Esterification product of castor oil fatty acids and pentaerythritol

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:

2017 October 11 - 2018 January 30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire DE72 2GD UK

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-2,2-bis(hydroxymethyl)propyl (R)-12-hydroxyoleate

EC Number:

201-095-3

EC Name:

3-hydroxy-2,2-bis(hydroxymethyl)propyl (R)-12-hydroxyoleate

Cas Number:

78-22-8

Molecular formula:

C23H44O6

IUPAC Name:

3-hydroxy-2,2-bis(hydroxymethyl)propyl 12-hydroxyoctadec-9-enoate

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0000150418
- Expiration date of the lot/batch: 14 August 2018
- Purity test date: Not reported

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Expected to be stable
- Solubility and stability of the test substance in the solvent/vehicle: Not reported
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS:

Method

Target gene:

histidine for the S. typhimurium strains, and tryptophan for E. coli.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver fraction (10% v/v), from male rats induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 ug/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

DMSO, FIsher Scientific, batch 1714907, >99% pure

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

concurrent

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:

METHOD OF APPLICATION: plate incorporation (first test) and preincubation (second test)
- Cell density at seeding (if applicable): 0.1 ml of a 10-h bacterial culture (having a density of at least 10E9/mL)

DURATION
- Preincubation period: 0.5 h
- Exposure duration: 48-72 h for plate incorporation; preincubation for 30 m before plating in top agar
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells): cells not fixed

SELECTION AGENT (mutation assays): The Ames assay employs, as an indicator of mutation, observable (and countable) growth (colonies) in agar deficient in histidine. Colonies of bacteria represent mutants which have back-reverted to a histidine auxotroph. An automated colony counter is used.


NUMBER OF REPLICATIONS: 3 (triplicate)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: reduction in bacterial lawn

Rationale for test conditions:

The first plate incorporation test included 7 concentrations, in triplicate. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and phosphate buffer. All plates were incubated at 34 to 39°C for 48-72 hours. After this period, the appearance of the background bacterial lawn were examined and revertant colonies counted using an automated colony counter. Colonies were counted manually if automated counting is not possible (e.g. if dense precipitate is present).
Any toxic effects of the test item were detected as thinning or absence of the background lawn of non-revertant colonies, and/or reduction in revertant colony numbers to ≤ 50% of the concurrent vehicle control count. In the absence of any toxic effects the maximum concentration used in the second test were the same as that used in the first. If toxic effects were observed at more than one concentration, a lower concentration was chosen. A minimum of four non-toxic concentrations were obtained. If this was not achieved then the first test was repeated using a more appropriate concentration range. If a negative or equivocal response was obtained a variation on the above procedure will be used, with a minimum of five concentrations of test item used.
If the required number of non-toxic concentrations was not obtained, the second test was repeated using a more appropriate concentration range. It may also have been repeated to confirm a positive response or to confirm a dose-response.

Evaluation criteria:

For a test material to be classified as toxic, the test item must cause a reduction in the number of spontaneous revertants (below a factor of 0.5 fold under the concurrent solvent control) and/or the bacterial lawn should exhibit evidence of thinning when viewed microscopically.

For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range of the laboratory unless otherwise justified by the Study Director.

The positive control compounds must produce an increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls.

Criteria for a positive result (any, one, or all of the following may be used to determine the overall results of the study, weighing the results in terms of their biological significance. The criteria are listed in order of priority:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase in mean revertant colony numbers at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Fold increase greater than two times thef the concurrent solvent control for any tester strain (especially if accompanied by an out-of historical range response (Cariello and Piegorsch, 1996).
5. Statistical analysis of data as determined by the UKEMS (Mahon et al., 1989)

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, the test data should be evalueated by expert judgment and or further possible investigations.

Fold-increase: (three times in the case of strains TA1535 and TA1537) those of the concurrent vehicle controls.

Statistics:

The automated scoring system (Delta Building Monitoring System, Ames Study Manager and Sorcerer Imaging System) automatically conducts statistical analysis during the scoring process using Dunnetts Regression Analysis. Statistical significance will be confirmed for those values that indicate statistically significant increases in the frequency of revertant colonies compared with the concurrent solvent control (* = p < 0.05). Values that the program concludes are statististically significant but are within the in-house historical vehicle/untreated control range will not be reported.
See (Mahon et al, 1989, Analysis of data from microbial colony assays in: Kirkland, D.J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part III. Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, Cambridge, pp.26-65.).

Results and discussion

Test results

Test results 1

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:

not applicable

Positive controls validity:

valid

Test results 2

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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

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:

not applicable

Positive controls validity:

valid

Test results 4

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:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Representative results:

Table 1            Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
72		29		24		18		10
98	(93)	25	(28)	18	(19)	18	(17)	9	(9)
108		29		15		14		9

Table 2            Test Results: Experiment 1 – Without Metabolic Activation(Plate Incorporation)

Test Period		From: 14 November 2017					To: 17 November 2017
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)	96 98 76	(90) 12.2#	23 38 21	(27) 9.3	32 24 16		(24) 8.0	21 21 15	(19) 3.5	9 9 12	(10) 1.7
	1.5 µg	108 97 95	(100) 7.0	40 24 26	(30) 8.7	29 20 22		(24) 4.7	9 9 13	(10) 2.3	14 8 13	(12) 3.2
	5 µg	94 83 90	(89) 5.6	33 24 23	(27) 5.5	19 26 26		(24) 4.0	21 20 19	(20) 1.0	14 13 13	(13) 0.6
	15 µg	87 88 82	(86) 3.2	42 33 25	(33) 8.5	29 25 23		(26) 3.1	19 11 12	(14) 4.4	8 13 12	(11) 2.6
	50 µg	93 89 91	(91) 2.0	42 33 38	(38) 4.5	23 24 23		(23) 0.6	19 19 18	(19) 0.6	15 12 15	(14) 1.7
	150 µg	89 92 98	(93) 4.6	25 33 30	(29) 4.0	33 21 22		(25) 6.7	20 19 16	(18) 2.1	15 11 14	(13) 2.1
	500 µg	77 89 89	(85) 6.9	28 31 30	(30) 1.5	25 25 23		(24) 1.2	21 25 24	(23) 2.1	12 13 11	(12) 1.0
	1500 µg	80 92 87	(86) 6.0	28 40 25	(31) 7.9	27 24 16		(22) 5.7	19 16 12	(16) 3.5	9 10 13	(11) 2.1
	5000 µg	81 F 74 F 79 F	(78) 3.6	32 F 18 F 13 F	(21) 9.8	24 F 22 F 16 F		(21) 4.2	19 F 27 F 10 F	(19) 8.5	15 F 2 F 6 F	(8) 6.7
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
		665 538 606	(603) 63.6	465 450 489	(468) 19.7	848 735 822		(802) 59.2	160 139 163	(154) 13.1	540 324 395	(420) 110.1

Table 3            Test Results: Experiment 1 – With Metabolic Activation(Plate Incorporation)

Test Period		From: 14 November 2017					To: 17 November 2017
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)	84 114 116	(105) 17.9#	22 36 28	(29) 7.0	27 26 25		(26) 1.0	34 19 35	(29) 9.0	12 18 14	(15) 3.1
	1.5 µg	106 93 93	(97) 7.5	30 25 32	(29) 3.6	25 25 31		(27) 3.5	15 19 21	(18) 3.1	19 5 12	(12) 7.0
	5 µg	105 98 105	(103) 4.0	25 32 35	(31) 5.1	30 32 26		(29) 3.1	26 30 27	(28) 2.1	13 19 8	(13) 5.5
	15 µg	103 91 96	(97) 6.0	27 21 21	(23) 3.5	25 26 28		(26) 1.5	17 21 19	(19) 2.0	14 19 13	(15) 3.2
	50 µg	97 98 102	(99) 2.6	30 20 30	(27) 5.8	26 25 28		(26) 1.5	18 15 16	(16) 1.5	16 19 12	(16) 3.5
	150 µg	92 106 112	(103) 10.3	40 23 28	(30) 8.7	28 36 25		(30) 5.7	12 23 25	(20) 7.0	12 15 9	(12) 3.0
	500 µg	116 94 96	(102) 12.2	25 23 26	(25) 1.5	30 28 30		(29) 1.2	22 22 14	(19) 4.6	10 10 11	(10) 0.6
	1500 µg	94 113 90	(99) 12.3	30 22 20	(24) 5.3	28 30 26		(28) 2.0	26 28 23	(26) 2.5	14 8 10	(11) 3.1
	5000 µg	103 F 97 F 113 F	(104) 8.1	15 F 17 F 17 F	(16) 1.2	18 F 26 F 18 F		(21) 4.6	19 F 11 F 24 F	(18) 6.6	4 F 12 F 7 F	(8) 4.0
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
		2395 2473 2494	(2454) 52.2	276 298 268	(281) 15.5	181 175 212		(189) 19.9	194 184 202	(193) 9.0	338 346 365	(350) 13.9

ENNG     N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO     4-Nitroquinoline-1-oxide

9AA        9-Aminoacridine

2AA   2 -Aminoanthracene

BP           Benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:

The substance was tested in a guideline Ames Assay (OECD 471) and found to be non-mutagenic. The substance does not meet the criteria for classification as a mutagen according to Regulation EC No. 1272/2008.