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EC number: 228-602-0 | CAS number: 6303-30-6
- Life Cycle description
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- Endpoint summary
- Appearance / physical state / colour
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- Ecotoxicological Summary
- Aquatic toxicity
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- Short-term toxicity to fish
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- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In an AMES test, performed according to OECD guideline 471 and in accordance with GLP principles, diisobutyl hydrogen phosphate was found not to be mutagenic with or 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:
- 23 July 2020 - 10 August 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:
- 26 June 2020
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 31 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment
- Version / remarks:
- 31 March 2011
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Salmonella typhimurium: histidine
Escherichia coli: tryptophan - 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:
- Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight)
- method of preparation of S9 mix : S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- volume of S9 mix and S9 in the final culture medium: 0.5 mL S9-mix - Test concentrations with justification for top dose:
- Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
First experiment (direct plate assay): 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (pre-incubation assay): 52, 164, 512, 1600 and 5000 μg/plate - Vehicle / solvent:
- - Solvent used: DMSO
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: 6-Chloro-9-(3-N-(2-Chloroethylamino) propylamino-2-methoxyacridine dihydrochloride ICR-191
- Remarks:
- Without metabolic activation
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (2AA)
- Remarks:
- With metabolic activation
- 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:
- Cell density: 10^9 cells/mL
- Test substance added in agar (plate incorporation) (experiment 1) and pre-incubated (experiment 2)
TREATMENT AND HARVEST SCHEDULE:
Experiment 1 (the dose-range finding study with two tester strains is reported as part of the direct plate assay, experiment 1)
- Duration of treatment: 48 ± 4 h
- Temperature during treatment: 37.0 ± 1.0°C
Experiment 2
- Preincubation period: 30 ± 2 minutes
- Temperature during preincubation period: 37 ± 1°C
- Harvest time after the end of treatment: 48 ± 4 h
- Temperature after end of treatment: 37.0 ± 1.0°C
CYTOTOXICITY:
- To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
FORMULATION ANALYSIS
- Sample collection: See under 'Any other information on materials and methods incl. tables'.
- Concentration and homogeneity analysis:
Storage conditions: At room temperature
Acceptance criteria: For concentration: mean sample concentration results within or equal to ± 10% of theoretical concentration. For homogeneity, relative standard deviation (RSD) of concentrations of ≤ 10% for each group.
- Stability analysis:
Conditions tested: 4 h at room temperature
Storage conditions: Temperature set to maintain room temperature
Acceptance criteria: Mean sample concentration results after storage that is within or equal to ±10% of the initial mean sample concentration results. - Evaluation criteria:
- In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
- The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
- The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
- The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
- In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment. - Key result
- Species / strain:
- S. typhimurium TA 1535
- 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
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle 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
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test item on the plates was not observed in any tester strain.
STUDY RESULTS
- Concurrent vehicle negative and positive control data : See table 1-3
Ames test:
EXPERIMENT 1
- Signs of toxicity : In strain TA100 (absence and presence) a slight reduction of the bacterial background lawn was observed. No biologically relevant decreases in the number of revertants were observed.
- Mutagenicity: In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
EXPERIMENT 2
- Signs of toxicity: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
- Mutagenicity: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
HISTORICAL CONTROL DATA
- The negative and strain-specific positive control values were within our laboratory historical cont rol data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly (see table 4 and 5).
FORMULATION ANALYSIS
- Accuracy: In the vehicle, no test item was detected. The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
- Homogeneity: The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%).
- Stability: Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours. - Conclusions:
- In an AMES test, performed according to OECD guideline 471 and in accordance with GLP principles, Diisobutyl hydrogen phosphate was found not to be mutagenic with or without metabolic activation.
- Executive summary:
In an AMES test, performed according to OECD guideline 471 and in accordance with GLP principles. The test item did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. In the second mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrAin the pre-incubation assay. In all three experiments the test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strain TA100 at the dose level of 5000μg/platein the absence and presence of S9-mix in the dose-range finding study. No toxicity was observed in any of the other strains at all conditions tested. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that diisobutyl hydrogen phosphate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation. Since all acceptability criteria were met, the study is considered valid.
Reference
Table 1 Dose-Range Finding Test: Mutagenic Response of Diisobutyl
hydrogen phosphate in theSalmonella typhimuriumReverse Mutation
Assay and in theEscherichia coliReverse Mutation Assay
Direct Plate Assay
(µg/plate) |
|
||
|
TA100 |
WP2uvrA |
|
Without S9-mix
Positive control |
798 |
± |
70 |
|
1234 |
± |
44 |
|
Solvent control |
100 |
± |
15 |
|
11 |
± |
6 |
|
1.7 |
107 |
± |
11 |
|
13 |
± |
2 |
|
5.4 |
99 |
± |
13 |
|
11 |
± |
3 |
|
17 |
99 |
± |
9 |
|
15 |
± |
3 |
|
52 |
95 |
± |
18 |
|
21 |
± |
8 |
|
164 |
120 |
± |
2 |
|
16 |
± |
5 |
|
512 |
100 |
± |
9 |
|
15 |
± |
1 |
|
1600 |
101 |
± |
12 |
n |
21 |
± |
5 |
|
5000 |
93 |
± |
10 |
s NP |
17 |
± |
8 |
n NP |
|
|
|
|
|
|
|
|
|
With S9-mix
Positive control |
1935 |
± |
562 |
|
400 |
± |
69 |
|
Solvent control |
96 |
± |
11 |
|
18 |
± |
5 |
|
1.7 |
93 |
± |
10 |
|
15 |
± |
4 |
|
5.4 |
89 |
± |
19 |
|
18 |
± |
5 |
|
17 |
108 |
± |
21 |
|
24 |
± |
3 |
|
52 |
89 |
± |
9 |
|
22 |
± |
6 |
|
164 |
92 |
± |
14 |
|
18 |
± |
1 |
|
512 |
106 |
± |
1 |
|
19 |
± |
11 |
|
1600 |
102 |
± |
16 |
n |
19 |
± |
4 |
|
5000 |
80 |
± |
6 |
s NP |
23 |
± |
9 |
n NP |
Table 2 Experiment 1: Mutagenic Response of Diisobutyl hydrogen phosphate in theSalmonella typhimuriumReverse Mutation Assay
Direct Plate Assay
(µg/plate) |
|
||
|
|
|
|
Without S9-mix
Positive control |
863 |
± |
118 |
|
968 |
± |
37 |
|
1377 |
± |
149 |
|
Solvent control |
8 |
± |
3 |
|
3 |
± |
2 |
|
18 |
± |
4 |
|
52 |
8 |
± |
1 |
|
3 |
± |
1 |
|
16 |
± |
4 |
|
164 |
10 |
± |
3 |
|
6 |
± |
4 |
|
14 |
± |
6 |
|
512 |
9 |
± |
1 |
|
5 |
± |
2 |
|
15 |
± |
5 |
|
1600 |
11 |
± |
3 |
|
4 |
± |
3 |
|
15 |
± |
4 |
|
5000 |
10 |
± |
2 |
n NP |
4 |
± |
0 |
n NP |
16 |
± |
2 |
n NP |
|
|
|
|
|
|
|
|
|
|
|
|
|
With S9-mix
Positive control |
248 |
± |
22 |
|
416 |
± |
39 |
|
1182 |
± |
156 |
|
Solvent control |
11 |
± |
4 |
|
5 |
± |
2 |
|
21 |
± |
5 |
|
52 |
14 |
± |
1 |
|
4 |
± |
1 |
|
17 |
± |
5 |
|
164 |
10 |
± |
5 |
|
5 |
± |
2 |
|
17 |
± |
6 |
|
512 |
12 |
± |
1 |
|
8 |
± |
3 |
|
19 |
± |
1 |
|
1600 |
22 |
± |
3 |
|
4 |
± |
1 |
|
21 |
± |
7 |
|
5000 |
19 |
± |
6 |
n NP |
4 |
± |
2 |
n NP |
21 |
± |
2 |
n NP |
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 3 Experiment 2: Mutagenic Response of Diisobutyl hydrogen phosphate in theSalmonella typhimuriumReverse Mutation Assay and in theEscherichia coliReverse Mutation Assay
Pre-incubation Assay
(µg/plate) |
|
||||
|
|
|
|
|
|
Without S9-mix
Positive control |
936 |
± |
73 |
|
198 |
± |
21 |
|
1697 |
± |
133 |
|
703 |
± |
30 |
|
1715 |
± |
67 |
|
Solvent control |
10 |
± |
5 |
|
4 |
± |
1 |
|
14 |
± |
3 |
|
109 |
± |
12 |
|
14 |
± |
6 |
|
52 |
11 |
± |
4 |
|
5 |
± |
1 |
|
14 |
± |
3 |
|
106 |
± |
13 |
|
17 |
± |
3 |
|
164 |
8 |
± |
2 |
|
8 |
± |
4 |
|
17 |
± |
10 |
|
85 |
± |
8 |
|
14 |
± |
2 |
|
512 |
14 |
± |
6 |
|
2 |
± |
1 |
|
10 |
± |
2 |
|
84 |
± |
12 |
|
17 |
± |
6 |
|
1600 |
13 |
± |
3 |
|
4 |
± |
1 |
|
15 |
± |
10 |
|
87 |
± |
7 |
|
17 |
± |
6 |
|
5000 |
10 |
± |
4 |
n NP |
5 |
± |
3 |
n NP |
14 |
± |
2 |
n NP |
93 |
± |
8 |
n NP |
16 |
± |
2 |
n NP |
With S9-mix1
Positive control |
259 |
± |
15 |
|
130 |
± |
6 |
|
709 |
± |
15 |
|
1560 |
± |
61 |
|
453 |
± |
49 |
|
Solvent control |
11 |
± |
7 |
|
5 |
± |
5 |
|
19 |
± |
1 |
|
95 |
± |
13 |
|
18 |
± |
11 |
|
52 |
15 |
± |
0 |
|
6 |
± |
5 |
|
19 |
± |
1 |
|
93 |
± |
13 |
|
25 |
± |
7 |
|
164 |
8 |
± |
4 |
|
4 |
± |
3 |
|
18 |
± |
10 |
|
98 |
± |
8 |
|
19 |
± |
8 |
|
512 |
14 |
± |
6 |
|
7 |
± |
4 |
|
20 |
± |
2 |
|
96 |
± |
10 |
|
21 |
± |
2 |
|
1600 |
12 |
± |
5 |
|
3 |
± |
2 |
|
25 |
± |
10 |
|
92 |
± |
14 |
|
19 |
± |
8 |
|
5000 |
10 |
± |
5 |
n NP |
3 |
± |
2 |
n NP |
18 |
± |
9 |
n NP |
98 |
± |
11 |
n NP |
19 |
± |
1 |
n NP |
Table 4 HCD Solvent control
|
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2uvrA |
|||||
S9-mix |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
Range |
3 – 29 |
4 – 25 |
2 – 24 |
2 – 18 |
5 – 61 |
8 – 60 |
61 – 188 |
55 - 176 |
11 – 61 |
12 - 68 |
Mean |
10 |
11 |
5 |
6 |
14 |
19 |
110 |
103 |
25 |
31 |
SD |
3 |
3 |
2 |
2 |
5 |
6 |
18 |
21 |
8 |
10 |
n |
2733 |
2694 |
2707 |
2712 |
2717 |
2754 |
2806 |
2724 |
2622 |
2596 |
Table 5 HCD Positive control
|
TA1535 |
TA1537 |
TA98 |
|||
S9-mix |
- |
+ |
- |
+ |
- |
+ |
Range |
107 – 1530 |
78 – 1481 |
58 – 1467 |
48 – 1239 |
365 – 2118 |
258 – 2033 |
Mean |
955 |
282 |
811 |
290 |
1220 |
903 |
SD |
174 |
114 |
372 |
138 |
221 |
367 |
n |
2598 |
2587 |
2223 |
2630 |
2670 |
2621 |
|
TA100 |
WP2uvrA |
||
S9-mix |
- |
+ |
- |
+ |
Range |
439 – 1993 |
397 – 2666 |
93 – 1999 |
109 - 1968 |
Mean |
879 |
1371 |
1167 |
421 |
SD |
170 |
412 |
527 |
169 |
n |
2654 |
2634 |
2516 |
2528 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
In an AMES test, performed according to OECD guideline 471 and in accordance with GLP principles, diisobutyl hydrogen phosphate was found not to be mutagenic with or without metabolic activation.
Therefore a classification according to CLP classification criteria (Regulation (EC) No 1272/2008) is not justified.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.