Registration Dossier
Registration Dossier
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EC number: 949-117-7 | CAS number: -
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 Dec 2018 - 08 Jan 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted in 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- adopted in 1998
- Qualifier:
- according to guideline
- Guideline:
- other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register
- Version / remarks:
- adopted in 2012
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- adopted in 2000
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Medicines & Healthcare products Regulatory Agency, Department of Health of the Government of the United Kingdom
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Reaction product of castor oil with glycerol
- EC Number:
- 949-117-7
- Molecular formula:
- not applicable, substance is UVCB
- IUPAC Name:
- Reaction product of castor oil with glycerol
- Test material form:
- liquid
Constituent 1
Method
- Target gene:
- his operon (for S. typhimurium strains) and trp operon (for E. coli strains)
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Trinova Biochem GmbH, Germany; obtained: 27 Jun 2017
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: British Industrial Biological Research Association; obtained: 17 Aug 1987
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male CD Sprague-Dawley rats (age: 6 - 8 weeks, weight: approx. 250 g) orally treated for 3 days with phenobarbitone/beta-naphthoflavone (80/100 mg/kg bw/day) in arachis oil as vehicle.
- date of preparation of S9 mix: 28 Oct 2018
- volume of S9 mix in the final culture medium: 0.5 mL
- quality controls of S9: enzymatic activity, sterility, metabolic capability: checked and certified - Test concentrations with justification for top dose:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
HIghest dose recommended in test guideline.
Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate
Concentrations selected based on the results of Experiment 1. - Vehicle / solvent:
- - Vehicle: dimethyl sulfoxide (DMSO)
- Supplier: ThermoFisher Scientific
- Batch number: 184106
- Purity: 99.9%
- Expiry: May 2023
- Justification for choice of solvent/vehicle: Due to the fact that the test substance was only partially miscible with water, DMSO was selected as the vehicle.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration and number of independent experiments: triplicates each in two independent experiments
METHOD OF TREATMENT / EXPOSURE:
- Test substance added in medium; in agar (plate incorporation, Experiment 1) and preincubation (Experiment 2)
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min at 37 ± 3 °C (only Experiment 1)
- Exposure duration/duration of treatment: 48 - 72 h at 37 ± 3 °C
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
METHODS FOR MEASUREMENTS OF GENOTOXICIY
Genotoxicity in this reverse mutation assay was assessed by counting the number of revertant colonies compared to the negative controls. - Evaluation criteria:
- Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response).
5. Statistical analysis of data as determined by UKEMS.
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. - Statistics:
- Statistical significance was confirmed by using Dunnetts Regression Analysis (p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at and above 1500 µg/plate in the presence of S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was only partially miscible with water.
- Precipitation and time of the determination: No precipitation was observed at any of the doses tested in either the presence or absence of metabolic activation.
STUDY RESULTS
- Concurrent vehicle negative and positive control data are reported in tabular form in the section 'Any other information on results incl. tables'.
Ames test:
- Mean number of revertant colonies per plate and standard deviation are reported in tabular form in the section 'Any other information on results incl. tables'.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive and negative (solvent/vehicle) historical control data are attached as pdf document in the section 'Attached background material'. Results obtained in the present study fell within the historical control data range.
Any other information on results incl. tables
Table 1: Test Results Experiment 1 – Without Metabolic Activation (Plate Incorporation)
Test Period |
From: 18 December 2018 |
To: 21 December 2018 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
118 101 100 |
(106) 10.1# |
20 13 9 |
(14) 5.6 |
16 28 15 |
(20) 7.2 |
20 11 20 |
(17) 5.2 |
9 8 5 |
(7) 2.1 |
||
1.5 µg |
120 135 106 |
(120) 14.5 |
9 12 21 |
(14) 6.2 |
20 20 18 |
(19) 1.2 |
16 12 16 |
(15) 2.3 |
11 6 10 |
(9) 2.6 |
||
5 µg |
85 103 95 |
(94) 9.0 |
11 11 10 |
(11) 0.6 |
19 18 20 |
(19) 1.0 |
17 16 19 |
(17) 1.5 |
10 6 12 |
(9) 3.1 |
||
15 µg |
109 98 84 |
(97) 12.5 |
20 11 14 |
(15) 4.6 |
21 21 18 |
(20) 1.7 |
16 11 16 |
(14) 2.9 |
10 11 12 |
(11) 1.0 |
||
50 µg |
116 93 77 |
(95) 19.6 |
10 12 12 |
(11) 1.2 |
27 20 21 |
(23) 3.8 |
12 17 23 |
(17) 5.5 |
4 16 9 |
(10) 6.0 |
||
150 µg |
106 77 111 |
(98) 18.4 |
19 26 7 |
(17) 9.6 |
28 28 18 |
(25) 5.8 |
24 24 15 |
(21) 5.2 |
6 16 9 |
(10) 5.1 |
||
500 µg |
136 118 103 |
(119) 16.5 |
10 13 10 |
(11) 1.7 |
32 22 31 |
(28) 5.5 |
15 14 21 |
(17) 3.8 |
7 18 3 |
(9) 7.8 |
||
1500 µg |
105 87 93 |
(95) 9.2 |
15 12 9 |
(12) 3.0 |
22 17 18 |
(19) 2.6 |
18 21 19 |
(19) 1.5 |
2 6 8 |
(5) 3.1 |
||
5000 µg |
99 72 91 |
(87) 13.9 |
12 12 11 |
(12) 0.6 |
36 19 15 |
(23) 11.2 |
19 14 20 |
(18) 3.2 |
9 8 3 |
(7) 3.2 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
752 662 766 |
(727) 56.4 |
210 204 187 |
(200) 11.9 |
550 737 986 |
(758) 218.7 |
147 185 163 |
(165) 19.1 |
206 371 293 |
(290) 82.5 |
|||
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
#: Standard deviation
Table 2: Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)
Test Period |
From: 18 December 2018 |
To: 21 December 2018 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
110 132 124 |
(122) 11.1# |
9 9 19 |
(12) 5.8 |
29 23 30 |
(27) 3.8 |
12 18 29 |
(20) 8.6 |
10 16 10 |
(12) 3.5 |
||
1.5 µg |
114 120 104 |
(113) 8.1 |
7 12 11 |
(10) 2.6 |
27 26 36 |
(30) 5.5 |
19 16 22 |
(19) 3.0 |
11 12 9 |
(11) 1.5 |
||
5 µg |
100 139 101 |
(113) 22.2 |
11 11 11 |
(11) 0.0 |
26 27 31 |
(28) 2.6 |
13 22 27 |
(21) 7.1 |
12 11 8 |
(10) 2.1 |
||
15 µg |
117 95 123 |
(112) 14.7 |
9 13 13 |
(12) 2.3 |
10 12 35 |
(19) 13.9 |
19 21 24 |
(21) 2.5 |
8 11 7 |
(9) 2.1 |
||
50 µg |
126 140 149 |
(138) 11.6 |
11 12 13 |
(12) 1.0 |
30 24 32 |
(29) 4.2 |
21 18 19 |
(19) 1.5 |
10 5 10 |
(8) 2.9 |
||
150 µg |
132 142 107 |
(127) 18.0 |
15 10 13 |
(13) 2.5 |
26 29 34 |
(30) 4.0 |
31 19 20 |
(23) 6.7 |
18 13 9 |
(13) 4.5 |
||
500 µg |
105 122 102 |
(110) 10.8 |
17 15 11 |
(14) 3.1 |
30 29 28 |
(29) 1.0 |
28 16 21 |
(22) 6.0 |
14 10 9 |
(11) 2.6 |
||
1500 µg |
92 114 112 |
(106) 12.2 |
11 13 9 |
(11) 2.0 |
28 20 25 |
(24) 4.0 |
16 21 22 |
(20) 3.2 |
3 5 6 |
(5) 1.5 |
||
5000 µg |
113 106 93 |
(104) 10.1 |
9 24 18 |
(17) 7.5 |
37 35 22 |
(31) 8.1 |
20 22 28 |
(23) 4.2 |
5 3 4 |
(4) 1.0 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
1693 1946 2005 |
(1881) 165.7 |
268 249 300 |
(272) 25.8 |
109 121 110 |
(113) 6.7 |
163 166 209 |
(179) 25.7 |
275 234 221 |
(243) 28.2 |
|||
BP: Benzo(a)pyrene
2AA: 2-Aminoanthracene
#: Standard deviation
Table 3: Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)
Test Period |
From: 04 January 2019 |
To: 07 January 2019 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
99 121 112 |
(111) 11.1# |
15 22 26 |
(21) 5.6 |
24 13 17 |
(18) 5.6 |
24 14 21 |
(20) 5.1 |
12 14 13 |
(13) 1.0 |
||
15 µg |
120 111 128 |
(120) 8.5 |
26 19 26 |
(24) 4.0 |
12 12 25 |
(16) 7.5 |
20 20 19 |
(20) 0.6 |
8 9 9 |
(9) 0.6 |
||
50 µg |
123 123 119 |
(122) 2.3 |
30 22 25 |
(26) 4.0 |
19 22 28 |
(23) 4.6 |
23 17 12 |
(17) 5.5 |
8 5 18 |
(10) 6.8 |
||
150 µg |
135 123 99 |
(119) 18.3 |
22 29 20 |
(24) 4.7 |
11 15 16 |
(14) 2.6 |
22 22 22 |
(22) 0.0 |
10 8 11 |
(10) 1.5 |
||
500 µg |
104 100 96 |
(100) 4.0 |
20 26 27 |
(24) 3.8 |
15 19 19 |
(18) 2.3 |
15 20 15 |
(17) 2.9 |
11 7 5 |
(8) 3.1 |
||
1500 µg |
119 90 86 |
(98) 18.0 |
15 19 20 |
(18) 2.6 |
18 17 14 |
(16) 2.1 |
21 19 13 |
(18) 4.2 |
2 5 5 |
(4) 1.7 |
||
5000 µg |
70 86 84 |
(80) 8.7 |
19 26 23 |
(23) 3.5 |
18 11 20 |
(16) 4.7 |
20 10 13 |
(14) 5.1 |
6 S 5 S 1 S |
(4) 2.6 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
742 645 799 |
(729) 77.9 |
2127 2127 2101 |
(2118) 15.0 |
976 937 973 |
(962) 21.7 |
242 293 214 |
(250) 40.1 |
277 335 492 |
(368) 111.2 |
|||
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
S: Sparse bacterial background lawn
#: Standard deviation
Table 4: Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)
Test Period |
From: 04 January 2019 |
To: 07 January 2019 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
122 112 137 |
(124) 12.6# |
17 21 25 |
(21) 4.0 |
31 23 32 |
(29) 4.9 |
29 26 24 |
(26) 2.5 |
14 11 12 |
(12) 1.5 |
||
15 µg |
119 120 123 |
(121) 2.1 |
20 24 20 |
(21) 2.3 |
32 28 22 |
(27) 5.0 |
13 26 23 |
(21) 6.8 |
12 9 12 |
(11) 1.7 |
||
50 µg |
120 107 138 |
(122) 15.6 |
13 16 22 |
(17) 4.6 |
23 21 23 |
(22) 1.2 |
33 25 13 |
(24) 10.1 |
9 10 9 |
(9) 0.6 |
||
150 µg |
125 94 121 |
(113) 16.9 |
18 23 17 |
(19) 3.2 |
34 29 29 |
(31) 2.9 |
30 22 22 |
(25) 4.6 |
9 10 11 |
(10) 1.0 |
||
500 µg |
125 137 125 |
(129) 6.9 |
17 29 11 |
(19) 9.2 |
20 24 19 |
(21) 2.6 |
21 21 24 |
(22) 1.7 |
17 15 16 |
(16) 1.0 |
||
1500 µg |
108 109 93 |
(103) 9.0 |
9 18 18 |
(15) 5.2 |
24 28 29 |
(27) 2.6 |
24 18 19 |
(20) 3.2 |
12 11 11 |
(11) 0.6 |
||
5000 µg |
71 84 80 |
(78) 6.7 |
24 16 20 |
(20) 4.0 |
25 22 18 |
(22) 3.5 |
19 24 32 |
(25) 6.6 |
3 3 8 |
(5) 2.9 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
949 1242 1082 |
(1091) 146.7 |
247 204 233 |
(228) 21.9 |
215 140 148 |
(168) 41.2 |
142 137 99 |
(126) 23.5 |
240 303 242 |
(262) 35.8 |
|||
BP: Benzo(a)pyrene
2AA: 2-Aminoanthracene
#: Standard deviation
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results: negative
- Executive summary:
The available data on gene mutation in bacteria for reaction product of castor oil with glycerol do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP). The data provide not sufficient evidence that would imply a classification & labelling with respect to mutagenicity/genotoxicity. However, as no information regarding gene mutation in mammalian cells and cytogenicity or chromosome aberration in mammalian cells are available, the overall conclusion for classification regarding mutagenicity/genotoxicity is ‘data lacking’.
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.
