Registration Dossier
Registration Dossier
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EC number: 701-177-3 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation in bacteria (OECD 471, Ames): negative in S. typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535, with and without metabolic activation
Chromosome aberration in mammalian cells (OECD 473): negative in peripheral human lymphocytes
Gene mutation in mammalian cells (OECD 476): negative in chinese hamster lung fibroblasts (V79), with and without metabolic activation
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 May - 25 May 2007
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland-Pfalz, Mainz, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102, TA 1535
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- First experiment: 50, 150, 500, 1501 and 5004 µg/plate with and without metabolic activation
Second experiment: 1250, 2500 and 5001 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitro-1,2-phenylene diamine (NPD; 80 µg/plate, -S9, TA98, TA97a and TA102); sodium azide (6 µg/plate, -S9, TA100 and TA1535), benzo-a-pyrene (BaP; 40 µg/plate, +S9, TA98), 2-Amino-anthracene (2-AA; 3 µg/plate, +S9, TA97a, TA100, TA102 and TA1535 )
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation, first experiment); preincubation (second experiment)
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 4 replications each in one independent experiment
DETERMINATION OF CYTOTOXICITY
- Method: titre determination; the titre control should reach a concentration of at least 10E9 bacteria/mL (100 colonies/plate) - Evaluation criteria:
- The test material may be considered as mutagen if at least one of the used strain (with or without metabolic activation) shows an increase of the revertant colonies over 2 times of the negative control (induction rate I ≥ 2) and/or a concentration-dependent relationship is apparent.
- Statistics:
- Mean values and standard deviation were calculated.
- Species / strain:
- S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA: The mutation rates of the positive controls were lower than the stated rates of Prof. Ames. However, the rates were within the range of the historical control data of the Laus GmbH. The rates of the spontaneous revertants were partly lower than the stated rates of Prof. Ames. However, the rates were within the ranges of the historical control data of the Laus GmbH.
- Conclusions:
- Interpretation of results:
negative - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Guideline study with acceptable restrictions. No E.coli strain or S. typhimurium TA102 is included.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- no E. coli or TA102 was included
- 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.
- Test concentrations with justification for top dose:
- First experiment: 20, 100, 500, 2500 and 5000 µg/plate with and without metabolic activation (plate incorporation)
Second and third experiment: 3, 6, 12, 25 and 50 µg/plate with metabolic activation (plate incorporation)
0.8, 4, 20, 100 and 500 µg/plate without metabolic activation (pre-incubation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-methyl-N´-nitro-N-nitroso-guanidine (MNNG; 5 µg/plate in water, -S9, TA100 and TA 1535); 4-nitro-o-phenylendiamine (10 µg/plate in DMSO, -S9, TA 98); 9-aminoacridine chloride monohydrate (100 µg/plate in DMSO, -S9, TA1537)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (10 µg/plate in DMSO, +S9, all strains)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation, experiment I and II) and preincubation (experiment III)
DURATION
- Preincubation period: 20 min
NUMBER OF REPLICATIONS: triplicates of each dose and control
DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn - Evaluation criteria:
- The test material may be considered positive in this test system if the following criteria are met:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results. - Statistics:
- Mean values and standard deviations were calculated.
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- from 50 µg/plate onwards
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results:
negative - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 Jul 2009 - 19 Feb 2010
- 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland-Pfalz, Mainz, Germany
- Type of assay:
- other: in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: in whole blood treated with heparin
- Details on mammalian cell type (if applicable):
- RPMI 1640 medium supplemented with
- 15% (v/v) fetal calf serum (FCS)
- 1% (v/v) penicillin/streptomycin (10000 U /10000 µg/mL)
- 4.8 µg/mL Phytohaemagglutinin solution in H2O
During exposure to the test substance, RPMI medium was used without FCS supplementation. - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Pre-Experiment I:
4h treatment: 55.3, 110.6, 221.3, 442.5, 885, 1770 and 3540 µg/mL, with and without metabolic activation.
Pre-Experiment II:
4h treatment: 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 µg/mL, without metabolic activation.
Experiment I:
4h treatment: 10, 15, 20, 25, 30, 35 and 40 µg/mL, with and without metabolic activation.
Experiment II:
4h treatment: 10, 15, 20, 25, 30, 35 and 40 µg/mL, with metabolic activation.
22h treatment: 0.5, 1, 5, 10, 15, 20, 25, 30, 40 an 50 µg/mL, without metabolic activation. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: cyclophosphamide, 35 µg/mL in 0.9% NaCl, +S9; methanesulphonate, 600 and 350 µg/mL (experiment I and II, respectively) in nutrient medium, –S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 22 h
- Fixation time (start of exposure up to fixation or harvest of cells): 22 h
SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.1 pg/mL
STAIN (for cytogenetic assays): Giemsa 10% (v/v) in Soerensen buffer
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes - Evaluation criteria:
- A test item is classified as non-mutagenic if: the number of induced structural chromosome aberrations in all evaluated dose groups is below 5.0 % aberrant cells, excluding gaps. No significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if: the number of induced structural chromosome aberrations is above 5.0 % aberrant cells, excluding gaps and either a concentration-related or a significant increase in the number of cells with structural chromosome aberrations is observed. - Statistics:
- Statistical significance was calculated by means of the Fisher's exact test.
- Species / strain:
- lymphocytes: in whole blood treated with heparin
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- from 30 µg/mL without and from 40 µg/mL with metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: was observed from 221.3 µg/mL and above with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. The applied concentrations ranged from 55.3 µg/mL to 3540 µg/mL with and without metabolic activation (exposure time 4 h, pre-experiment I). In pre-experiment II concentrations from 0.78 to 50 µg/mL were used.
ADDITIONAL INFORMATION ON CYTOTOXICITY: All concentraitons of the pre-experiment I showed cytotoxicity. - Conclusions:
- Interpretation of results:
negative - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- other: mammalian cell gene mutation assay
- Target gene:
- HPRT locus
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM medium supplemented with 10% fetal bovine serum and 1% neomycin
- Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/ß-naphtoflavone
- Test concentrations with justification for top dose:
- Experiment I
Without S9 mix: 0.25, 0.5, 1, 2 and 4 µg/mL (4 h)
With S9 mix: 4, 8, 16, 32 and 48 µg/mL (4 h)
Experiment II
Without S9 mix: 4, 8, 16, 64 and 96 µg/mL (24 h)
With S9 mix: 4, 8, 16, 48 and 56 µg/mL (4 h) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ethylmethane sulfonate, 150 µg/mL in nutrient medium, without S9; 7,12-dimethylbenz(a)anthracene, 1.1 µg/mL in DMSO, with S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
4 h exposure with and without S9 mix
4 h exposure with S9 mix and 24 h without S9 mix
- Expression time (cells in growth medium): The expression time in growth medium was 7 days. For the selection of mutant cells the complete medium was supplemented with 11 µg/mL thioguanine.
- Selection time (if incubation with a selection agent): 2 days (experiment I) or 3 days (experiment II) after sub-cultivation stock cultures were trypsinized.
- Fixation time (start of exposure up to fixation or harvest of cells): 9-10 days
SELECTION AGENT (mutation assays): 11 µg/mL thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution
NUMBER OF REPLICATIONS: 5 replications each in two independent experiments
DETERMINATION OF CYTOTOXICITY: cloning efficiency and relative total growth
- Method: mitotic index - Evaluation criteria:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. 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 non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations 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. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration. - Statistics:
- A linear regression 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 is < 0.05.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at ≥4 µg/mL (experiment I, -S9), ≥ 48 µg/mL (experiment I,+ S9); 96 µg/mL (experiment II, -S9, 24 h), 56 µg/mL (experiment II, +S9, 4 h)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the preliminary test, the pH of the culture medium was adjusted at concentrations of 1900 and 3800 µg/mL with 2 N NaOH. In the main experiments no pH adjustment was necessary.
- Precipitation: precipitation occurred at 237.5 µg/mL an above after 24 h without metabolic activation. Following 4 h treatment no precipitation was noted.
RANGE-FINDING/SCREENING STUDIES: Concentrations between 29.7 and 3800 µg/mL were used to evaluate toxicity in the presence (4 h) and absence (4 h and 24 h treatment) of metabolic activation. After 24 h treatment without metabolic activation the cell growth was completely inhibited at 118.8 mg/L and above. Following 4 htreatment with and without metabolic activation the cell growth was already completely inhibited at a concentration of 29.7 µg/mL. Therefore, the preliminary experiment was repeated with concentrations of 0.23 to 30.0 µg/mL with and without metabolic activation. In the repeated preliminary experiment strong cytotoxic effects occurred only in the absence of metabolic activation at 7.5 µg/mL and above.
COMPARISON WITH HISTORICAL CONTROL DATA: All mutant frequencies were within the historical range of solvent controls. A single exception occurred in experiment I at 2.0 µg/mL. The induction factor (4.0) exceeded the threshold of three times the corresponding solvent control and the range of the historical solvent control data is exceeded (44.0 mutant colonies/10E6 cells compared to 0.6 – 32.4 mutant colonies/10E6 cells of the historical solvent control data). However, the induction factor of the parallel culture remained below the threshold and this event is therefore considered to be not reproducible and of no biological relevance. - Conclusions:
- Interpretation of results:
negative
Referenceopen allclose all
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 ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
TA102 |
TA98 |
TA97a |
||
|
Water |
154 ± 20.2 |
16 ±8.4 |
n.d. |
n.d. |
n.d. |
– |
DMSO |
132 ± 25.4 |
16 ± 5.8 |
153 ± 18.2 |
12 ± 2.1 |
120 ± 18.1 |
– |
50 |
126 ± 19 |
12 ± 6 |
165 ±15 |
8 ±1 |
108 ± 3 |
– |
150 |
155 ± 9 |
10 ± 3 |
161 ±45 |
15 ± 4 |
89 ± 10 |
– |
500 |
135± 22 |
6 ± 5 |
146 ± 21 |
9 ± 4 |
128 ±10 |
– |
1501 |
140±12 |
9 ± 1 |
161 ± 13 |
7 ± 2 |
127 ±32 |
– |
5004 |
158 ± 22 |
14 ± 4 |
82 ± 55 |
9 ± 5 |
121 ± 54 |
Positive controls, –S9 |
Name |
SA |
SA |
4NPD |
4NPD |
4NPD |
Concentrations (μg/plate) |
6 |
6 |
80 |
80 |
80 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1001 |
1001 |
1001 |
1001 |
1001 |
|
|
Water |
n.d. |
n.d. |
n.d. |
n.d. |
n.d. |
+ |
DMSO |
140 ± 17.4 |
13 ± 3.4 |
145 ± 37.3 |
15 ±1.4 |
120 ± 18.1 |
+ |
50 |
125 ± 7 |
12 ± 6 |
158 ± 42 |
10 ± 3 |
115 ± 12 |
+ |
150 |
146± 13 |
12 ± 4 |
105 ± 61 |
8 ± 1 |
143 ± 18 |
+ |
500 |
166 ± 30 |
13 ± 5 |
152 ± 21 |
8 ± 2 |
149 ± 36 |
+ |
1500 |
143 ± 43 |
11 ± 6 |
141 ± 27 |
8 ± 2 |
151 ± 24 |
+ |
5000 |
0 148 ± 28 |
11 ± 3 |
166 ± 28 |
8 ± 2 |
144 ± 28 |
Positive controls, +S9 |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
Concentrations (μg/plate) |
3 |
3 |
3 |
40 |
3 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1001 |
1001 |
1001 |
150 ± 7 |
1001 |
|
1001 = more than 1000 colonies
4NPD = 4-Nitro-1,2-phenylendiamin
2AA = 2-Aminoanthracene
BP = Benzo(a)pyrene
Table 2. Results of experiment 2 (pre-incubation).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
TA102 |
TA98 |
TA97a |
||
|
Water |
137 ± 30 |
12 ± 1 |
n.d. |
n.d. |
n.d. |
– |
DMSO |
180 ± 9.5 |
12 ± 2.4 |
147 ± 29.6 |
9 ± 2.9 |
161 ± 65.9 |
– |
1250 |
122 ± 32 |
9 ± 2 |
126 ± 9 |
9 ± 1 |
125 ± 22 |
– |
2500 |
95 ± 15 |
11 ± 3 |
132 ± 46 |
6 ± 1 |
138 ± 27 |
– |
5001 |
113 ± 12 |
11 ± 4 |
106 ± 48 |
7 ± 2 |
129 ± 15 |
Positive controls. –S9 |
Name |
SA |
SA |
4NPD |
4NPD |
4NPD |
Concentrations (μg/plate) |
6 |
6 |
80 |
80 |
80 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1001 |
1001 |
1001 |
1001 |
1001 |
|
|
Water |
n.d. |
n.d. |
n.d. |
n.d. |
n.d. |
+ |
DMSO |
174 ± 31.7 |
13 ± 2.9 |
117 ± 36.8 |
11 ± 3.7 |
132 ± 35.3 |
+ |
1250 |
143 ± 18 |
9 ± 1 |
136 ± 17 |
7 ± 3 |
159 ± 11 |
+ |
2500 |
142 ± 27 |
9 ± 2 |
154 ± 12 |
7 ± 1 |
116 ± 6 |
+ |
5001 |
130 ± 13 |
10 ± 2 |
68 ± 56 |
7 ± 2 |
131 ± 13 |
Positive controls. +S9 |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
Concentrations (μg/plate) |
3 |
3 |
3 |
40 |
3 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1001 |
1001 |
1001 |
1001 |
1001 |
|
1001 = more than 1000 colonies
4NPD = 4-Nitro-1,2-phenylendiamin
2AA = 2-Aminoanthracene
BP = Benzo(a)pyrene
Table 1. Test results of experiment 1 (plate incorporation).
With or without S9-Mix |
Test substance concentration |
Mean number of revertant colonies per plate |
||||
(μg/plate) |
(average of 3 plates ± Standard deviation) |
|||||
|
Base-pair substitution type |
Frameshift type |
||||
|
TA 100 |
TA1535 |
TA98 |
TA1537 |
||
– |
Solvent |
135 ± 5 |
15 ± 4 |
28 ± 2 |
8 ± 3 |
|
– |
20 |
95 ± 6 |
14 ± 2 |
27 ± 6 |
14 ± 4 |
|
– |
100 |
13 ± 3 |
B |
20 ± 7 |
3 ± 2 |
|
– |
500 |
B |
B |
7 ± 1 |
6 ± 4 |
|
– |
2500 |
B |
B |
2 ± 1 |
B |
|
– |
5000 |
B |
B |
B |
B |
|
Positive controls, –S9 |
Name |
MNNG |
MNNG |
NPD |
AAC |
|
Concentrations (μg/plate) |
5 |
5 |
10 |
100 |
||
Mean No. of colonies/plate (average of 3 ± SD) |
1630 ± 270 |
1340 ± 61 |
614 ± 49 |
973 ± 188 |
||
+ |
Solvent |
119 ± 8 |
17 ± 3 |
38 ± 5 |
9 ± 2 |
|
+ |
20 |
115 ± 6 |
12 ± 3 |
38 ± 7 |
14 ± 1 |
|
+ |
100 |
114 ± 1 |
13 ± 4 |
46 ± 10 |
10 ± 5 |
|
+ |
500 |
28 ± 18 |
2 ± 2 |
30 ± 8 |
4 ± 2 |
|
+ |
2500 |
B |
B |
21 ± 2 |
3 ± 0 |
|
+ |
5000 |
B |
B |
10 ± 7 |
B |
|
Positive controls, –S9 |
Name |
2AA |
2AA |
2AA |
2AA |
|
Concentrations (μg/plate) |
10 |
10 |
10 |
10 |
||
Mean No. of colonies/plate (average of 3 ± SD) |
1880 ± 101 |
128 ± 20 |
1617 ± 64 |
125 ± 5 |
||
B = Reduced background lawn
MNNG = N-methyl-N´-nitro-N-nitroso-guanidine
2AA = 2-Aminoanthracene
NPD = 4-nitro-o-phenylendiamine
AAC = 9-aminoacridine
Table 2. Test results of experiment 2 (plate incorporation).
With or without S9-Mix |
Test substance concentration |
Mean number of revertant colonies per plate |
|||
(μg/plate) |
(average of 3 plates ± Standard deviation) |
||||
|
Base-pair substitution type |
Frameshift type |
|||
|
TA 100 |
TA1535 |
TA98 |
TA1537 |
|
– |
Solvent |
106 ± 8 |
21 ± 5 |
28 ± 2 |
9 ± 2 |
– |
3 |
102 ± 8 |
18 ± 8 |
30 ± 4 |
9 ± 2 |
– |
6 |
109 ± 5 |
20 ± 3 |
30 ± 3 |
15 ± 4 |
– |
12 |
98 ± 4 |
20 ± 3 |
40 ± 3 |
9 ± 3 |
– |
25 |
104 ± 7 |
17 ± 5 |
32 ± 5 |
12 ± 1 |
– |
50 |
57 ± 7 |
10 ± 2 |
29 ± 4 |
7 ± 0 |
Positive controls, –S9 |
Name |
MNNG |
MNNG |
NPD |
AAC |
Concentrations (μg/plate) |
5 |
5 |
10 |
100 |
|
|
|||||
Mean No. of colonies/plate (average of 3 ± SD) |
1977 ± 45 |
1843 ± 5 |
832 ± 44 |
483 ± 109 |
|
+ |
Solvent |
110 ± 5 |
15 ± 1 |
43 ± 3 |
13 ± 2 |
+ |
0.8 |
94 ± 10 |
14 ± 2 |
43 ± 2 |
12 ± 2 |
+ |
4 |
101 ± 11 |
13 ± 2 |
46 ± 1 |
16 ± 3 |
+ |
20 |
101 ± 9 |
14 ± 3 |
43 ± 5 |
11 ± 3 |
+ |
100 |
95 ± 9 |
15 ± 3 |
49 ± 9 |
11 ± 3 |
+ |
500 |
22 ± 6 |
2 ± 1 |
41 ± 4 |
6 ± 1 |
Positive controls, –S9 |
Name |
2AA |
2AA |
2-AA |
2AA |
Concentrations (μg/plate) |
10 |
10 |
10 |
10 |
|
|
|||||
Mean No. of colonies/plate (average of 3 ± SD) |
2050 ± 50 |
121 ± 9 |
1713 ± 90 |
127 ± 10 |
|
B = Reduced background lawn
MNNG = N-methyl-N´-nitro-N-nitroso-guanidine
2AA = 2-Aminoanthracene
NPD = 4-nitro-o-phenylendiamine
AAC = 9-aminoacridine
Table 3. Test results of experiment 3 (pre-incubation).
With or without S9-Mix |
Test substance concentration |
Mean number of revertant colonies per plate |
|||
(μg/plate) |
(average of 3 plates ± Standard deviation) |
||||
|
Base-pair substitution type |
Frameshift type |
|||
|
TA 100 |
TA1535 |
TA98 |
TA1537 |
|
– |
Solvent |
98 ± 8 |
23 ± 2 |
21 ± 7 |
11 ± 2 |
– |
3 |
99 ± 10 |
22 ± 7 |
17 ± 4 |
13 ± 3 |
– |
6 |
94 ± 6 |
24 ± 2 |
21 ± 3 |
14 ± 6 |
– |
12 |
87 ± 20 |
25 ± 5 |
22 ± 3 |
9 ± 3 |
– |
25 |
76 ± 12 |
26 ± 3 |
25 ± 4 |
11 ± 2 |
– |
50 |
78 ± 10 |
24 ± 4 |
24 ± 5 |
12 ± 1 |
Positive controls, –S9 |
Name |
MNNG |
MNNG |
NPD |
AAC |
Concentrations (μg/plate) |
5 |
5 |
10 |
100 |
|
|
|||||
Mean No. of colonies/plate (average of 3 ± SD) |
1340 ± 193 |
1370 ± 30 |
833 ± 32 |
450 ± 61 |
|
± |
Solvent |
110 ± 16 |
20 ± 2 |
37 ± 1 |
12 ± 2 |
± |
0.8 |
116 ± 18 |
19 ± 1 |
35 ± 4 |
16 ± 5 |
± |
4 |
118 ± 11 |
20 ± 6 |
37 ± 4 |
15 ± 2 |
± |
20 |
95 ± 9 |
24 ± 1 |
41 ± 5 |
15 ± 2 |
± |
100 |
116 ± 8 |
27 ± 3 |
39 ± 4 |
12 ± 4 |
± |
500 |
76 ± 6 |
23 ± 4 |
35 ± 6 |
6 ± 2 |
Positive controls, –S9 |
Name |
2AA |
2AA |
2AA |
2AA |
Concentrations (μg/plate) |
10 |
10 |
10 |
10 |
|
|
|||||
Mean No. of colonies/plate (average of 3 ± SD) |
1310 ± 272 |
240 ± 10 |
923 ± 223 |
333 ± 21 |
|
B = Reduced background lawn
MNNG = N-methyl-N´-nitro-N-nitroso-guanidine
2AA = 2-Aminoanthracene
NPD = 4-nitro-o-phenylendiamine
AAC = 9-aminoacridine
Table 1. Results of cytogenicity experiments.
Test item |
Concentration |
Mitotic Index |
Aberrant cells in % |
||||
|
in µg/mL |
in % |
with gaps |
without gaps |
with exchanges |
||
|
Exposure period 4h, fixation time 22h, without S9 mix |
||||||
DMSO |
0.5% (v/v) |
100.0 |
5 |
1 |
0 |
||
EMS |
600 |
48.4 |
23 |
21.5* |
2.5 |
||
Test substance |
10 |
94.0 |
2.5 |
1.5 |
0 |
||
20 |
70.2 |
3 |
2 |
0.5 |
|||
30 |
50.4 |
0.5 |
0 |
0 |
|||
|
Exposure period 4h, fixation time 22h, with S9 mix |
||||||
DMSO |
0.5% (v/v) |
100 |
2 |
2 |
0 |
||
CPA |
35 |
77.2 |
21.5 |
16.5* |
0.5 |
||
Test substance |
15 |
82.6 |
2.5 |
2 |
0 |
||
30 |
39.1 |
2.5 |
1.5 |
0.5 |
|||
40 |
18.8 |
3 |
2 |
0 |
|||
|
Exposure period 22h, fixation time 22h, without S9 mix |
||||||
DMSO |
0.5% (v/v) |
100.0 |
0 |
0 |
0 |
||
EMS |
350 |
44.8 |
39.5 |
33.5* |
4.5 |
||
Test substance |
50 |
97.1 |
5.5 |
2 |
0 |
||
100 |
77.6 |
3.5 |
2 |
0 |
|||
125 |
36.8 |
2 |
1 |
0 |
|||
|
Exposure period 4h, fixation time 22h, with S9 mix |
||||||
DMSO |
0.5% (v/v) |
100.0 |
1.5 |
1.5 |
0 |
||
CPA |
35 |
70.1 |
33 |
22* |
1 |
||
Test substance |
25 |
94.8 |
3.5 |
2 |
0 |
||
35 |
69.7 |
3.5 |
3 |
0 |
|||
40 |
29.5 |
1 |
1 |
0 |
|||
*:Aberration frequency statistically significant higher than corresponding control values
CPA: Cyclophosphamide
EMS: Ethyl methanesulphonate
Table 1. Results of experiment I without metabolic activation.
Concentration |
Cloning efficiency relative [%] (Survival) |
Cell density in % of control [%] |
Mutants per 1E+06 surviving cells |
Cloning efficiency relative [%] |
Cell density in % of control [%] |
Mutants per 1E+06 surviving cells |
|
Culture I |
Culture II |
||||
0 (Acetone) |
100 |
100 |
11 |
100 |
100 |
12,9 |
EMS 150 |
95,8 |
75,0 |
209,9 |
96,5 |
104,9 |
117,0 |
0,25 |
98,6 |
88,0 |
5,1 |
92,5 |
73,7 |
7,5 |
0,5 |
99,2 |
96,6 |
11,7 |
97,6 |
97,0 |
17,4 |
1 |
100 |
112,5 |
7,3 |
97,8 |
78,8 |
14,8 |
2 |
97,2 |
108,9 |
44,0 |
97,2 |
104,8 |
11,2 |
4 |
91,4 |
10,4 |
0,0 |
89,7 |
8,3 |
4,0 |
6 |
-* |
-* |
||||
8 |
-* |
- |
||||
*: culture was not continued due to strong toxic effects.
**: culture was not continued since a minimum of only four analysable concentrations is required.
Table 2. Results of experiment I with metabolic activation.
Concentration |
Cloning efficiency relative [%] (Survival) |
Cell density in % of control [%] |
Mutants per 1E+06 surviving cells |
Cloning efficiency relative [%] |
Cell density in % of control [%] |
Mutants per 1E+06 surviving cells |
|
Culture I |
Culture II |
||||
0 (Acetone) |
100 |
100 |
29,1 |
100 |
100 |
20,7 |
DMBA 1.1 |
80,7 |
80,2 |
665,9 |
86,6 |
95,5 |
411,3 |
2 |
96,0 |
116,6 |
** |
98,0 |
105,4 |
** |
4 |
92,7 |
98,2 |
28,1 |
95,0 |
100,3 |
10,5 |
8 |
88,5 |
110,7 |
5,2 |
89,4 |
118,5 |
22,5 |
16 |
90,6 |
141,1 |
11,6 |
94,5 |
100,3 |
18,4 |
32 |
87,3 |
95,0 |
13,0 |
85,3 |
88,8 |
23,6 |
48 |
41,3 |
40,7 |
29,8 |
12,1 |
12,5 |
16,8 |
64 |
-* |
-* |
||||
*: culture was not continued due to strong toxic effects.
**: culture was not continued since a minimum of only four analysable concentrations is required.
Table 3. Results of experiment II without metabolic activation.
Concentration |
Cloning efficiency relative [%] (Survival) |
Cell density in % of control [%] |
Mutants per 1E+06 surviving cells |
Cloning efficiency relative [%] |
Cell density in % of control [%] |
Mutants per 1E+06 surviving cells |
|
Without S9 mix |
Without S9 mix |
||||
0 (Acetone) |
100 |
100 |
14,6 |
100 |
100 |
13,5 |
EMS 150 |
81,0 |
133,5 |
224,7 |
110,0 |
129,3 |
260,1 |
2 |
102,3 |
98,9 |
** |
109,7 |
107,6 |
** |
4 |
102,8 |
136,3 |
9,7 |
111,8 |
82,0 |
11,1 |
8 |
103,1 |
115,3 |
17,9 |
111,3 |
93,5 |
16,3 |
16 |
101,9 |
114,3 |
17,3 |
100,9 |
104,9 |
17,8 |
64 |
91,2 |
164,5 |
11,8 |
102,6 |
60,1 |
9,5 |
96 |
24,1 |
32,9 |
15,0 |
25,5 |
23,9 |
22,1 |
128 |
- |
-* |
||||
*: culture was not continued due to strong toxic effects.
**: culture was not continued since a minimum of only four analysable concentrations is required.
Table 4. Results of experiment II with metabolic activation.
Concentration |
Cloning efficiency relative [%] (Survival) |
Cell density in % of control [%] |
Mutants per 1E+06 surviving cells |
Cloning efficiency relative [%] |
Cell density in % of control [%] |
Mutants per 1E+06 surviving cells |
|
With S9 mix |
With S9 mix |
||||
0 (Acetone) |
100 |
100 |
10,5 |
100 |
100 |
10,2 |
DMBA 1.1 |
81,0 |
68,4 |
863,3 |
28,0 |
73,7 |
743,3 |
2 |
102,3 |
62,0 |
** |
91,0 |
92,4 |
** |
4 |
102,8 |
63,2 |
5,4 |
91,7 |
90,7 |
11,2 |
8 |
103,1 |
91,2 |
8,4 |
93,1 |
97,6 |
4,4 |
16 |
101,9 |
101,5 |
15,4 |
90,4 |
100,6 |
8,6 |
48 |
91,2 |
43,5 |
17,4 |
0 |
70,9 |
10,0 |
56 |
24,1 |
3,2 |
7,1 |
0 |
4,3 |
4,1 |
64 |
-* |
-* |
||||
*: culture was not continued due to strong toxic effects.
**: culture was not continued since a minimum of only four analysable concentrations is required.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Genetic toxicity (mutagenicity) in bacteria in-vitro
Two bacterial gene mutation assays (Ames test) were performed with N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) according to OECD guideline 471 and in compliance with GLP.
In the first experiment (LAUS, 2007), the strains Salmonella typhimurium TA97a, TA98, TA 100, TA 102 and TA 1535 were tested in two independent experiments according to the plate incorporation and preincubation procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiments were conducted each in 4 replications at concentrations from 50 to 5004 µg/plate (vehicle: DMSO). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and negative controls showed the expected results. Under the conditions of the study, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.
A further experiment in the tester strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 is available (BASF, 1990). In concentrations from 0.8 to 5000 µg/plate with and without a metabolic activation system, no increase in the number of revertant colonies was observed in any of the strains tested. However, in concentrations from 50 µg/plate onwards cytotoxicity was observed. The included positive and negative controls showed the expected results. Thus, under the conditions of the study, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.
Genetic toxicity (cytogenicity) in mammalian cells in-vitro
An in vitro mammalian chromosome aberration test was conducted with N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) in accordance with OECD guideline 473 under GLP conditions (LAUS, 2010). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of fresh heparinised human whole blood cultures, incubated for 4 h with and without and for 22 h without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). Concentrations of 10-40 µg/mL (4 h incubation) and 0.5-50 µg/mL (22 h incubation) of the test substance in the vehicle DMSO were applied. The negative as well as the positive controls showed the expected results. In the pre-experiment cytotoxicity was observed from 55.3 µg/mL with and without metabolic activation. In the main experiments a reduction in the mitotic index was observed from 30 µg/mL without and from 40 µg/mL with metabolic activation. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed. Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.
Genetic toxicity (mutagenicity) in mammalian cells in-vitro
The in vitro mammalian cell gene mutation study of N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) was carried out according to OECD guideline 476 under GLP conditions (Harlan, 2010). Gene mutations in the HPRT locus were investigated in Chinese hamster lung fibroblasts (V79) in the presence and absence of a metabolic activation system (Phenobarbital/beta-naphtoflavone-induced rat liver, S9). V79 cells were incubated with the test material at 0.25, 0.5, 1, 2 and 4 µg/mL for 4 h in the absence and at 4, 8, 16, 32 and 48 µg/mL in the presence of a metabolic activation system. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 4 to 96 µg/mL, and concentrations with metabolic activation for an exposure period of 4 h ranged from 4 to 56 µg/mL. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data of the laboratory. Cytotoxicity was apparent at concentrations from 4 µg/mL and 48 µg/mL in experiment I without and with metabolic activation, respectively. In the second experiment, cytotoxicity was observed from 96 µg/mL and 56 µg/mL without and with metabolic activation, respectively. In the second experiment, precipitation was observed from 237.5 µg/mL after 24 h without metabolic activation. There was no significant increase in the number of forward mutations at the HPRT locus of V79 cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study, the test substance did not show gene mutation activity in this test performed in V79 cells in vitro.
Justification for classification or non-classification
The available data on genetic toxicity with N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.
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