frequency was judged as irrelevant precipitation artefact. The minor increase at 18.8 µg/mL was judged as biologically irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in both cultures of the first experiment without metabolic activation. The trend observed in culture I however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. The trend observed in culture II was judged irrelevant as it was based on a precipitation artefact at 150 µg/mL as described above.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.7 up to 35.2 mutants per 106cells; the range of the groups treated with the test item was from 2.6 up to 84.9 mutants per 106cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

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
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
Experiment II:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
The cultures at the lowest concentration of 4.7 µg/mL in the presence and absence of metabolic activation (experiment I and II) were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Solvent used: Tetrahydrofuran (THF) (99.9%)
- Justification for choice of solvent/vehicle: Solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
- Effects of pH: Not effected pH 7.32 in the solvent control versus pH 7.31 at 1200 µg test item/mL
- Effects of osmolality: Not increased (357 mOsm in the solvent control versus 342 mOsm at 1200 µg test item/mL
- Evaporation from medium: Not examined
- Water solubility: Not indicated
- Precipitation:
In the first experiment precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 1200 µg/mL limited by the solubility of the test item in THF and aqueous medium. Test item concentrations between 9.4 µg/mL and 1200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 1200 µg/mL following 24 hours treatment without metabolic activation. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 75.0 µg/mL and above in the presence (4 hours treatment) and absence (4 and 24 hours treatment) of metabolic activation. .


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Summary Table
  relative relative relative mutant   relative relative relative mutant  
conc. P S9 cloning cell cloning colonies/ induction cloning cell cloning colonies/ induction
µg/mL mix efficiency I density efficiency II 106cells factor efficiency I density efficiency II 106cells factor
        % % %     % % %    
Column 1 2 3 4 5 6 7 8 9 10 11 12 13
Experiment I / 4 h treatment       culture I          culture II
Solvent control with THF - 100.0 100.0 100.0 12.3 1.0 100.0 100.0 100.0 13.8 1.0
Positive control (EMS) 150.0 - 84.1 132.6 113.8 70.9 5.8 80.4 76.7 63.8 114.8 8.3
Test item 4.7 - 97.9 culture was not continued# 87.8 culture was not continued#
Test item 9.4 - 84.6 104.9 118.8 13.4 1.1 79.6 106.1 55.7 24.3 1.8
Test item 18.8 - 88.6 142.2 100.7 9.8 0.8 98.8 105.7 31.0 47.0 3.4
Test item 37.5 - 80.4 155.1 139.6 12.3 1.0 83.9 76.0 39.0 25.3 1.8
Test item 75.0 P - 68.5 70.8 113.2 8.3 0.7 82.0 96.4 42.7 25.1 1.8
Test item 150.0 P - 80.7 90.3 126.2 7.4 0.6 69.8 80.6 35.6 84.9 6.1
Experiment I / 4 h treatment       culture I          culture II
Solvent control with THF + 100.0 100.0 100.0 11.9 1.0 100.0 100.0 100.0 10.7 1.0
Positive control (DMBA) 1.1 + 58.3 70.4 70.2 802.4 67.3 85.0 48.2 119.1 274.5 25.5
Test item 4.7 + 81.4 culture was not continued# 113.4 culture was not continued#
Test item 9.4 + 77.8 120.4 92.1 28.2 2.4 96.0 60.9 109.9 13.3 1.2
Test item 18.8 + 80.5 105.1 100.7 10.3 0.9 119.3 87.5 92.3 17.5 1.6
Test item 37.5 + 78.5 130.0 100.3 11.5 1.0 119.0 97.9 105.2 8.5 0.8
Test item 75.0 P + 72.4 100.9 119.0 9.5 0.8 128.4 77.6 109.0 7.4 0.7
Test item 150.0 P + 72.6 98.2 107.7 6.7 0.6 131.9 76.2 121.7 15.1 1.4
Experiment II / 24 h treatment       culture I          culture II
Solvent control    - 100.0 100.0 100.0 20.4 1.0 100.0 100.0 100.0 9.7 1.0
Positive control (EMS) 150.0 - 108.1 79.6 86.7 381.1 18.7 105.1 126.4 103.5 422.4 43.7
Test item 4.7 - 109.1 culture was not continued# 106.8 culture was not continued#
Test item 9.4 - 98.6 108.0 84.6 26.2 1.3 105.6 95.6 117.2 23.1 2.4
Test item 18.8 - 98.8 92.3 92.4 34.1 1.7 109.3 75.7 98.4 20.2 2.1
Test item 37.5 P - 102.2 93.8 85.5 14.7 0.7 106.1 93.8 99.1 9.9 1.0
Test item 75.0 P - 97.2 104.0 84.7 24.0 1.2 105.8 144.5 113.3 13.2 1.4
Test item 150.0 P - 99.0 115.4 79.0 30.3 1.5 103.9 94.1 95.9 2.6 0.3
Experiment II / 4 h treatment          
Solvent control with THF   + 100.0 100.0 100.0 35.2 1.0 100.0 100.0 100.0 21.0 1.0
Positive control (DMBA) 1.1 + 103.0 120.7 67.8 732.9 20.8 100.0 105.0 93.9 348.7 16.6
Test item 4.7 + 104.4 culture was not continued# 101.6 culture was not continued#
Test item 9.4 + 123.9 109.0 98.6 14.8 0.4 108.7 107.6 95.2 4.5 0.2
Test item 18.8 + 133.2 89.6 93.2 29.6 0.8 112.2 109.0 98.7 10.8 0.5
Test item 37.5 + 94.0 127.6 79.8 26.9 0.8 100.9 93.4 101.3 16.4 0.8
Test item 75.0 P + 97.8 95.3 74.5 23.4 0.7 105.7 123.3 107.4 15.7 0.7
Test item 150.0 P + 106.3 104.3 74.7 15.7 0.4 105.3 99.5 94.7 14.2 0.7

#   culture was not continued since a minimum of only four analysable concentrations is required

P  precipitation observed at the end of treatment

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The test item (Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts) was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration was limited by the solubility of the test item in THF and aqueous medium. Precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL in the first experiment with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control and the range of the historical solvent control data in the second culture of the first experiment without metabolic activation at 18.8 and 150.0 µg/mL. However, the increase at 18.8 µg/mL was marginal (induction factor of 3.4) and was not reproduced in the parallel culture under identical conditions. The increase at 150 µg/mL was substantial (induction factor of 6.1) but again, not reproduced in the parallel culture under identical experimental conditions. Furthermore, the concentration of 150 µg/mL was the second precipitating concentration so, the irreproducible increase of the mutation frequency was judged as irrelevant precipitation artefact. The minor increase at 18.8 µg/mL was judged as biologically irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in both cultures of the first experiment without metabolic activation. The trend observed in culture I however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. The trend observed in culture II was judged irrelevant as it was based on a precipitation artefact at 150 µg/mL as described above.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.7 up to 35.2 mutants per 106cells; the range of the groups treated with the test item was from 2.6 up to 84.9 mutants per 106cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Target gene:
not applicable
Species / strain / cell type:
primary culture, other: human lymphocytes
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 (500 mg/kg bw)
Test concentrations with justification for top dose:
Preliminary toxicity test:
with and without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL

First experiment (and repeat tests):
without metabolic activation: 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
without metabolic activation (repeat): 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Only slides from cultures of the following dose groups were selected for metaphase analyses:

First experiment:
without metabolic activation: 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL

Second experiment:
without metabolic activation (repeat): 40, 80 and 160 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL



Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycin (0.015 µg/mL, -S9), cyclophosphamide (6 µg/mL, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Preliminary test and first main test:
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

Second main test:
- Exposure duration: +S9: 3 h, -S9: 20 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (0.1 µg/mL)
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: at least 100 metaphases (when possible) and 1000 cells for the determination of mitotic index

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (calculated as percentage of cells in metaphases)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, defined as metaphases with multiples of the haploid chromosome number other than diploid (e.g. 3n, 4n etc.) and determined in 200 metaphases
- Determination of endoreplication: yes, defined by the presence of chromosomes with 4, 8 chromatids and determined in 200 metaphases
Evaluation criteria:
EVALUATION OF RESULTS
The study was considered as valid when:
- the negative control cultures showed a low frequency of metaphases with chromosome aberrations, normally 0 - 3% (excluding gaps)
- the positive control cultures showed a clear increase in the frequency of metaphases with chromosome aberrations

For the evaluation of the results, the number of metaphases with chromosome aberrations of each test condition were compared to the concurrent negative control. Gaps were recorded but excluded from the analyses.

The test material was considered clastogenic in this test system if all of the following criteria were met:
1. increases in the frequency of chromosome aberrations were determined at one or more test concentrations
2. reproducible increases in aberrant chromosomes between replicates
3. statistically significance in the increases of chromosome aberrations
4. increases exceed the historical negative control range
5. increases were not associated with large changes in pH or osmolarity
The evidence of a dose-response relation ship was considered to support the conclusion.

The test material was considered non-clastogenic in this test system when the increase in chromosomal aberrations was not statistically significanct and/or no reproducibility was observed.

Results which failed to meet the above mentioned criteria were considered as equivocal.

Statistics:
When appropriate, Fischer´s Exact Test was performed to evaluate statistical significance.
Key result
Species / strain:
primary culture, other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dose-related toxicity which reduced the mitotic index in the high-dose group (5000 µg/mL) to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation (table 1)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
First main test
As the frequencies of metaphases with chromosomal aberrations were in general unacceptably high (4% for the duplicates without metabolic activation and 7 or 3% for the duplicates with metabolic activation), the repetition of the first experiment was conducted with the identical experimental design as the initial test. The values for chromosome aberrations within the test samples were between 1 and 9%, but they were not reproducible between the replicates nor did they show any dose-related effect (the data from the initial experiment are not included in the study report).
Scoring of slides prepared from the repeat of the initial experiment revealed no appropriate increases in chromosomal aberrations within the positive control samples without metabolic activation. Thus, this part of the test was considered as invalid and therefore repeated.

Second main test
As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups (data not shown), this part of the test was repeated with lower dosages in the second test (table 3).

Polyploid and endoreduplicated metaphases
Single polyploid metaphases were observed at few test points without showing a dose-relation ship. Therefore, this effect is considered as incidental and not treatment-related. In contrast, no endoreduplicated metaphases were observed.

In each test group despite the two positive controls treated with cyclophosphamide, 100 metaphases were counted. In the cyclophosphamide treated samples only 59 and 30 scorable metaphases were detected.

Test validity
The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values (table 4). The positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations in the valid parts of the tests, thereby demonstrating the sensitivity of the test and the efficacy of the S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Test results of the preliminary toxicity test

 

Treatment (µg/mL)

Mitotic index (MI)

Without S9

With S9

Individual values

Relative mean MI (%)

Individual values

Relative mean MI (%)

0

4.8

5.3

100

3.8

2.9

100

313

4.8

4.3

90

3.1

3.5

99

625

4.3

4.1

83

2.9

3.5

96

1250

3.4

3.7

70

3.4

2.8

93

2500

3.7

3.3

69

2.1

1.8

58

5000

3.6

2.9

64

0.4

0.3

10

Mitotic index: percentage of cells at metaphase

Individual values: values for each of the duplicate     

Relative mean MI: relative mean mitotic index for the duplicates

 

 

Table 2. Test results of the first main test (-S9: second repeat test, +S9: repeat test)

Treatment (µg/mL)

S9 mix

Relative mean MI (%)

No. aberrant metaphases

Number and types of aberrations

Number of polyploid metaphases

Gaps

Breaks

Exchanges

 

0

-

100

1 / 0

2 / 1

1 / 0

 

1 / 0

1250

-

110

1 / 1

0 / 2

2 / 1

 

 

2500

-

75

1 / 4

1 / 3

1 / 4

 

 

5000

-

70

2 / 3

2 / 1

2 / 4

 

 

Daunomycin (0.015 µg/mL)

-

91

17 / 14 **

9 / 5

20 / 14

2 / 1

 

0

2%

100

0 / 0

1 / 2

0 / 0

 

 

625

2%

67

0 / 1

0 / 0

0 / 1

 

 

1250

2%

51

0 / 0

2 / 1

0 / 0

 

 

2500

2%

42

0 / 1

1 / 1

0 / 1

 

 

Cyclophosphamide (6 µg/mL)

2%

20

33a /19b **

16 / 3

36 / 20

15 / 9

 

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 3. Test results of the second main test (repeat test)

Treatment (µg/mL)

S9 mix

Relative mean MI (%)

No. aberrant metaphases

Number and types of aberrations

Number of polyploid metaphases

Gaps

Breaks

Exchanges

 

0

-

100

0 / 3

1 / 5

0 / 3

 

 

40

-

84

1 / 4

3 / 6

1 / 4

 

1 / 0

80

-

59

1 / 2

0 / 2

1 / 2

 

 

160

-

49

0 / 2

3 / 1

0 / 2

 

0 / 1

Daunomycin (0.015 µg/mL)

-

102

13 / 14 **

6 / 9

13 / 13

1 / 1

 

0

4%

100

2 / 1

3 / 2

1 / 1

1 / 0

 

625

4%

66

2 / 4

3 / 1

5 / 1

 

0 / 1

1250

4%

61

1 / 1

3 / 4

1 / 1

 

 

2500

4%

33

5 / 1

3 / 0

3 / 4

 

 

Cyclophosphamide (6 µg/mL)

4%

77

36 / 33 **

8 / 8

37 / 33

11 / 11

 

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 4. Historical Data (n = 9 previous study)

Treatment (µg/mL)

S9 mix

Frequency of metaphases with aberrant chromosomes excluding gaps (%)

Number of cultures

Mean

SD

Mimimum

Maximum

 

Negative control

-

0.8

0.8

0

3

32

Daunomycin (0.015 µg/mL)

-

15.1

7.4

7

34

32

Negative control

+

0.7

0.9

0

3

32

Cyclophosphamide (6 µg/mL)

+

43.0

13.6

23

70

32

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Target gene:
his operon
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 the livers of rats treated with Phenobarbital (PB) and 5,6-benzoflavone (BF)
Test concentrations with justification for top dose:
0.61, 1.2, 2.4, 4.9, 10, 20, 39, and 78 µg/plate without metablic activation: TA 100, TA1535, TA98 and TA1537
10, 20, 39, 78, 156, and 313 µg/plate with metabolic activation: TA 100, TA1535, TA98 and TA1537
313, 625, 1250, 2500, and 5000 µg/plate with and without metabolic activation: E. coli WP2 uvr A
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test substance was insoluble in water, a solubility test was performed with DMSO and acetone. The test substance was insoluble at 50 mg/mL in DMSO, and was dissolved at 100 mg/mL in acetone, and neither exothermic reaction nor generation of gas was observed. Therefore acetone was used as solvent for preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: +S9: Benzo(a)pyrene (5.0 µg/plate for TA100, TA98 and TA1537); 2-Aminoanthracene (2.0 and 10.0 µg/plate for TA1535 and WP2 uvrA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 and 0.1 µg/plate for TA100, WP2 uvrA and TA98, respectively); 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (1.0 µg/plate for TA1537); sodium azide (0.5 µg/plate for TA1535)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation);

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Two replications each in 3 independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition




Evaluation criteria:
If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates ( based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged postive.
Statistics:
The results at each concentration were demonstrated with the mean and the standard deviation.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation:Yes, at 1250 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary test, the growth inhibition by the test substance was observed at 20 µg/plate and in S. typhimurim TA98, TA1537 without metabolic activation, and at 78 µg/plate and in S. typhimurim TA100, TA1535 without metabolic activation, and at 313 µg/plate and in S. typhimurim TA strains with metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 20 µg/plate dose was selected for S. typhimurim TA98, TA1537 without metabolic activation, and the 78 µg/ plate was selected for S. typhimurim TA100, TA1535 without metabolic activation, and the 313 µg/plate dose was selected for S. typhimurim TA strains with metabolic activation, and these highest dose was diluted 5 times (using a ratio of 2) to provide a total of 6 dose levels. And the 5000 µg/plate dose was selected for E.coli WP2uvrA both with and without metabolic activation, and this highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.

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

1st experiment

number of revertants: mean value of negative control

number of revertants: mean value of positive control

max. number of revertants: mean value of test material [µg/plate]

                without S9-mix

 

 

 

TA 1535

14 ±2

399 ±25

 12±2.5 [39]

TA 100

103±5.0

825 ± 34.6

113 ± 9.7 [4.9]

TA 1537

11±1.5

 1989± 52.0

 13±0.6 [10]

TA 98

19 ±1.7

561 ± 19

18 ±4.4 [1.2]

WP2uvrA

25 ±4.0

243 ±24.7

30 ±2.6 [5000]

with S9-mix

 

 

 

TA 1535

 11± 0.6

399 ±25

12 ±2.5 [20]

TA 100

111 ± 6.1

825 ± 34.6

126 ±7.5 [39]

TA 1537

17 ±4.7

 1989± 52.0

 20±3.8 [20]

TA 98

29±1.5

561 ± 19

28 ±5.0 [39]

WP2uvrA

28 ±0.6

243 ±24.7

 33±7.0 [5000]

2nd experiment

number of revertants: mean value of negative control

number of revertants: mean value of positive control

max. number of revertants: mean value of test material [µg/plate]

          without S9-mix

 

 

 

TA 1535

 10±2.3

 388± 16.4

8 ±0.6 [10]

TA 100

121 ± 6.0

780 ±7.1

114 ±7.8 [20]

TA 1537

11 ±1.0

 2057±200.9

15 ±0 [4.9]

TA 98

17 ±1.2

577 ±32.8

20±1.5  [1.2]

WP2uvrA

20 ±2.0

219 ±28:1

27 ±4.6 [1250]

with S9-mix

 

 

 

TA 1535

9 ±2.1

388 ±16.4

11 ±3.2 [78]

TA 100

114 ±12.7

780 ±7.1

 117±9.3 [10]

TA 1537

23 ±4.7

2057 ±200:9

 21±2.3 [10]

TA 98

25±3.8

577 ±32.8

29 ±2.6 [39]

WP2uvrA

 25± 3.2

219 ±28:1

35 ±2.1 [1250]

 

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Target gene:
Not applicable
Species / strain / cell type:
other: CHL/IU
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
6h treatment with and without S9-mix: 0.02, 0.39, 0.078 and 0.156 mg/mL
24 and 48h treatment without S9-mix: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor, the test substance was insoluble in water and physiological saline. And the solubility test was performed with DMSO and acetone. The test substance was insoluble at 500.0 mg/mL in DMSO, and was dissolved at 500.0 mg/mL in acetone, and neither generation of gas nor exothermic reaction was observed. Therefore acetone was selected as solvent in this study.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix Migrated to IUCLID6: 0.1 µg/mL ( 6 h treatment), 0.05 µg/mL (24 and 48 h treatment)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9-mix Migrated to IUCLID6: 7 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6, 24 and 48 h
- Expression time (cells in growth medium): 18 h after 6 h treatment

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid
STAIN (for cytogenetic assays): 0.1% crystal violet solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells carrying greater than 38 chromosomes including triploid were recorded as polyploidy


Evaluation criteria:
The following criteria were set to evaluate the frequency of aberrant cells in each dose group:
A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps.
Negative (-) less than 5%
Equivocal (+-) 5% or more, less than 10%
Positive (+) 10% or more
Key result
Species / strain:
other: CHU/IU
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 0.078 µg/mL and higher

RANGE-FINDING/SCREENING STUDIES: In the results of the cell growth inhibition test, the dose of 50% cell growth inhibition was 5.0 mg/mL and
more all of without S9 mix, with S9 mix, 24 h and 48 h exposure. In addition, the cell growth inhibition was observed, though the cell growth rate was more than 50% at the 0.156~1.25 mg/mL doses of the 24 h exposure and the 0,313~1.25 mg/mL doses of 48 h exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Table 1. Effects of the test substance on viability and chromosome aberrations.

Test item

Concentration

Cell viability

Aberrant cells in %

 

in µg/mL

in %

Numerical

Structural including gaps

Exposure period 6h without S9 mix

Untreated

--

--

0.0

0.0

Solvent

--

100

0.0

1.5

MMC

0.010

105.5

0.0

39.5

Test substance

0.020

101.0

0.5

0.0

0.039

98.0

1.0

1.5

0.078

88.0

0.0

0.0

0.156

82.5

0.5

1.0

Exposure period 6h with S9 mix

Untreated

--

--

0.0

2.0

Solvent

--

100

0.0

0.5

B(a)P

7.000

96.5

0.0

28.5

Test substance

0.020

96.0

0.0

2.0

0.039

92.0

0.0

0.0

0.078

86.0

1.0

0.5

0.156

84.5

0.5

1.0

Exposure period 24h without S9 mix

Untreated

--

--

1.0

0.5

Solvent

--

100

0.5

0.0

MMC

0.050

102.0

0.0

41.5

Test substance

0.078

90.5

0.0

0.5

0.156

82.5

0.0

1.0

0.313

76.0

1.0

1.0

0.625

66.0

0.0

0.5

1.250

66.0

0.5

2.0

2.500

84.5

0.5

0.0

5.000

92.0

0.0

1.0

Exposure period 48h without S9 mix

Untreated

--

--

0.0

0.0

Solvent

--

100

0.5

0.0

MMC

0.050

94.0

0.0

58.5

Test substance

0.078

99.0

0.0

1.0

0.156

86.5

0.0

0.5

0.313

74.5

1.0

0.0

0.625

70.0

0.0

0.0

1.250

79.5

0.5

0.0

2.500

87.5

0.5

0.0

5.000

92.5

0.5

1.0

MMC: Mitomycin

B(a)P: Benz(a)pyrene

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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 (500 mg/kg bw)
Test concentrations with justification for top dose:
First mutation assay (plate incorporation method) = 50, 160, 500, 1600 and 5000 µg/plate in the absence and presence of S9-mix in tester strains TA 1535, TA 1537, TA100 and TA 98.
Second mutation assay (plate incorporation method) = 50, 160, 500, 1600 and 5000 µg/plate in the absence of S9-mix in tester strain TA1537.
Third mutation assay (plate incorporation method) = 800, 1600, 2400, 3200 and 4000 µg/plate in the absence and presence of S9-mix in tester strain TA1537.
Fourth mutation assay (pre-incubation method) = 50, 160, 500, 1600 and 5000 µg/plate in the absence and presence of S9-mix in tester strains TA 1535, TA 1537, TA100 and TA 98.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofurane (THF)
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and Tetrahydrofurane (THF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix: 2-Nitroflurene (2.5 µg/plate) for TA98, Sodium azide (5.0 + 2.5 µg/plate) for TA100 and TA 1535, respectively and 9-Amino-acridine (25 µg/plate) for TA1537. +S9 mix: 2 Amino-anthracene (2.5 µg/plate) for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation


DURATION
- Preincubation period: 30 min
- Exposure duration: 96 h

NUMBER OF REPLICATIONS: 3 replications in 4 independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined

OTHER EXAMINATIONS:
- Other: Determination of the frequency of induced or spontaneous reversion to histidine independence with negative controls (H2O), solvent controls (DMSO), test substance concentrations and positive controls; determination of the titers of overnight cultures
Evaluation criteria:
For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.
Statistics:
Mean values and standard deviation were calculated
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes. at 5000 µg/plate with and without metabolic activation in all strains.

RANGE-FINDING/SCREENING STUDIES: not performed.

ADDITIONAL INFORMATION ON CYTOTOXICTY: None

ADDITIONAL INFORMATION ON MUTAGENICITY: In the first plate incorporation test with TA 13537 (-S9 mix), all plates treated with either the test compound or controls revealed a total absence of the bacterial background lawn. This test was therefore not accepted by the study director and was repeated. In this repeat (second) experiment, a small increase in the revertant frequency was observed at the two top dose levels of 1600 and 5000 µg/plate. At the top dose, this effect however was accompanied by a reduced bacterial background lawn and precipitation of the test compound. For clarification, another repeat (third) experiment with adjusted dose levels was therefore performed. Finally, in the fourth pre-incubation experiment, a precipitation of the test compound was observed at the top dose levels with all bacterial tester strains. This was however not accompanied by any overt signs of toxicity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. 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

TA98

TA1537

0

146±16

8±3

16±3

0±0A

50

166±1

6±1

14±2

0±0A

160

136±16

7±1

18±1

0±0A

500

182±13

10±3

15±7

0±0A

1600

175±11

7±1

18±7

0±0A

5000

166±18P

7±2P

21±4P

0±0A,P

Positive controls, –S9

Name

SA

SA

2NF

9-AC

Concentrations

(μg/plate)

5.0

2.5

2.5

25

Mean No. of colonies/plate

(average of 3)

499±38

242±34

134±25

0A

+

0

168±10

12±3

48±10

13±3

+

50

163±8

9±2

39±8

16±5

+

160

168±3

15±4

50±5

15±8

+

500

172±9

12±2

 49±8

15±4

+

1600

163±15

12±5

59±9

21±2

+

5000

179±8P

18±4P

55±4P

20±4

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

2.5

2.5

2.5

Mean No. of colonies/plate

(average of 3)

2028±39

215±14

1561±88

154±13

SA = Sodium azide

9NF = 2-Nitro-fluorene

2AA = 2-Aminoanthracene

9-AC = 9-Amino-acridine

P = Precipitate

A = Absence of background lawn

Table 2. Test results of experiment 2 (plate incorporation)

Without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Frameshift type

TA1537

0

7±1

50

10±3

160

11±2

500

11±1

1600

15±3

5000

13±1B,P

Positive controls, –S9

Name

9-AC

Concentrations

(μg/plate)

25

Mean No. of colonies/plate

(average of 3)

49±14

9-AC = 9-Amino-acridine

P = Precipitate

B = Background lawn reduced 

Table 3. Test results of experiment 3 (plate incorporation)

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Frameshift type

TA1537

0

15±3

800

12±4

1600

16±6

2400

12±4

3200

11±3

4000

16±2P

Positive controls, –S9

Name

9-AC

Concentrations

(μg/plate)

25

Mean No. of colonies/plate

(average of 3)

40±10

+

0

14±5

+

800

11±5

+

1600

13±3

+

2400

12±4

+

3200

15±4

+

4000

23±4P

Positive controls, +S9

Name

2AA

Concentrations

(μg/plate)

2.5

Mean No. of colonies/plate

(average of 3)

233±35

 

9-AC = 9-Amino-acridine

2-AA = 2-Amino-anthracene

P = Precipitate

Table 4. Test results of experiment 4 (pre-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

TA98

TA1537

0

81±8

9±2

21±5

8±2

50

101±16

7±3

24±4

7±2

160

88±10

6±3

19±4

6±2

500

66±2

4±2

24±4

10±3

1600

82±4

7±3

24±3

7±1

5000

80±10P

10±6P

23±9P

6±4P

Positive controls, –S9

Name

SA

SA

2NF

9-AC

Concentrations

(μg/plate)

5.0

2.5

2.5

25

Mean No. of colonies/plate

(average of 3)

539±17

327±46

103±17

55±22

+

0

84±9

6±3

22±6

11±4

+

50

77±11

9±2

23±6

9±3

+

160

81±11

8±2

17±6

11±3

+

500

82±8

9±2

 20±3

13±1

+

1600

93±15

9±3

17±4

14±2

+

5000

104±20P

11±1P

19±3P

11±3

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

2.5

2.5

2.5

Mean No. of colonies/plate

(average of 3)

1033±163

220±49

1059±330

143±35

SA = Sodium azide

9NF = 2-Nitro-fluorene

2AA = 2-Aminoanthracene

9-AC = 9-Amino-acridine

P = Precipitate

 

Conclusions:
Interpretation of results (migrated information):
negative

Additional information

Justification for read-across

Data on the genetic toxicity of glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3) are not available. The assessment was therefore based on studies conducted with analogue substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. 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 analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

In vitro bacterial mutagenicity

CAS 91052-13-0

A bacterial gene mutation assay with glycerides, C8-18 and C18-unsatd. mono- and di-, acetates was performed according to OECD guideline 471 and GLP (Sarada, 2010). Two independent experiments were performed both in the presence or absence of metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and in E. coli WP2 uvrA. In the preliminary toxicity screening, growth inhibitory effects were observed at ≥ 20 µg/plate in S. typhimurium TA 98 and TA 1537 (without metabolic activation), at ≥ 78 µg/plate in S. typhimurium TA 100 and TA 1535 (without metabolic activation), and at ≥ 313 µg/plate in all S. typhimurium strains with metabolic activation. Based on these results, concentrations ranging from 0.61 to 78 µg/plate were used for the tester strains TA 100, TA 1535, TA98 and TA 1537 in the absence of metabolic activation, whereas concentrations ranging from 10 to 313 µg/plate were applied for treatment of the tester strains TA 100, TA 1535, TA 98 and TA 1537 in the presence of metabolic activation. Since no cytotoxicity was seen in E. coli WP2 uvrA, the maximum test concentration of 5000 µg/plate and concentrations of 2500, 1250, 625 and313 µg/plate were selected for treatment in the main assay. Precipitation of the test substance was observed on the plates with E. coli WP2 uvrA at test concentrations ≥ 1250 µg/plate without metabolic activation and at ≥ 2500 µg/plate with metabolic activation in both experiments. No increase in mean revertant number was observed in any bacterial strain after exposure to the test substance in the presence or absence of metabolic activation. The positive and negative controls revealed the expected results. Under the conditions of this assay, the test substance did not induce gene mutations in the selected strains of S. typhimurium and in E. coli WP2 uvrA in the absence and presence of metabolic activation, respectively.

CAS 91744-23-3

A bacterial gene mutation assay with glyceryl citrate/lactate/linoleate/oleate was performed according to OECD guideline 471 and GLP (Ebert, 1996). Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were treated with the test substance by the Ames test plate incorporation as well as the pre-incubation method test. Five dose levels covering the range between 50 and 5000 µg/plate in solvent (tetrahydrofurane), in triplicate both with and without the addition of a metabolising system (Acolor 1254 induced rat liver S9 mix) was used. All four bactieral strains exhibited mutagenic responses to the appropriate positive control substances. Solvent controls were also tested with each strain and the mean numbers of spontaneous revertants were in the acceptable range. Mutagenicity activity of the test substance to one or more of the tester strains was not observed in either experiment with and without metabolic activation. It is therefore concluded, that glyceryl citrate/lactate/linoleate/oleate is not a bacterial mutagen.

In vitro mammalian mutagenicity

CAS 91744-38-6

The test item (glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts) was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum concentration was limited by the solubility of the test item in THF and aqueous medium. Precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL in the first experiment with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control and the range of the historical solvent control data in the second culture of the first experiment without metabolic activation at 18.8 and 150.0 µg/mL. However, the increase at 18.8 µg/mL was marginal (induction factor of 3.4) and was not reproduced in the parallel culture under identical conditions. The increase at 150 µg/mL was substantial (induction factor of 6.1) but again, not reproduced in the parallel culture under identical experimental conditions. Furthermore, the concentration of 150 µg/mL was the second precipitating concentration so, the irreproducible increase of the mutation frequency was judged as irrelevant precipitation artefact. The minor increase at 18.8 µg/mL was judged as biologically irrelevant fluctuation. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in both cultures of the first experiment without metabolic activation. The trend observed in culture I however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. The trend observed in culture II was judged irrelevant as it was based on a precipitation artefact at 150 µg/mL as described above. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.7 up to 35.2 mutants per 106cells; the range of the groups treated with the test item was from 2.6 up to 84.9 mutants per 106cells. EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies

CAS 736150-63-3

An in vitro mammalian cell gene mutation assay was performed with glycerides, castor-oil, mono, hydrogenated, acetates according to OECD guideline 476 and under GLP conditions (Edwards, 2002). In the first experiment, mutations at theTK locusof mouse-lymphoma L5178Y cells were investigated at concentrations of 625, 1250, 2500, 3600 and 5000 µg/mL. In this experiment, cells were exposed to the test material for a period of 3 h in the presence and for 4 h in the absence of metabolic activation (S9-mix). At 3600 µg/mL, the relative total growth was 10-20% compared to the negative controls, thus providing an appropriate maximum concentration for treatment in the second experiment. In this experiment, cells were exposed without S9-mix to concentrations ranging from 313 to 3600 µg/ for a period of 24 h, whereas in the presence of S9-mix, cells were treated with concentrations of 156-3600 µg/mL for a period of 4 h. Since the relative growth with S9-mix was very low (0-2%) at all test concentrations, the 24-h treatment of cells in the absence of S9-mix was repeated with concentrations ranging from 2.5-320 µg/mL, which resulted in appropriate levels of cytotoxicity (10-20% relative growth) at 160 µg/mL. In the presence of metabolic activation, the relative total growth was 9% at 2500 µg/mL in the first experiment and 37 and 0% at 2500 and 3600 µg/mL in the second experiment, respectively. After a 3-day expression period of the cultures, the resistance to 5-trifluorothymidine (TFT) was determined in all experiments. The test substance did not induce a significant increase in the mutant frequency at any preparation time and dose concentration. The positive controls significantly increased mutant frequency. In conclusion, the test substance did not induce mutations in mouse-lymphoma L5178Y cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.

In vitro mammalian cytogenicity

CAS 91052-13-0

The clastogenic potential of glycerides, C8-18 and C18-unsatd. mono- and di-, acetates was assessed in a GLP-study in Chinese hamster lung cells (CHL/IU) according to OECD guideline 473 (Seki, 2010). In a preliminary cell growth inhibition test with concentrations ranging from 9.8 to 5000 µg/plate, no significant inhibition of cell growth was observed after 4 h exposure in the presence and absence of metabolic activation (S9 mix). However, a moderate reduction of cell growth of ca. 30-40% compared to controls was observed at concentrations ranging from 156-1250 µg/mL after 24 and 48 h continuous exposure. Based on the results of this study, concentrations of 20, 39, 78 and 156 µg/mL (± S9 mix) were used for the analysis of chromosomal aberrations after short-term exposure (6 h), whereas concentrations of 78, 156, 313, 625, 1250, 2500 and 5000 µg/mL (± S9 mix) were chosen for analysis chromosomal aberrations after continuous exposure (24 and 48 h). No increase in the number of cells with chromosomal aberrations was observed compared to controls in any of the experiments performed. No cytotoxic effects were observed in any of the experiments performed. An oily precipitation of the test substance was observed at concentrations ≥ 78 µg/mL, but did not interfere with chromosomal analysis of the cells. The positive controls included during short-term and continuous exposure showed the expected results. Under the conditions of this experiment, the test substance was considered to be not clastogenic in Chinese hamster lung cells (CHL/IU) in the presence and absence of metabolic activation.

 

In vivo genetic toxicity

CAS 736150-63-3

An in vivo Mammalian Erythrocyte Micronucleus Test was performed with glycerides, castor-oil, mono, hydrogenated, acetates according to OECD guideline 474 and under GLP conditions (Durward, 2008). The test was conducted as a limit test using groups of seven mice (males) at the maximum recommended dose of 2000 mg/kg bw. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of arachis oil (each of 7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

There were no premature deaths seen at any of the dose groups. Clinical sings were not observed in animals dosed with the test material. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to their concurrent control groups. The positive control groups showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenicity activity of cyclophosphamide under the conditions of the test. Overall, glycerides, castor-oil.mono, hydrogenated, acetates was considered to be non-genotoxic under the conditions of the test. 

Short-, medium- and long-chain triglycerides (SCT, MCT, LCT)

An in vivo Mammalian Erythrocyte Micronucleus Test was performed with the SALATRIM (short- and long-chain acyl triglyceride molecules) family of triacylglycerols in Crl:CD BR VAF/Plus rats similarly to OECD Guideline 474 (Hayes et al., 1994a and b). Groups of 20 male and 20 female animals were exposed to either of two SALATRIM fats or corn oil at 10% (w/w) in the diet (equivalent to 7000 mg/kg bw/day) for at least 13 weeks. Erythrocytes were scored to determine the frequency of micro-nucleated erythrocytes. No signs of systemic toxicity in any of the treated animals were observed. No increases in the frequency of micronuclei in polychromatic erythrocytes of the femoral bone marrow of Crl:CD BR VAF/Plus rats exposed to 7000 mg/kg bw/day SALATRIM occurred. Because these data were collected from a 13-week sub-chronic toxicity study, a positive control for micronuclei formation was not included. Under the conditions of this experiment, the test substance was negative in in the micronucleus assay Crl:CD BR VAF/Plus rats.

Overall conclusion for genetic toxicity

There are no available studies on the genetic toxicity of the target substance glycerides, C16-18 and C18-unsaturated mono-and di-citrates. Therefore analogue read-across from source substances was applied from in vitro and in vivo studies on cytogenicity and in vitro gene mutation in bacteria and mammalian cells, using 5 source substances. The results of the available in vitro and in vivo studies were negative. Based on the available data and following the analogue approach, glycerides, C16-18 and C18-unsaturated mono-and di-citrates is considered to be not mutagenic and clastogenic.


Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across from structural analogues. All available in vitro genetic toxicity studies were negative. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
Genetic toxicity in vitro:
Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 100, TA 98, TA 1538 and E. coli WP2 uvrA.
Chromosome aberration (OECD 473): negative in Chinese hamster lung cells (CHL/IU) with and without metabolic activation.
Gene mutation in mammalian cells (OECD 476): negative in L5178Y mouse lymphoma cells and V79 Chinese hamster cells with and without metabolic activation.
Genetic toxicity in vivo:
Mammalian erythrocyte micronucleus test (OECD 474): negative with and without metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” 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 analogue concept is applied to glycerides, C16-18 and C18-unsaturated mono-and di-citrates, data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.