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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacteria:

-        OECD 471 (Ames test), GLP: S. typhimurium TA1535, TA1537, TA98, TA100, TA102; 3 – 5000 µg/plate; with and without metabolic activation; negative in all strains

Mammalian cells:

-        OECD 473 (Chromosome Aberration), GLP: Cultured peripheral human lymphocytes; 19.01 – 800 µg/mL; with and without metabolic activation; negative

-        OECD 476 (Gene mutation), GLP: 15.625 – 1000 µg/mL; with and without metabolic activation; negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Oct - 10 Dec 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1996
Qualifier:
according to guideline
Guideline:
other: UKEMS Guidelines (1990)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI medium containing 20% (v/v) foetal calf serum and 50 µg/mL gentamycin. Phytohaemagglutinin (PHA) was included at a concentration of approximately 10 µg/mL of culture to stimulate the lymphocytes to divide.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: no

Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
KCl-buffered post-mitochondrial fraction (S9-mix) supplemented with co-factors (glucose-6-phosphate, NADP), prepared from male Sprague Dawley rats treated with Aroclor 1254 (MolToxTM S-9, Molecular Toxicology Incorporated, Boone, USA)
Test concentrations with justification for top dose:
Experiment I – normal sampling time:
Cytotoxicity testing: 19.01, 25.34, 33.79, 45.05, 60.07, 80.09, 106.8, 142.4, 189.8, 253.1, 337.5, 450, 600 and 800 µg/mL
Chromosome aberration analysis: 253.1*, 450, 600 and 800 µg/mL (*this concentration was additionally tested in the absence of S9 mix)

Experiment II – normal sampling time:
Cytotoxicity testing: 106.8, 142.4, 189.8, 253.1, 337.5, 450, 600 and 800 µg/mL
Chromosome aberration analysis: 253.1*, 450, 600 and 800 µg/mL (*this concentration was additionally tested in the absence of S9 mix)

Experiment II – delayed sampling time:
Cytotoxicity testing: 106.8*, 142.4*, 189.8*, 253.1, 337.5, 450, 600 and 800 µg/mL (*this concentrations were additionally tested in the absence of S9 mix)
Chromosome aberration analysis: 800 µg/mL


Vehicle / solvent:
- Vehicle/solvent used: DMSO (0.1 mL per culture)
- Justification for choice of solvent/vehicle: Solubility data indicated that the test article was soluble (with warming to 37 °C) in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h (+S9), 20 and 44 h (-S9)
- Fixation time (start of exposure up to fixation or harvest of cells):
3 h treatment (+S9): 20 and 44 h;
20 h treatment (-S9): 20 h;
44 h treatment (-S9): 44 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine 1 µg/mL medium
STAIN (for cytogenetic assays): 4% (v/v) filtered Giemsa stain in pH 6.8 buffer

NUMBER OF REPLICATIONS: duplicate cultures for the treatment groups and quadruplicate culture for the solvent, two independent experiments

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
The test article was to be considered as positive if:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations, and
2) the proportion of cells with structural aberrations at such doses exceeded the normal range, and
3) the results were confirmed in the second experiment.
A positive result only at the delayed harvest in Experiment 2 was to be taken as evidence of clastogenicity, provided criteria 1 and 2 were met. Increases in numbers of cells with gaps or increases in the proportions of cells with structural aberrations, not exceeding the normal range or occurring only at very high or very toxic concentrations, were likely to be concluded as "equivocal".
Statistics:
Heterogeneity between replicate cultures was determined using the binomial dispersion test. The proportion of cells with structural aberrations (excluding gaps) for each test treatment condition was compared with the proportion in concurrent negative controls using Fisher's exact test. Statistical significance was indicated at p ≤ 0.05.
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: up to 337.5 µg/mL (-S9, 20 h) and ≥ 450 µg/mL (+S9, 3 h); Experiment II: ≥ 253 µg/mL (-S9, 20 h), up to 337.5 µg/mL (-S9, 44 h), 337.5 - 600 µg/mL (+S9, 3 h + 17 h recovery); 600 µg/mL (+S9, 3 h + 41 h recovery)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In experiment 1, precipitation of the test substance was observed at ≥ 253.1 µg/mL (with or without S9). In experiment 2, precipitation of the test substance was observed at ≥ 253.1 µg/mL (with S9) and at ≥ 337.5 µg/mL (without S9).
- Biological significance of the effects: a small, but statistically significant (p ≤ 0.05) increase in cells with aberrations at the highest concentration (800 µg/mL) following treatment in the absence of S9 for 20 h in Experiment II was considered to be of no biological significance because:
(A) no statistically significant increase in numbers of cells with aberrations were observed under the same conditions in Experiment I
(B) no dose-response relationship was observed and
(C) the numbers of aberrant cells observed fell within the historical negative control range.

RANGE-FINDING/SCREENING STUDIES: A maximum concentration of 800 µg/mL was chosen as an appropriate top dose for the assay as being close to, but in excess of, the solubility limit (approximately 518 µg/mL) in culture medium. Further treatment concentrations for chromosome analysis were selected by evaluating the effect of the test substance on the mitotic index. The top dose for chromosome analysis from the 20 + 0 h (-S9) and 3 + 17 h (+S9) treatments in Experiments 1 and 2 was to be one at which a 50-80% reduction in mitotic index occurred.

COMPARISON WITH HISTORICAL CONTROL DATA: The proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within normal ranges of the historical control data and thus within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The absence of a clear dose-relationship for mitotic inhibition was a reflection of an accumulation of cells in mitosis at > 337.5 µg/mL (-S9) in Experiment I.


Remarks on result:
other: no clear dose-response relationship for cytotoxic effects; highest concentrations did not yield 50% reduction in mitotic index; precipitation was observed at higher doses; no data on the number of accumulated cells in metaphase were given

Table 1. Test results of experiment I

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

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

DMSO

1.0% (v/v)

100

0.5

0

NQO

2.5

n.d.

8.5

7

Test substance

253.1

39

1.5

1

450

68

2

1

600

60

1.5

1.5

800

58

3

1.5

Exposure period 3 h, fixation time 20 h, with S9 mix

DMSO

1.0% (v/v)

100

0.5

0

CP

12.5

n.d.

26

24

Test substance

450

62

1.5

0.5

600

78

1

0.5

800

69

1

0.5

n.d.: not determined;

NQO: 4-Nitroquinoline-1-oxide; CP: Cyclophosphamide (positive controls)

Table 2. Test results of experiment II

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

Exposure period 3 h, fixation time 20 h, with S9 mix

DMSO

1.0% (v/v)

100

1

0

CP

12.5

n.d.

18.5

13

Test substance

450

62

1.5

0.5

600

85

1.5

0.5

800

100

1

0.5

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

DMSO

1.0% (v/v)

100

0.5

0

NQO

1.25

n.d.

12.5

9.5

Test substance

253.1

34

1.1

0.6

450

67

1.5

0

600

69

2

0.5

800

84

3.5

2.5

Exposure period 3 h, fixation time 44 h, with S9 mix

DMSO

1.0% (v/v)

100

1.5

1

Test substance

800

100

1.5

1.5

Exposure period 44 h, fixation time 44 h, without S9 mix

DMSO

1.0% (v/v)

100

0.5

0.5

Test substance

800

100

2

1.5

n.d.: not determined

NQO: 4-Nitroquinoline-1-oxide; CP: Cyclophosphamide (positive controls)

Conclusions:
Under the conditions chosen, the test substance was concluded not to be clastogenic in peripheral human lymphocytes.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Oct - 25 Nov 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UKEMS Guidelines (1990)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests (1995)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA Toxic Substances Control Act Test Guidelines; Final Rule (August 1997) Section 799.9530 in vitro mammalian cell gene mutation test
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing gentamycin and amphotericin B. Three types of RPMI 1640 medium were prepared: RPMI A (with pluronic), RPMI 10 (with pluronic and 10% v/v heat-inactivated horse serum), RPMI 20 (with 20% v/v heat-inactivated horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
KCl-buffered post-mitochondrial fraction (S9-mix) supplemented with co-factors (glucose-6-phosphate, NADP), prepared from male Sprague Dawley rats treated with Aroclor 1254 (MolToxTM S-9, Molecular Toxicology Incorporated, Boone, USA)
Test concentrations with justification for top dose:
Range-finder experiment
With and without S9 mix: 31.25, 62.5, 125, 250, 500, 1000 µg/mL

Experiment I
With and without S9 mix: 15.625, 31.25, 62.5, 125, 250, 500 µg/mL (3 h)

Experiment II
Without S9 mix: 15.625, 31.25, 62.5, 125, 250, 500 µg/mL (3 h)
With S9 mix: 15.625, 31.25, 62.5, 125, 250, 375, 500 µg/mL (3 h)

Vehicle / solvent:
- Vehicle/solvent used: sterile DMSO (1:100-diluted in treatment medium)
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that RPA 107382 was soluble in sterile DMSO up to at least 450 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Cytotoxicity range-finding experiment: 4 days exposure with and without S9 mix
- 1st experiment: 3 h exposure with and without S9 mix
- 2nd experiment: 3 h exposure with and without S9 mix
- Expression time (cells in growth medium): 2 days
- Selection time: 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-15 days

SELECTION AGENT (mutation assays): 3 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicates each in two independent experiments in 96-well microtitre plates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

OTHER:
Small and large colonies were differentiated, as small colonies were capable to indicate chromosomal mutations.
Evaluation criteria:
The test article was considered to be mutagenic if the following criteria were met:
1) the assay was valid (mutant frequencies in the solvent control fell within the normal range; at least one concentration of each positive control chemicals induced a clear increase in frequency; plating efficiencies of the solvent controls from the mutation experiments were between the range of 60-140% on Day 0 and 70-130% on Day 2)
2) the mutant frequency at one or more doses was significantly greater than that of the negative control (p < 0.05)
3) there was a significant dose-relationship as indicated by the linear trend analysis (p < 0.05)
4) the effects described above were reproducible
Statistics:
The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration using Dunnett's test. Data were checked for a linear trend in mutant frequency with treatment concentration using one-tailed weighted regression.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 250 µg/mL without S9 and ≥ 500 µg/mL without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In experiment 1, precipitation of the test substance was observed at 500 µg/mL (with or without S9). In experiment 2, precipitation of the test substance was observed at 375 µg/mL (with S9) and at 500 µg/mL (with or without S9).
- Solubility in medium: the test substance was soluble in tissue culture medium (RPMI 1640) in the range of 250 to 500 µg/mL.
- Biological significance of the effects: in Experiments 1 and 2, a weak linear trend was observed in the presence of S9 (p < 0.05). This linear trend was solely due to a very small and not statistically significant increase in mutant frequency at the top dose tested (500 µg/mL). Reanalysis of the data with the top dose excluded confirmed that without this dose there was no evidence of a linear trend in Experiment 1 or 2.

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity range-finding experiment, six concentrations (31.25, 62.5, 125, 250, 500 and 1000 µg/mL) were tested. Marked toxicity was observed at the top three concentrations tested in the absence of S9 and the top two concentrations tested in the presence of S9. At the top concentration tested (1000 µg/mL) heavy post-treatment precipitate was observed in both the absence and presence of S9. Hence, a top dose of 500 µg/mL was considered to be appropriate for Experiments 1 and 2.

COMPARISON WITH HISTORICAL CONTROL DATA:
Mutant frequencies in solvent control cultures fell within normal ranges of the historical control data and thus within the acceptability criteria of this assay.

Table 1. Experiment I - 3 h exposure - With Metabolic Activation

Concentration

[µg/mL]

Cloning efficiency [%]

Relative

Total

Growth

Mutants per

1E+06

surviving cells

Mutation factor

0 (DMSO)

100

1.00

116.24

1

15.625

97.35

1.13

99.02

0.85

31.25

99.84

1.04

79.3

0.68

62.5

88.38

0.95

93.16

0.80

125

75.37

0.75

86.31

0.74

250

53.92

0.48

92.37

0.79

500

10.44

0.22

146.04

1.26

B[a]P, 2.0

41.54

0.39

1555.9

13.39

B[a]P, 3.0

46.37

0.33

1130.71

9.73

B[a]P = Benzo[a]pyrene

Table 2. Experiment I - 3 h exposure - Without Metabolic Activation

Concentration

[µg/mL]

Cloning efficiency [%]

Relative

Total

Growth

Mutants per

1E+06

surviving cells

Mutation factor

0 (DMSO)

100

1.00

95.90

1

15.625

89.04

0.96

79.92

0.83

31.25

94.5

0.87

101.71

1.06

62.5

71.74

0.65

100.52

1.05

125

69.95

0.42

107.49

1.12

250

28.38

0.42

127.22

1.33

500

17.98

0.12

102.30

1.07

NQO, 0.05

88.32

0.74

401.31

4.18

NQO, 0.1

67.15

0.60

736.77

7.68

NQO = 4-nitroquinoline-N-oxide

Table 3. Experiment II - 3 h exposure - With Metabolic Activation

Concentration

[µg/mL]

Cloning efficiency [%]

Relative

Total

Growth

Mutants per

1E+06

surviving cells

Mutationfactor

0 (DMSO)

100

1.00

81.74

1

15.625

100.19

1.13

68.60

0.84

31.25

91.56

1.08

80.01

0.98

62.5

78.38

0.80

82.82

1.01

125

60.71

0.44

75.21

0.92

250

48.17

0.25

82.11

1.00

375

16.89

0.57

81.03

0.99

500

17.33

0.24

112.74

1.38

B[a]P, 2.0

61.33

0.48

788.43

9.65

B[a]P, 3.0

55.26

0.50

725.36

8.87

B[a]P = Benzo[a]pyrene

Table 4. Experiment II - 3 h exposure - Without Metabolic Activation

Concentration

[µg/mL]

Cloning efficiency [%]

Relative

Total

Growth

Mutants per

1E+06

surviving cells

Mutation factor

0 (DMSO)

100

1.00

104.94

1.00

15.625

94.27

0.99

80.30

0.77

31.25

90.68

1.00

94.99

0.91

62.5

68.10

0.79

115.05

1.10

125

48.67

0.46

100.68

0.96

250

33.97

0.41

124.57

1.19

500

19.13

0.28

100.05

0.95

NQO, 0.05

84.16

0.68

353.15

3.37

NQO, 0.1

66.20

0.55

576.43

5.49

NQO = 4-nitroquinoline-N-oxide

Conclusions:
Under the conditions chosen, the test substance was not mutagenic in mammalian cells.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Jul 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : performing testing facility
- method of preparation of S9 mix: Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the SOP for rat liver S9 preparation.
- concentration or volume of S9 mix and S9 in the final culture medium : 29.7 mg/mL
- quality controls of S9: metabolic capability. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
Test concentrations with justification for top dose:
Pre-experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation; Experiment I); preincubation (Experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 Minutes
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY : spontaneous reversion rates
Rationale for test conditions:
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used. Since precipitation of the test item was observed in experiment I seven concentrations were tested in experiment II. 5000 µg/plate were chosen as maximal concentration.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twofold or above (strains TA 98, TA 100, and 102) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate with and without S9 mix in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 µg/plate in the absence of S9 mix and from 1000 to 5000 µg/plate in the presence of S9 mix and in experiment II from 1000 to 5000 µg/plate in the presence and absence of S9 mix. The undissolved particles had no influence on the data recording.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Table 1: Summary of Experiment I

                                                                                                                                

 

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without

DMSO

 

9 ± 3

10 ± 5

30 ± 3

100 ± 21

533 ± 29

Activation

Untreated

 

9 ± 4

12 ± 4

33 ± 5

114 ± 15

569 ± 16

 

Test

3 µg

8 ± 3

10 ± 5

32 ± 6

96 ± 5

543 ± 57

 

substance

10 µg

10 ± 3

11 ± 4

23 ± 6

106 ± 22

522 ± 18

 

 

33 µg

9 ± 4

13 ± 3

31 ± 6

115 ± 4

565 ± 20

 

 

100 µg

11 ± 3

14 ± 3

26 ± 7

108 ± 11

557 ± 16

 

 

333 µg

11 ± 4

15 ± 3

28 ± 3

101 ± 2

522 ± 11

 

 

1000 µg

7 ± 0

12 ± 5

28 ± 7

116 ± 4

545 ± 35

 

 

2500 µg

8 ± 2P

15 ± 4P

25 ± 4PM

110 ± 8P

509 ± 6P

 

 

5000 µg

8 ± 3PM

9 ± 3PM

24 ± 5PM

96 ± 9PM

489 ± 32PM

 

NaN3

10 µg

1099 ±

 

 

1903 ± 73

 

 

 

 

45

 

 

 

 

 

4-NOPD

10 µg

 

 

353 ± 20

 

 

 

4-NOPD

50 µg

 

83 ± 5

 

 

 

 

MMS

2.0 µL

 

 

 

 

4681 ± 561

With

DMSO

 

13 ± 4

13 ± 4

37 ± 5

113 ± 9

673 ± 11

Activation

Untreated

 

11 ± 5

15 ± 1

33 ± 3

117 ± 6

672 ± 37

 

test

3 µg

10 ± 2

16 ± 0

37 ± 8

99 ± 5

685 ± 21

 

substance

10 µg

12 ± 4

10 ± 3

36 ± 5

91 ± 10

681 ± 6

 

 

33 µg

10 ± 2

16 ± 3

46 ± 3

112 ± 11

703 ± 12

 

 

100 µg

12 ± 5

18 ± 1

36 ± 6

111 ± 10

681 ± 21

 

 

333 µg

11 ± 4

16 ± 6

33 ± 5

100 ± 7

709 ± 7

 

 

1000 µg

7 ± 2P

13 ± 3P

44 ± 10P

105 ± 16P

726 ± 36P

 

 

2500 µg

8 ± 1PM

11 ± 4PM

33 ± 4PM

96 ± 7PM

659 ± 31PM

 

 

5000 µg

8 ± 2PM

12 ± 1PM

31 ± 9PM

92 ± 4PM

597 ± 26PM

 

2-AA

2.5 µg

278 ± 11

365 ± 12

3559 ± 236

3474 ± 112

 

 

2-AA

10.0 µg

 

 

 

 

1432 ± 273

 

Key toPositiveControls                                                       Key to Plate PostfixCode

4-NOPD 4-nitro-o-phenylene-diamine                                   P Precipitate

NaN3 sodium azide

2 -AA 2-aminoanthracene                                                 M Manual count

MMS methyl methane sulfonate

Table 2: Summary of Experiment II

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without

DMSO

 

11 ± 5

12 ± 2

31 ± 4

112 ± 2

455 ± 14

Activation

Untreated

 

12 ± 6

13 ± 2

36 ± 1

112 ± 14

549 ± 44

 

test

10 µg

11 ± 5

10 ± 2

27 ± 5

105 ± 19

492 ± 28

 

substance

33 µg

11 ± 3

12 ± 5

35 ± 12

114 ± 5

490 ± 10

 

 

100 µg

12 ± 2

12 ± 2

25 ± 3

118 ± 8

488 ± 19

 

 

333 µg

12 ± 3

9 ± 3

32 ± 8

117 ± 8

485 ± 31

 

 

1000 µg

10 ± 3P

10 ± 1P

30 ± 8P

107 ± 6P

496 ± 17P

 

 

2500 µg

14 ± 4P

11 ± 3P

31 ± 3P

104 ± 16P

474 ± 19P

 

 

5000 µg

9 ± 3PM

8 ± 2PM

21 ± 1PM

89 ± 5PM

449 ± 22PM

 

NaN3

10 µg

1315 ±

 

 

1972 ±

 

 

 

 

69

 

 

134

 

 

4-NOPD

10 µg

 

 

355 ± 48

 

 

 

4-NOPD

50 µg

 

76 ± 8

 

 

 

 

MMS

2.0 µL

 

 

 

 

4016 ± 170

With

DMSO

 

12 ± 4

13 ± 3

37 ± 3

106 ± 9

629 ± 30

Activation

Untreated

 

10 ± 2

13 ± 2

41 ± 3

115 ± 16

664 ± 15

 

test

10 µg

10 ± 2

12 ± 3

38 ± 3

106 ± 8

636 ± 22

 

substance

33 µg

11 ± 1

15 ± 1

34 ± 2

110 ± 15

649 ± 19

 

 

100 µg

8 ± 1

16 ± 3

36 ± 9

113 ± 9

648 ± 24

 

 

333 µg

9 ± 1

13 ± 1

39 ± 12

102 ± 13

664 ± 28

 

 

1000 µg

11 ± 7P

15 ± 5PM

39 ± 8P

109 ± 17P

647 ± 33P

 

 

2500 µg

10 ± 1PM

7 ± 2PM

28 ± 2PM

90 ± 11PM

721 ± 202PM

 

 

5000 µg

9 ± 2PM

9 ± 5PM

31 ± 3PM

101 ± 7PM

549 ± 28PM

 

2-AA

2.5 µg

250 ± 14

279 ± 25

2781 ±

3840 ±

 

 

 

 

 

 

165

529

 

 

2-AA

10.0 µg

 

 

 

 

1237 ± 7


Key toPositiveControls                                                       Key to Plate PostfixCode

4-NOPD4-nitro-o-phenylene-diamine                                  P Precipitate

NaN3 sodium azide

2 -AA 2-aminoanthracene                                                M Manual count

MMS methyl methane sulfonate

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- OECD 474 (Micronucleus test), GLP: CD-1 mouse; oral gavage; 500, 1000, and 2000 mg/kg bw/day; not cytogenic

- OECD 486 (UDS), GLP: Han Wistar rat; oral gavage; 800 and 2000 mg/kg bw/day; negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Jul - 20 Oct 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UKEMS Guidelines (1990)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests (1995)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd, Margate, UK
- Age at study initiation: 35 days
- Weight at study initiation: 25-32 g (males), 21-27 (females)
- Assigned to test groups randomly: yes, under following basis: male and female mice were randomised to groups of five using a system of randomly generated numbers.
- Housing: animals were housed in groups of no more than four animals of the same sex in appropriate caging.
- Diet: diet (Special Diets Services Ltd, RM1.[E].SQC.), ad libitum
- Water: mains water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 45-65
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% methyl cellulose
- Concentration of test material in vehicle: 25, 50 and 100 mg/mL for dose levels of 500, 1000 and 2000 mg/kg bw/day, respectively
- Amount of vehicle: 20 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosing preparations were made by suspending the test substance (with homogenisation) in 0.5% methyl cellulose (0.5% MC) to give the top concentrations of 25, 50 and 100 mg/mL for dose levels of 500, 1000 and 2000 mg/kg bw/day, respectively, and dilutions were made using 0.5% MC. The test article preparations were protected from light, maintained as an even suspension (by multiple inversion) and used within 6.5 h of initial formulation (following analysis of achieved concentration).
Duration of treatment / exposure:
not applicable
Frequency of treatment:
Range-finding study and main study: single administration
Post exposure period:
Range-finding study: 4 days after treatment
Main study: 24, 48 and 72 h after treatment with the test substance; 24 h after treatment with the positive control CPA
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Range-finding study: 3
Main study: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw (2 mg/mL in saline)
Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: a range finding study was performed to find the maximum tolerated dose. The test article in 0.5% CM was administered as a single dose to groups of three male and three female mice at 2000 mg/kg/day via oral gavage. Observations were made over a 4-day period following administration and signs of toxicity and body weight recorded. These data were used to obtain a maximum acceptable dose (2000 mg/kg/day) which was used as the top dose in the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): sampling was performed 24, 48 and 72 h after single administration of the test substance and 24 h after single administration of the positive control CPA.

DETAILS OF SLIDE PREPARATION: slides were fixed with methanol and then stained with filtered Giemsa solution (1:6 (v/v) diluted in distilled water) for 10 min.

METHOD OF ANALYSIS: slides were microscopically analysed and relative proportions of polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed. PCE/NCE ratios were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity. Counting continued (but of PCE only) until at least 2000 PCE had been observed. All PCE containing micronuclei observed during these two phases of counting were recorded. The vernier coordinates of all cells containing micronuclei were recorded to a maximum of six per 2000 cells scored.
Evaluation criteria:
The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the frequency of micronucleated PCE occurred at least at one dose, at one sampling time, was observed and
2) the frequency of micronucleated PCE at such a point exceeded the historical vehicle control range.
If statistically insignificant increases in frequencies of micronucleated PCE at one dose, at one sampling time in conjunction with either 1 or 2 of the above at the other sampling time were observed, additional testing or analysis was needed.
Statistics:
The ratio of PCE/NCE for each animal and the mean for each group was calculated and the frequency of micronucleated PCE/1000 PCE was determined. The individual and group mean frequencies of micronucleated PCE/1000 cells (± standard deviation) were also determined. The group mean frequencies of micronucleated PCE in vehicle control animals were compared with historical negative control ranges to determine whether or not the assay was acceptable. For each group, interindividual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity χ2 test. The numbers of micronucleated PCE in each treated group (males and females, separately and combined) were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine χ2. Probability values of p < 0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
1000 mg/kg bw/day: mortalities were observed in 1/5 females 24 h after treatment and in 1/5 males 48 h after treatment; 2000 mg/kg bw/day: mortalities were observed in 1/5 females 24 h after treatment and in 2/5 females 48 h after treatment
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: based on the results of the range finding study, dose levels of 500, 1000 and 2000 mg/kg bw were selected for the main study.
- Solubility: the test substance was well soluble in 0.5% CM.
- Clinical signs of toxicity in test animals: abnormal gait was exhibited by both sexes between 2 and 4 hours after administration. No other clinical signs or loss of body weight were observed.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: the test substance did not induce an increase in the frequency of micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 2000 mg/kg bw. Group mean frequencies of micronucleated PCE were similar to those seen in vehicle control groups and were not significantly different after statistical analysis. All micronuclei frequencies fell within the historical vehicle control range.
- Ratio of PCE/NCE: groups of mice treated with the test substance exhibited PCE/NCE ratios which were similar to those treated with the vehicle at all sampling times.
- Appropriateness of dose levels and route: dose levels and route of exposure were appropriate, since at least eight animals (males plus females) out of each group at each kill time were available for analysis. Thus, the acceptance criteria for the micronucleus assay were fulfilled (see “Any other information on materials and methods incl. tables”).
- Statistical evaluation: the numbers of micronucleated PCE in each treated group (males and females, separately and combined) were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine χ2. Probability values of p < 0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships. No statistically significant differences were observed in the frequency of micronucleated polychromatic erythrocytes between treated and control animals.

VALIDITY OF THE STUDY

The acceptance criteria outlined under “Any other information on materials and methods incl. tables” were fulfilled, as shown by the following results:

1) in most cases, the heterogeneity χ2 test provided evidence of acceptable variability in the number of micronucleated PCE between animals within each group. Heterogeneity of marginal significance (p ≤ 0.05) was seen among animals treated with the vehicle and sampled at 48 hours and was attributable to one mouse with 4 micronucleated PCE/2000 cells. Insofar as such a frequency fell within the historical vehicle control range and has previously been seen among control animals, and other animals in the group had low frequencies of cells with micronuclei, it was considered to be of no biological significance.

2) The incidence of micronucleated PCE in vehicle control groups fell within the historical vehicle control range.

3) At least eight animals (males plus females) out of each group at each kill time were available for analysis.

4) The positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCE.

RESULTS OF THE MAIN ASSAY

Table 1. Results of the in vivo micronucleus assay in male animals

Male treatment group

Mean ratio PCEs / NCEs
at sampling time

Mean frequency of micronucleated PCEs (per 1000 cells) at sampling time

Group

Number of animals

Dose [mL/kg]

24 h

48 h

72 h

24 h

48 h

72 h

Vehicle control (0.5% methyl cellulose)

5

20

1.21

0.79

1.16

0.30

0.00

0.25

Positive control (CPA)

5

20

0.98

n.d.

n.d.

12.50

n.d.

n.d.

500 mg/kg bw

5

20

0.96

0.65

0.96

0.69

0.30

0.20

1000 mg/kg bw

5

20

1.15

0.61

0.80

0.30

0.25

0.30

2000 mg/kg bw

5

20

1.38

0.71

0.97

0.10

0.20

0.10

n.d. = not determined; CPA = cyclophosphamide

Table 2. Results of the in vivo micronucleus assay in female animals.

Female treatment group

Mean ratio PCEs / NCEs
at sampling time

Mean frequency of micronucleated PCEs (per 1000 cells) at sampling time

Group

Number of animals

Dose [mL/kg]

24 h

48 h

72 h

24 h

48 h

72 h

Vehicle control (0.5% methyl cellulose)

5

20

0.99

0.81

0.89

0.70

0.79

0.50

Positive control (CPA)

5

20

0.80

n.d.

n.d.

12.18

n.d.

n.d.

500 mg/kg bw

5

20

1.14

0.98

0.90

0.60

0.70

0.10

1000 mg/kg bw

5

20

1.14

0.80

1.04

0.12

0.20

0.20

2000 mg/kg bw

5

20

0.98

0.76

0.82

0.25

0.12

0.25

n.d. = not determined; CPA = cyclophosphamide

Table 3. Results of the in vivo micronucleus assay (per treatment group)

Treatment group

Dose [mg(kg bw]

Sampling time [h]

Mean frequency of PCE with MN (per 1000 cells) (± SD)

PCE/NCE ratio

Vehicle control

0

24

0.50 ± 0.6

1.10

48

0.44 ± 0.7

0.80

72

0.39 ± 0.3

1.03

Test substance

500

24

0.64 ± 0.6

1.05

48

0.50 ± 0.5

0.82

72

0.15 ± 0.2

0.93

Test substance

1000

24

0.22 ± 0.4

1.15

48

0.22 ± 0.3

0.71

72

0.25 ± 0.4

0.92

Test substance

2000

24

0.17 ± 0.3

1.18

48

0.17 ± 0.3

0.74

72

0.17 ± 0.4

0.90

Positive control (CPA)

40

24

12.34 ± 3.7*

0.89

CPA = cyclophosphamid; MN = Micronuclei; *p ≤ 0.001

Conclusions:
Under the conditions chosen, the test substance was not cytogenic in mice.
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Aug - 29 Oct 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Version / remarks:
Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.5550 (1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UKEMS Guidelines (1993)
Deviations:
no
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
other: Han Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, UK
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 205-240 g
- Assigned to test groups randomly: yes, under following basis: 16 male rats were ear-tagged and allocated to groups of four using a system of computer generated random numbers.
- Fasting period before study: no
- Housing: animals were housed in groups of no more than four animals in solid floored cages with wood shavings for bedding.
- Diet: diet (Special Diets Services Ltd, RM1.(E).SQC.), ad libitum
- Water: from public supply, ad libitum
- Acclimation period: 6-8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 47-60
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% (w/v) methyl cellulose
- Justification for choice of solvent/vehicle: the vehicle used in the range-finder experiment, 0.5% w/v methylcellulose (0.5% MC), was tested in a homogeneity trial and found to produce an acceptable homogeneous formulation. Therefore, this was also used as the vehicle in the main study experiments.
- Concentration of test material in vehicle: 80 and 200 mg/mL for dose levels of 800 and 2000 mg/kg bw/day, respectively
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosing preparations were made by suspending the test substance in 0.5% methyl cellulose (MC), with the aid of a Silverson homogeniser and stirring, to give the concentrations of 80 and 200 mg/mL for dose levels of 800 and 2000 mg/kg bw/day, respectively. The test article preparations were protected from light, stored at 1-10°C, and used within 89 h of initial formulation.
Duration of treatment / exposure:
not applicable
Frequency of treatment:
Range-finding study and main studies (experiment I and II): single administration
Post exposure period:
Range-finding study: 2 days after treatment
Main study – Experiment I: 12-14 h after treatment with the test substance and the positive control
Main study – Experiment II: 2-4 h after treatment with the test substance and the positive control
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Range-finding study: 3 males
Main study: 4 males per group
Control animals:
yes, concurrent vehicle
Positive control(s):
Main assay – Experiment I: 2-acetamidofluorene (2-AAF)
Main assay – Experiment II: dimethylnitrosamine (DMN)
- Route of administration: oral gavage
- Doses / concentrations: 75 mg/kg bw acetamidofluorene (7.5 mg/mL in corn oil); 10 mg/kg bw dimethylnitrosamine (1.0 mg/mL in purified water)
Tissues and cell types examined:
Tissue: liver
Cell type: primary hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: data from a preliminary acute oral toxicity test indicated that the LD50 value for the test substance was greater than 2000 mg/kg bw, which is the recommended maximum dose for the unscheduled DNA synthesis (UDS) assay. Accordingly, a confirmatory toxicity range-finding experiment was performed in which three male rats were dosed once with 2000 mg/kg bw. Since previous studies did not show any substantial inter-sex differences in toxicity of the test substance or likely sex-specific human exposure, only male animals were tested in this study.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): for Experiments I and II, animals were killed a nominal 12-14 h and 2-4 h after dosing, respectively.

DETAILS OF SLIDE PREPARATION: hepatocytes were isolated from the livers of three of the four animals of each treatment and control group by perfusion with suitable buffers. Then, cells were seeded in 6-well multiplates and allowed to attach for at least 90 min at 37 ± 1 °C and 5% CO2. Thereafter, cells were incubated with medium containing 10 µCi/mL [3H] thymidine for a period of 4 h. After incubation, medium was removed and cells were washed with medium containing 0.25 mM thymidine. Cultures were then incubated overnight with 3 mL of the same medium. To prepare for autoradiography, coverslips were washed with 2 mL phosphate buffered saline (PBS) and cells were fixed with three changes of 2 mL glacial acetic acid:ethanol (1:3 v/v). The coverslips were then washed four times with purified water, allowed to dry and mounted onto previously labelled microscope slides, cells side up, with DPX. Six slides from each animal were prepared with fixed hepatocytes and of these, three were dipped in photographic emulsion to prepare autoradiograms. After gelling over ice, for 10 minutes face upwards, the slides were incubated in a light-tight box at room temperature for approximately 90 min to let the emulsion dry. The slides were then packed in light-tight boxes containing desiccant, sealed with tape and refrigerated for 14 days. At the end of this time, the emulsion was developed in Kodak D19 developer and fixed using Ilford Hypam fixer. The cell nuclei and cytoplasm were then stained with Meyers haemalum/eosinY. Slides were then dehydrated in ethanol, cleared in xylene and mounted with coverslips for microscopic examination.

METHOD OF ANALYSIS: repairable DNA damage in cultured primary rat hepatocytes induced by the test substance was measured as unscheduled DNA synthesis (UDS) by the uptake of radio-labelled thymidine assayed by autoradiography of the hepatocytes and grain counting. Normal S-phase synthesis is rare in hepatocytes and can readily be distinguished from UDS using autoradiography, since it detects [3H] thymidine which is incorporated into newly synthesised DNA. For analysis, slides were examined microscopically after development of the photographic emulsion and staining, and the net nuclear grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for each of two of the three slides, each animal and dose group.

OTHER: The following criteria were used for cell analysis (grain counting):
1. only cells with normal morphology were scored
2. isolated nuclei with no surrounding cytoplasm were not scored
3. cells without nuclear and/or cytoplasmic graining were not scored
4. cells with unusual staining artefacts were not scored
5. heavily labelled cells in S-phase were not scored
6. all other normal cells, 100 per animal were scored
7. all slides were analysed blind (coded).
Evaluation criteria:
The test article would be considered as positive in this assay if, at any dose and at either time point:
1. the test article yielded group mean NNG values greater than 0 NNG and 20% or more of cells responding (mean NNG values > 5)
2. an increase above solvent control levels was seen in both NNG and the percentage of cells in repair.
Cytoplasmic and nuclear grain count values as well as the concurrent negative control data would be considered in relation to the overall NNG values of cultures from treated animals. If the test article failed to induce UDS at any dose tested after both 2-4 and 12-14 hours exposure, it would be considered clearly negative in this system.
Statistics:
The following were calculated for each slide, animal and dose point:
1. the population average NNG and standard deviation (SD)
2. the percent of cells responding or in repair (i.e. > 5 NNG)
3. the population average cytoplasmic and nuclear grain count.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2000 mg/kg bw/day (range-finding and Experiment I (12-14 h experiment) of the main study): slight loss of body weight
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: during a 2 day post-dose observation period only weight loss was observed, therefore 2000 mg/kg was selected as the maximum dose for the main study experiments. A lower dose level of 800 mg/kg (40% of the maximum dose level) was also selected.
- Solubility: the vehicle used in the range-finder experiment, 0.5% w/v methylcellulose (0.5% MC), was tested in a homogeneity trial and found to produce an acceptable homogeneous formulation.
- Clinical signs of toxicity in test animals: except for a slight loss in body weight, no further clinical signs were observed in the animals after single administration of 2000 mg/kg bw of the test substance.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: except for a slight loss in body weight at 2000 mg/kg bw, no further clinical signs were observed in the animals after single administration of the test substance at dose levels of 800 and 2000 mg/kg bw, respectively.
- Induction of unscheduled DNA synthesis (UDS) in hepatocytes as analysed by net nuclear grain count values (NNG): hepatocytes of vehicle control animals gave a group mean NNG value of less than zero with only 1% cells in repair. Group mean NNG values were increased by 2-AAF and DMN treatment to more than 19.5 and more than 50% cells found to be in repair. Treatment with 800 or 2000 mg/kg bw of the test substance did not produce a group mean NNG value greater than -2.0 nor were any more than 1% cells found in repair at either dose.
- Appropriateness of dose levels and route: in this study, the vehicle control NNG value was consistent with both published and historical control data, and the system was shown to be sensitive to two known DNA damaging agents requiring metabolism for their action. The assay was therefore accepted as valid (see “Any other information on materials and methods incl. tables”).

VALIDITY OF THE STUDY

The acceptance criteria outlined under “Any other information on materials and methods incl. tables” were fulfilled, as shown by the following results:

1. the group mean net grain count for vehicle-treated animals was less than the upper limit of the historical control range (-1.4 and -3.2 for Experiments 1 and 2 respectively), with only 0 and 1% cells in repair.

2. the positive control chemicals 2-AAF and DMN induced increases in group mean net grain count of five or more (19.5 and 28.0 respectively), and 50% or more of cells (96.3% and 96% respectively) had net grain counts of five or more. This result showed that the test system was sensitive to two known DNA damaging agents requiring metabolism for their action and that the experiment was valid.

HISTORICAL CONTROL DATA

Table 1. Historical vehicle control data

No. of groups*

Parameter

Mean value

Standard error

Calculated range (99% confidence limits)

Observed range

51

NNG

-1.05

0.96

-3.52 to 1.43

-4.2 to 0.4

% cells in repair

0.77

1.01

0 to 3.39

0 to 4.3

NNG = net nuclear grain count

* All values are calculated using the group animal mean values from studies performed using male Han Wistar rats between August 1998 and February 2001.

 

Table 2. Historical positive control data for 2-AAF (75 mg/kg), 12-14 h sampling time

No. of groups*

Parameter

Mean value

Standard error

Calculated range (99% confidence limits)

Observed range

25

NNG

15.36

7.08

-2.87 to 33.59

3.7 to 29.4

% cells in repair

83.70

18.15

36.94 to 100

35.3 to 100

NNG = net nuclear grain count

* All values are calculated using the group animal mean values from studies performed using male Han Wistar rats between August 1998 and February 2001.

 

Table 3. Historical positive control data for DMN (10 mg/kg), 2-4 h sampling time

No. of groups*

Parameter

Mean value

Standard error

Calculated range (99% confidence limits)

Observed range

26

NNG

14.65

5.87

-0.48 to 29.77

5.8 to 26.9

% cells in repair

81.81

11.93

51.07 to 100

53 to 99.3

NNG = net nuclear grain count

* All values are calculated using the group animal mean values from studies performed using male Han Wistar rats between August 1998 and February 2001.

RESULTS OF THE MAIN ASSAY

Table 4. Experiment I (12-14 h experiment) - Individual net nuclear grain (NNG) values (mean values ± standard deviation (SD) of two slides per animal)

Substance [mg/kg bw]

Animal No.

NNG

% cells in repair (NNG ≥ 5)

NNG of cells in repair

No. of cells scored

Mean

SD

Mean

SD

0.5% MC [0]

803

-2.08

1.42

0.00

-

-

100

808

-1.15

1.25

0.00

-

-

100

809

-1.02

0.44

0.00

-

-

100

Test item [800]

804

-1.58

0.59

0.00

-

-

100

810

-2.19

0.61

0.00

-

-

100

812

-2.19

0.10

3.00

6.33

-

100

Test item [2000]

802

-2.77

2.45

0.00

-

-

100

807

-1.24

0.35

0.00

-

-

100

815

-2.64

0.19

2.00

6.67

-

100

2-AAF [75]

801

25.49

13.91

92.00

26.65

12.27

100

805

17.34

1.25

100.00

17.34

1.25

100

806

15.78

2.06

97.00

16.12

1.57

100

NNG = net nuclear grain count; MC = methyl cellulose; 2-AAF = 2-acetamidofluorene

 

Table 5. Experiment II (2-4 h experiment) - Individual net nuclear grain (NNG) values (mean values ± standard deviation (SD) of two slides per animal)

Substance [mg/kg bw]

Animal No.

NNG

% cells in repair (NNG ≥ 5)

NNG of cells in repair

No. of cells scored

Mean

SD

Mean

SD

0.5% MC [0]

821

-3.19

0.64

0.00

-

-

100

824

-2.70

0.54

2.00

5.33

-

100

830

-3.75

0.35

1.00

7.33

-

100

Test item [800]

818

-4.78

0.02

0.00

-

-

100

819

-3.77

0.18

1.00

5.00

-

100

820

-4.53

0.46

0.00

-

-

100

Test item [2000]

817

-6.10

1.67

2.00

6.00

-

100

822

-8.35

0.42

1.00

11.00

-

100

823

-6.51

0.68

0.00

-

-

100

DMN [10]

825

25.77

3.00

96.00

26.74

2.34

100

827

31.81

0.87

96.00

33.10

1.74

100

828

26.30

8.80

96.00

27.35

9.17

100

NNG = net nuclear grain count; MC = methyl cellulose; DMN = dimethylnitrosamine

 

Table 6. Experiment I (12-14 h experiment) – Group net nuclear grain (NNG) values (mean values ± standard deviation (SD) of 3 animals per group)

Substance

[mg/kg bw]

NNG

% cells in repair (NNG ≥ 5)

NNG of cells in repair

Mean

SD

Mean

SD

Mean

SD

0.5% MC [0]

-1.4

0.6

-

-

-

-

Test item [800]

-2.0

0.4

1.0

1.7

6.3

-

Test item [2000]

-2.2

0.8

0.7

1.2

6.7

-

2-AAF [75]

19.5

5.2

96.3

4.0

20.0

5.8

NNG = net nuclear grain count; MC = methyl cellulose; 2-AAF = 2-acetamidofluorene

 

Table 7. Experiment II (2-4 h experiment) – Group net nuclear grain (NNG) values (mean values ± standard deviation (SD) of 3 animals per group)

Substance

[mg/kg bw]

NNG

% cells in repair (NNG ≥ 5)

NNG of cells in repair

Mean

SD

Mean

SD

Mean

SD

0.5% MC [0]

-3.2

0.5

1.0

1.0

6.3

1.4

Test item [800]

-4.4

0.5

0.3

0.6

5.0

-

Test item [2000]

-7.0

1.2

1.0

1.0

8.5

3.5

DMN [10]

28.0

3.3

96.0

0.0

29.1

3.5

NNG = net nuclear grain count; MC = methyl cellulose; DMN = dimethylnitrosamine

Conclusions:
Under the conditions chosen, the test substance was not mutagenic in rats.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No data available.

Additional information

In vitro

- Gene mutation in bacteria

A bacterial reverse mutation assay (Ames test) was conducted with the test substance in compliance with OECD guideline 471 and under GLP conditions (M-686546-01-1, 2020). In this study, the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 was investigated.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.
The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:  3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:  10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate with and without S9 mix in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 µg/plate in the absence of S9 mix and from 1000 to 5000 µg/plate in the presence of S9 mix and in experiment II from 1000 to 5000 µg/plate in the presence and absence of S9 mix. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In addition, a supporting study is available. This bacterial gene mutation assay (Ames test) was conducted with the test substance in compliance with OECD guideline 471 and under GLP conditions (M-192448 -01 -2, 1998a). In a preliminary cytotoxicity test with S. typhimurium TA 100 at concentrations ranging from 8 to 5000 µg/plate, evidence of cytotoxicity and precipitation of the test substance were observed at the maximum test concentration and only in in the presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). However, this concentration was included in the main study as being the best estimate of the precipitation and toxicity limit. The main study included two series of experiments, both performed in the absence and in the presence of S9-mix. In the first experiment, the direct plate incorporation procedure was conducted with Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 as well as with Escherichia coli WP2 uvrA using the same concentrations as in the preliminary test. In the second experiment, concentrations ranging from 312.5 to 5000 µg/plate were used in the same strains of both species, with the exception that a preincubation period was applied. Furthermore, an additional concentration of 156.25 µg/plate was tested in the presence of the metabolic activation system. In the first experiment (plate incorporation), cytotoxic effects were only observed in S. typhimurium TA 98 and TA 1537 and at the highest concentration used (with or without S9-mix). In the second experiment (with preincubation), cytotoxic effects only occurred in the S. typhimurium strains at concentrations ≥ 625 µg/plate (with S9-mix) and concentrations ≥ 2500 µg/plate (without S9-mix). Precipitation of the test substance was observed in the presence or absence of S9-mix in both experiments (5000 µg/plate in experiment 1 and ≥ 625 µg/plate in experiment 2). The positive and negative controls included showed the expected results in each experiment, but only one positive control substance was tested in the presence of metabolic activation and not in all strains. However, each batch of the metabolic activation system was checked for sterility, purity and functionality by the manufacturer. Therefore, these deviations were not considered to prejudice the validity of this study. The test substance did not induce mutations in the bacterial mutation tests in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E.coli (WP2 uvrA).

- Gene mutation in mammalian cells

The genotoxic potential of the test substance was further assessed using a gene mutation assay in cultured mammalian cells (mouse lymphoma L5178Y cells) according to OECD guideline 476 and complying with GLP (M-158105 -01 -2, 1999). A preliminary cytotoxicity study in this cell line was performed with concentrations ranging from 31.25 to 1000 µg/mL. After a 4-day incubation period with the test substance, marked cytotoxicity was observed at ≥ 250 or ≥ 500 µg/mL in the absence or presence of metabolic activation (Aroclor 1254-induced rat liver S9-mix). Precipitation of the test substance was observed at the highest concentration tested (1000 µg/mL), thus a top concentration of 500 µg/mL was selected for the main study experiments. Two independent assays with test concentrations ranging from 15.625 to 500 µg/mL were conducted with and without metabolic activation. In these experiments, cells were exposed to the test material for 3 h. Two days after treatment, viability and 5-trifluorothymidine (TFT) resistance were determined. In the first assay, marked cytotoxicity was observed at the highest concentration (500 µg/mL), resulting in a relative survival of 18 and 10% in the absence or presence of S9-mix, respectively. An additional concentration of 375 µg/mL was included in the second assay in the presence of the S9-mix. In this experiment, similar results for cytotoxicity were obtained compared to the first experiment, yielding 19% and 17% relative growth at 500 µg/mL in the absence and presence of S9-mix, respectively. The negative (solvent) controls included in these experiments were within the expected ranges for mutant frequencies and the positive controls induced clear increases in mutation. In both assays, a weak linear trend was observed in the presence of S9-mix (p < 0.05). This linear trend was solely due to a very small, but not statistically significant increase in mutant frequency at the highest concentration tested (500 µg/mL). No increase in mutant frequency was observed at any other concentration tested. Reanalysis of the data under exclusion of the highest concentration tested confirmed that no linear trend was seen in both assays. Therefore, it was concluded that under the conditions used in the study, the test material was not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the absence and presence of metabolic activation.

- Chromosome aberrations

The test substance was assayed in an in vitro mammalian chromosome aberration test conducted in accordance with GLP and similar to OECD guideline 473 (M-191907-01-2, 1998b). In two independent experiments, human lymphocytes from two healthy donors were treated with the test substance at concentrations up to 800 µg/mL, with and without metabolic activation (S9-mix from Aroclor 1254-induced rat liver). In the first experiment, continuous treatment for 20 h was performed in the absence of metabolic activation, whereas treatment in the presence of S9-mix included the incubation with the test substance for 3 h followed by a 17 h-recovery period. Concentrations for chromosome analysis were selected based on the cytotoxicity data after these treatments. At the highest concentration tested, the mitotic index was reduced by 31% (with S9-mix) and 42% (without S9-mix) compared to that of the solvent control. In contrast, the mitotic index in the absence of metabolic activation was reduced by 61and 73% at concentrations of 253.1 and 337.5 µg/mL, respectively. These results demonstrated no clear dose-response relationship for cytotoxic effects in the absence of S9-mix. The author suggested that the absence of a clear dose-response relationship was due to an accumulation of cell in mitosis at higher concentrations. However, this assumption was not substantiated by respective data. Moreover, test substance precipitation was observed at concentrations ≥ 337.5 µg/mL (with or without S9-mix). Thus, the decrease in cytotoxicity at higher concentrations may have been a result of result of low test substance availability in the culture medium. According to OECD guideline 473, the highest exposure concentrations for chromosome analyses should be those, which reduce the mitotic index by at least 50%. Since the highest concentrations applied for chromosome analysis in the absence of S9-mix resulted in less than 50% reduction of mitotic index at 450, 600 and 800 µg/mL, the test concentration of 253.1 µg/mL (corresponding to 61% mitotic inhibition) was included in this experiment. In the presence of S9-mix, no effects on the mitotic index were observed up to 337.5 µg/mL. Thus, concentrations ≥ 450 µg/mL (corresponding to 22-38% mitotic inhibition) were selected for chromosome analysis in the presence of metabolic activation.

The second experiment was performed with an additional group of cells treated for 44 h in the absence of S9-mix. An additional test group was also included in the presence of S9-mix (3 h treatment and a 41 h-recovery period). Similar patterns for the reduction in mitotic inhibition were observed in the absence of S9-mix compared to the first experiment, showing no dose-response relationship at higher concentrations. Moreover, little or no reduction in the mitotic index was observed at the highest concentration used in all treatments. Test substance precipitation occurred at ≥ 253.1 µg/mL (with S9-mix) and ≥ 337.5 µg/mL (without S9-mix). Based on the results of mitotic inhibition after 20 h incubation with test substance and S9-mix, chromosomes were analysed at concentrations ≥ 450 µg/mL. An additional concentration of 253.1 µg/mL (corresponding to 66% mitotic inhibition) was included in the absence of S9-mix. The effects on chromosomes at the delayed sampling time were only investigated at a single concentration of 800 µg/mL (with and without S9-mix), since no or only minor effects on the mitotic index were seen at the lower concentrations. In the first experiment, the test substance did not induce a statistically significant increase in the number of cells with chromosomal aberrations at any of the concentration analysed. In the second experiment, a small, but statistically significant (p ≤ 0.05) increase in cells with aberrations at the highest concentration (800 µg/mL) following treatment in the absence of S9-mix for 20 h was observed. Since no dose-response relationship was seen and the numbers of aberrant cells fell within the historical range of the solvent control, this result was not considered to be biologically significant. The positive control substances yielded the expected results. Under the conditions of this chromosome aberration assay, it was concluded that the test substance did not show clastogenic activity in cultured human lymphocytes.

In vivo

The in vivo clastogenicity of the test substance was investigated in a GLP-conform Mammalian Erythrocyte Micronucleus Test in male and female CD-1 mice according to OECD guideline 474 (M-158055 -01 -1, 1999). Based on a preliminary range-finding toxicity test, the test substance diluted in 0.5% methyl cellulose was administered as a single dose to groups of five animals per sex at dose levels of 0, 500, 1000 and 2000 mg/kg bw via oral gavage. At 1000 mg/kg bw, mortalities were observed in 1/5 females 24 h after treatment and in 1/5 males 48 h after treatment. Further mortalities were observed in 1/5 females 24 h after treatment and in 2/5 females 48 h after treatment. After a post-exposure period of 24, 48 and 72 h, femoral bone marrow was taken from animals of each treatment and control groups. To determine the frequency of micro-nucleated erythrocytes, 1000 erythrocytes per animal were scored. No increases in the frequency of micronuclei in polychromatic erythrocytes of the femoral bone marrow of CD 1 mice exposed to dose levels up to 2000 mg/kg bw compared to vehicle control values were observed. The mean ratios of polychromatic to normochromatic erythrocytes, being indicative of possible bone marrow toxicity, were similar to the values for vehicle control groups at all sampling times and dose levels and also fell within normal ranges of historical control data. The positive control substance (40 mg/kg bw cyclophosphamide in saline) significantly increased the number of polychromatic erythrocytes with micronuclei in male and female animals, thus verifying the sensitivity of the assay. Under the conditions of this Mammalian Erythrocyte Micronucleus Test, the test substance was not clastogenic in male and female CD1 mice up to dose levels of 2000 mg/kg bw.

A further GLP-conform in vivo genotoxicity test is available, in which the ability of the test substance to induce to unscheduled DNA synthesis (UDS) in isolated hepatocytes of treated rats was investigated according to OECD guideline 486 (M-210455 -01 -1, 2001). Based on a preliminary range-finding toxicity test, groups of 4 male Han Wistar rats were treated with the test substance diluted in 0.5% methyl cellulose at dose levels of 800 and 2000 mg/kg bw via oral gavage. A similar group of animals received the vehicle alone and served as vehicle control group. Furthermore, two positive controls groups, each consisting of 4 animals, were administered a single oral dose of 75 mg/kg 2-acetamidofluorene (2-AAF) suspended in corn oil (12-14 hour experiment) or 10 mg/kg dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour experiment) via gavage. The main study was performed in two independent experiments, comprising two different sampling times (12-14 h and 2-4 h) of hepatocytes after single administration of the test substance. At each sampling time, hepatocytes were isolated from the livers of three of four animals of each treatment and control group by collagenase perfusion and then radioactively labelled with [3H] thymidine. The incorporation of [3H] thymidine into newly synthesised DNA, being indicative of DNA damage and subsequent repair, was analysed using autoradiography. Slides were examined microscopically after development of the photographic emulsion and staining, and the net nuclear grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for each of two of the three slides, for each animal and dose group. The vehicle control animals showed a group mean NNG value of less than zero with only 1% cells in repair. Group mean NNG values of positive control animals were increased by 2-AAF and DMN treatment to more than 19.5 and more than 50% cells found to be in repair. The vehicle and positive control NNG values were within the respective historical control data, and thus verified the validity and sensitivity of the assay. Treatment with 800 or 2000 mg/kg bw of the test substance did not produce a group mean NNG value greater than -2.0 nor were any more than 1% cells found in repair at either dose in isolated hepatocytes approximately 12-14 or 2-4 hours after dosing. No clinical signs of toxicity were noted, except for a slight reduction in body weights at 2000 mg/kg bw/day 12-14 h after test substance administration. Based on these results, test substance did not induce UDS in isolated hepatocytes of male Han Wistar rats treated with single doses up to 2000 mg/kg bw.

Justification for classification or non-classification

The available data on genetic toxicity of ethiprole do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.