Registration Dossier
Registration Dossier
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EC number: 701-298-1 | CAS number: -
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 July to 20 October 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Dialkyl C18 and C18-unsaturated phosphonates
- EC Number:
- 701-298-1
- Cas Number:
- 64051-29-2
- Molecular formula:
- Not applicable for a UVCB Substance
- IUPAC Name:
- Dialkyl C18 and C18-unsaturated phosphonates
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Alkenyl phosphonate
- Expiration date of the lot/batch: 09 September 2017
- Purity test date: 08/06/2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%)
- Stability under test conditions: yes
Method
- Target gene:
- Histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA.
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Dose range finding test: dose range: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate with and without S9-mix (TA98 and TA100)
Experiment 1, with and without metabolic activation: 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate
Experiment 2, with and without metabolic activation: 1.581, 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate. - Vehicle / solvent:
- In the study three vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive control chemicals. The following chemicals were used for vehicle (solvent) control groups:
1- Dimethyl sulfoxide (puriss. p.a., dried, water ≤ 0.02%):
- Supplier: Sigma-Aldrich Co.
- Batch No.: STBG8411
- Expiry date: 29 February 2020
- Purity: >99.99%
2- Distilled water:
- Manufacturer: Hungaro-Gal Kft.
- Batch No.: 802 0117 / 8080617
- Expiry date: 23 July 2017 / 06 December 2017
3- N,N-dimethylformamide (DMF)
- Supplier: VWR
- Batch No.: 17C034009
- Expiry date: 28 February 2022
Controls
- Untreated negative controls:
- yes
- Remarks:
- Dimethyl sulfoxide, Distilled water and N,N-dimethylformamide (DMF).
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulfoxide, Distilled water and N,N-dimethylformamide (DMF).
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-1,2-phenylenediamine (NDP) and 2-aminoanthracene (2AA)
- Remarks:
- with and without metabolic activation.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation method
DURATION
- Preincubation period: 20 minutes at 37°C
NUMBER OF REPLICATIONS: 3 plates/dose/strain
DETERMINATION OF CYTOTOXICITY
- Method: background lawn of microcolonies observation - Evaluation criteria:
- The test article was considered to be positive for mutagenicity if the mean number of revertant colonies in the test article group was no less than 2 times that of the negative control group, and if the number of revertant colonies increased with increasing dose of the test article.
- Statistics:
- Statistical methods were not used in the determination of test results.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was on the plates in the Preliminary Concentration Range Finding Test. Precipitate/slight precipitate/ microdrops was/were detected on the plates in the main tests at 5000 μg/plate concentration without metabolic activation.
RANGE-FINDING: yes
Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 15.81 and 5 μg/plate, in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
COMPARISON WITH HISTORICAL CONTROL DATA: yes - Remarks on result:
- other:
- Remarks:
- Slightly reduced background lawn was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA100 and TA1537 strains without metabolic activation on the plates at 5000 and 1581 µg/plate.
Any other information on results incl. tables
Table 1: Summary Table of the Preliminary Concentration Range Finding Test
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimuriumtester strains |
||||
TA 98 |
TA 100 |
|||||
-S9 |
+S9 |
-S9 |
+S9 |
|||
Untreated control |
Mean |
24.0 |
27.3 |
95.0 |
100.3 |
|
MF |
1.09 |
1.17 |
1.04 |
1.03 |
||
Distilled water control |
Mean |
-- |
-- |
90.7 |
-- |
|
MF |
-- |
-- |
0.99 |
-- |
||
DMSO control |
Mean |
21.0 |
27.0 |
-- |
104.3 |
|
MF |
0.95 |
1.16 |
-- |
1.08 |
||
DMF control |
Mean |
22.0 |
23.3 |
91.7 |
97.0 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
||
5000 |
Mean |
24.0 |
27.0 |
90.0 |
108.7 |
|
MF |
1.09 |
1.16 |
0.98 |
1.12 |
||
2500 |
Mean |
18.0 |
25.0 |
99.3 |
102.0 |
|
MF |
0.82 |
1.07 |
1.08 |
1.05 |
||
1000 |
Mean |
27.7 |
25.0 |
108.0 |
114.3 |
|
MF |
1.26 |
1.07 |
1.18 |
1.18 |
||
316 |
Mean |
25.0 |
27.7 |
107.0 |
105.0 |
|
MF |
1.14 |
1.19 |
1.17 |
1.08 |
||
100 |
Mean |
22.7 |
26.3 |
110.0 |
113.0 |
|
MF |
1.03 |
1.13 |
1.20 |
1.16 |
||
31.6 |
Mean |
21.3 |
24.0 |
104.0 |
124.0 |
|
MF |
0.97 |
1.03 |
1.13 |
1.28 |
||
10 |
Mean |
21.3 |
22.3 |
107.7 |
148.3 |
|
MF |
0.97 |
0.96 |
1.17 |
1.53 |
||
NPD (4mg) |
Mean |
396.7 |
-- |
-- |
-- |
|
MF |
18.89 |
-- |
-- |
-- |
||
2AA (2mg) |
Mean |
-- |
2401.3 |
-- |
2412.7 |
|
MF |
-- |
88.94 |
-- |
23.12 |
||
SAZ (2mg) |
Mean |
-- |
-- |
1212.7 |
-- |
|
MF |
-- |
-- |
13.38 |
-- |
||
Table 2: Summary Table of the Initial Mutation Test
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella thyphimuriumtester strains |
Escherichia coli |
|||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||
Untreated control |
Mean |
20.7 |
24.3 |
92.7 |
95.7 |
12.3 |
11.0 |
12.3 |
14.7 |
50.7 |
56.0 |
|
MF |
1.02 |
1.16 |
1.07 |
1.01 |
1.19 |
0.97 |
0.93 |
1.05 |
0.92 |
1.06 |
||
Distilled water control |
Mean |
-- |
-- |
92.0 |
-- |
11.3 |
-- |
-- |
-- |
56.3 |
-- |
|
MF |
-- |
-- |
1.07 |
-- |
1.10 |
-- |
-- |
-- |
1.02 |
-- |
||
DMSO control |
Mean |
19.0 |
26.7 |
-- |
90.7 |
-- |
9.0 |
13.7 |
14.7 |
-- |
56.0 |
|
MF |
0.93 |
1.27 |
-- |
0.96 |
-- |
0.79 |
1.03 |
1.05 |
-- |
1.06 |
||
DMF control |
Mean |
20.3 |
21.0 |
86.3 |
94.3 |
10.3 |
11.3 |
13.3 |
14.0 |
55.3 |
53.0 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
||
5000 |
Mean |
21.3 |
25.0 |
38.7 |
74.0 |
10.7 |
10.7 |
19.7 |
15.7 |
44.0 |
55.3 |
|
MF |
1.05 |
1.19 |
0.45 |
0.78 |
1.03 |
0.94 |
1.48 |
1.12 |
0.80 |
1.04 |
||
1581 |
Mean |
21.0 |
22.7 |
54.0 |
79.0 |
16.0 |
11.7 |
17.0 |
19.3 |
47.0 |
51.0 |
|
MF |
1.03 |
1.08 |
0.63 |
0.84 |
1.55 |
1.03 |
1.28 |
1.38 |
0.85 |
0.96 |
||
500 |
Mean |
19.0 |
21.0 |
65.3 |
78.7 |
12.3 |
10.7 |
16.7 |
20.0 |
43.5 |
46.7 |
|
MF |
0.93 |
1.00 |
0.76 |
0.83 |
1.19 |
0.94 |
1.25 |
1.43 |
0.79 |
0.88 |
||
158.1 |
Mean |
20.7 |
21.0 |
72.0 |
80.0 |
9.0 |
12.0 |
16.7 |
17.0 |
47.7 |
50.0 |
|
MF |
1.02 |
1.00 |
0.83 |
0.85 |
0.87 |
1.06 |
1.25 |
1.21 |
0.86 |
0.94 |
||
50 |
Mean |
21.0 |
22.0 |
70.7 |
79.7 |
11.0 |
8.7 |
18.3 |
21.7 |
50.0 |
52.0 |
|
MF |
1.03 |
1.05 |
0.82 |
0.84 |
1.06 |
0.76 |
1.38 |
1.55 |
0.90 |
0.98 |
||
15.81 |
Mean |
20.7 |
22.7 |
81.3 |
88.0 |
16.0 |
12.7 |
21.0 |
17.0 |
47.7 |
48.0 |
|
MF |
1.02 |
1.08 |
0.94 |
0.93 |
1.55 |
1.12 |
1.58 |
1.21 |
0.86 |
0.91 |
||
5 |
Mean |
19.7 |
23.3 |
83.7 |
82.3 |
10.0 |
7.0 |
16.7 |
21.0 |
40.7 |
46.3 |
|
MF |
0.97 |
1.11 |
0.97 |
0.87 |
0.97 |
0.62 |
1.25 |
1.50 |
0.73 |
0.87 |
||
NPD (4mg) |
Mean |
429.3 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
MF |
22.60 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||
2AA (2mg) |
Mean |
-- |
2481.3 |
-- |
2482.7 |
-- |
201.3 |
-- |
200.7 |
-- |
-- |
|
MF |
-- |
93.05 |
-- |
27.38 |
-- |
22.37 |
-- |
13.68 |
-- |
-- |
||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
282.7 |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.05 |
||
SAZ (2mg) |
Mean |
-- |
-- |
1158.7 |
-- |
1156.0 |
-- |
-- |
-- |
-- |
-- |
|
MF |
-- |
-- |
12.59 |
-- |
102.00 |
-- |
-- |
-- |
-- |
-- |
||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
404.0 |
-- |
-- |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
29.56 |
-- |
-- |
-- |
||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
986.7 |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
17.51 |
-- |
||
Table 3: Summary Table of the Confirmatory Mutation Test
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella thyphimuriumtester strains |
Escherichia coli |
|||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||
Untreated control |
Mean |
24.7 |
32.3 |
99.0 |
99.0 |
12.3 |
14.7 |
12.7 |
14.3 |
53.0 |
57.7 |
|
MF |
0.84 |
1.03 |
1.03 |
0.99 |
0.82 |
1.26 |
0.86 |
0.98 |
0.95 |
1.09 |
||
Distilled water control |
Mean |
-- |
-- |
97.0 |
-- |
15.0 |
-- |
-- |
-- |
55.3 |
-- |
|
MF |
-- |
-- |
1.01 |
-- |
1.00 |
-- |
-- |
-- |
0.99 |
-- |
||
DMSO control |
Mean |
28.3 |
29.7 |
-- |
99.3 |
-- |
14.7 |
13.3 |
12.0 |
-- |
53.7 |
|
MF |
0.97 |
0.95 |
-- |
1.00 |
-- |
1.26 |
0.91 |
0.82 |
-- |
1.02 |
||
DMFcontrol |
Mean |
29.3 |
31.3 |
95.7 |
99.7 |
15.0 |
11.7 |
14.7 |
14.7 |
56.0 |
52.7 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
||
5000 |
Mean |
20.0 |
31.3 |
56.7 |
97.7 |
12.7 |
14.7 |
11.7 |
16.0 |
47.7 |
57.7 |
|
MF |
0.68 |
1.00 |
0.59 |
0.98 |
0.84 |
1.26 |
0.80 |
1.09 |
0.85 |
1.09 |
||
1581 |
Mean |
18.0 |
29.3 |
57.3 |
92.7 |
16.0 |
16.0 |
11.3 |
14.3 |
48.0 |
55.0 |
|
MF |
0.61 |
0.94 |
0.60 |
0.93 |
1.07 |
1.37 |
0.77 |
0.98 |
0.86 |
1.04 |
||
500 |
Mean |
18.3 |
27.0 |
60.0 |
92.3 |
16.3 |
15.0 |
11.0 |
13.7 |
44.3 |
56.0 |
|
MF |
0.63 |
0.86 |
0.63 |
0.93 |
1.09 |
1.29 |
0.75 |
0.93 |
0.79 |
1.06 |
||
158.1 |
Mean |
23.7 |
27.0 |
72.3 |
92.0 |
16.3 |
15.3 |
12.3 |
14.7 |
48.3 |
55.7 |
|
MF |
0.81 |
0.86 |
0.76 |
0.92 |
1.09 |
1.31 |
0.84 |
1.00 |
0.86 |
1.06 |
||
50 |
Mean |
20.7 |
30.0 |
76.3 |
93.0 |
18.0 |
16.3 |
11.7 |
15.0 |
48.3 |
59.0 |
|
MF |
0.70 |
0.96 |
0.80 |
0.93 |
1.20 |
1.40 |
0.80 |
1.02 |
0.86 |
1.12 |
||
15.81 |
Mean |
18.0 |
27.7 |
74.7 |
95.7 |
16.3 |
18.7 |
13.3 |
13.0 |
51.7 |
58.7 |
|
MF |
0.61 |
0.88 |
0.78 |
0.96 |
1.09 |
1.60 |
0.91 |
0.89 |
0.92 |
1.11 |
||
5 |
Mean |
20.7 |
26.3 |
70.0 |
98.0 |
16.7 |
16.7 |
14.0 |
14.3 |
52.3 |
63.0 |
|
MF |
0.70 |
0.84 |
0.73 |
0.98 |
1.11 |
1.43 |
0.95 |
0.98 |
0.93 |
1.20 |
||
1.581 |
Mean |
21.3 |
24.3 |
88.3 |
87.7 |
18.0 |
16.3 |
11.3 |
14.3 |
56.3 |
59.0 |
|
MF |
0.73 |
0.78 |
0.92 |
0.88 |
1.20 |
1.40 |
0.77 |
0.98 |
1.01 |
1.12 |
||
NPD (4mg) |
Mean |
400.7 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
MF |
14.14 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||
2AA (2mg) |
Mean |
-- |
2432.0 |
-- |
2352.0 |
-- |
202.7 |
-- |
204.0 |
-- |
-- |
|
MF |
-- |
81.98 |
-- |
23.68 |
-- |
13.82 |
-- |
17.00 |
-- |
-- |
||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
318.7 |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.94 |
||
SAZ (2mg) |
Mean |
-- |
-- |
1202.7 |
-- |
1172.0 |
-- |
-- |
-- |
-- |
-- |
|
MF |
-- |
-- |
13.36 |
-- |
78.13 |
-- |
-- |
-- |
-- |
-- |
||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
452.0 |
-- |
-- |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
33.90 |
-- |
-- |
-- |
||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1038.7 |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
18.77 |
-- |
||
Applicant's summary and conclusion
- Conclusions:
- Under the test conditions of this study, Alkenyl phosphonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
- Executive summary:
In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 guideline, Alkenyl phosphonate was tested in S. typhimurium TA1535, TA1537, TA100 and TA98 and in E. coli WP2 uvr A in the presence and the absence of mammalian metabolic activation (S9) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats,using the preincubation method.
Based on the results of the Compatibility Test, the test item was dissolved in N,N-Dimethylformamide (DMF) at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 15.81 and 5 μg/plate, in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
No precipitate was on the plates in the Preliminary Concentration Range Finding Test. Precipitate/slight precipitate/ microdrops was/were detected on the plates in the main tests at 5000 μg/plate concentration without metabolic activation. Inhibitory, cytotoxic effect of the test item was not detected in thePreliminary Concentration Range Finding Test and in theInitial Mutation Test. Slightly reduced background lawn was observed in the Confirmatory Mutation Test inSalmonella typhimuriumTA100 and TA1537 strains without metabolic activation on the plates at 5000 and 1581 µg/plate. In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.
The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid.
Under the test conditions of this study, Alkenyl phosphonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
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