Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate

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:

09 Aug 2016 - 20 Jan 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Medicines & Healthcare products Regulatory Agency, United Kingdom

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of hydroxyethyl laurate and ethylenedilaurate

EC Number:

908-917-6

Molecular formula:

C14H28O3 C26H50O4

IUPAC Name:

Reaction mass of hydroxyethyl laurate and ethylenedilaurate

Specific details on test material used for the study:

The test item dilutions were prepared in acetone on the day of each experiment. No correction for purity was required. Acetone is toxic to the bacterial cells at 0.1 mL (100 μL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 μL) aliquots (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined.

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of California, Berkeley, on culture discs, on 04 August 1995, British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987

MEDIA USED
- Type and identity of media: Top agar was prepared using 0.6% Bacto agar (lot number 5223985 06/20) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 43372 02/17 and 43406 02/17).
- Properly maintained: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

microsomal preparations (S9 mix), prepared from rat livers treated with a mixture known to induce an elevated level of these enzymes.

Test concentrations with justification for top dose:

1st Experiment: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
2nd Experiment:
Salmonella strain TA100 (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
Salmonella strains TA1535, TA98 and TA1537 (absence of S9): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Salmonella strains TA1535 and TA100 (presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
E.coli strain WP2uvrA (absence and presence of S9) and Salmonella strains TA98 and TA1537 (presence of S9): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
The top dose was selected based on cytotoxicity in each strain with or without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide and dimethyl formamide at 50 mg/mL but was fully soluble in acetone at 100 mg/mL in solubility checks performed in-house

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

positive controls in the absence of S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Remarks:

positive controls in the presence of S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation (in buffer without metabolic activation or instead in S9-mix for metabolic activation)

All of the plates were incubated at 37 ± 3°C for approximately 48 h and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were required at 5000 µg/plate because of test item precipitation.

DURATION
- Exposure duration: 48 h after 20 min preincubation

NUMBER OF REPLICATIONS: 3 (triplicates)
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method with and without metabolic activation.

Without metabolic activation:
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 min (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method.

With metabolic activation:
The procedure was the same as without metabolic activation, except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media.

Evaluation criteria:

A test item was considered non-mutagenic (negative) in the test system if the following criteria were not met:
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. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

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 results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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
- Precipitation: A test item precipitate (white and particulate in appearance) was noted under a low power microscope at 1500 μg/plate and by eye at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
The results of the range finding study were included as experiment 1.

CYTOTOXICITY
First experiment: maximum dose of 5000 µg/plate. The test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains initially from 150 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA in either the absence or presence S9-mix. Consequently, the toxic limit or the maximum recommended dose concentration of the test item was employed as the maximum dose in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. The test item induced a much stronger toxic response in the second mutation test (employing the pre-incubation method), with weakened bacterial background lawns noted in the absence of S9-mix from 15 μg/plate (TA100), 50 μg/plate (TA1535, TA98 and TA1537) and 150 μg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were noted to the Salmonella strains only from 500 μg/plate (TA100, TA1535 and TA98) and 1500 μg/plate (TA1537). No toxicity was noted to Escherichia coli strain WP2uvrA dosed in the presence S9-mix in the second mutation test. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.


HISTORICAL CONTROL DATA
- Positive historical control data: provided for 2015 and 2016
- Negative (solvent/vehicle) historical control data: provided for 2015 and 2016
see attached document

Any other information on results incl. tables

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA98 revertant colony frequency was observed in the presence of S9-mix at 50 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Table 1: Mean number of revertants in experiment 1

Experiment I	without S9
	TA100	TA1535	WP2 uvrA	TA98	TA1537
Vehicle (acetone)	87	12	23	13	10
1.5 µg	89	14	13	15	12
5 µg	76	12	13	18	12
15 µg	101	14	17	18	13
50 µg	93	12	19	17	11
150 µg	94 S	16	17	15	7
500 µg	78 S	8 S	18	14 S	7 S
1500 µg	23 S	8 S	12	13 S	0 V
5000 µg	0 VP	0 VP	11 P	8 SP	0 VP
N-ethyl-N'-nitro-N-nitrosoguanidine	524	128	553
4-Nitroquinoline-1-oxide				173
9-Aminoacridine					610
Experiment I	with S9
	TA100	TA1535	WP2 uvrA	TA98	TA1537
Vehicle (acetone)	94	13	21	22	19
1.5 µg	97	11	20	21	15
5 µg	88	13	21	20	15
15 µg	94	11	19	25	12
50 µg	97	17	18	22	15
150 µg	98	14	19	20	12
500 µg	77	11	23	22	11
1500 µg	74 S	7 S	19	18	8
5000 µg	32 SP	7 SP	22 P	12 SP	3 SP
2-aminoanthracene	747	214	224		448
Benzo(a)pyrene				111

P = precipitation; S = sparse bacterial background law, V = very weak bacterial background lawn

 

Table 2: Mean number of revertants in experiment II

Experiment II	without S9
	TA100	TA1535	WP2 uvrA	TA98	TA1537
Vehicle (acetone)	82	18	20	15	8
0.05 µg	82	NT	NT	NT	NT
0.15 µg	80	8	NT	17	9
0.5 µg	75	11	NT	21	7
1.5 µg	82	13	17	19	8
5 µg	85	13	20	14	6
15 µg	81 S	12	20	14	5
50 µg	0 T	0 T	17	18 S	0 T
150 µg	0 T	0 T	14	0 T	0 T
500 µg	NT	0 T	18 S	0 T	0 T
1500 µg	NT	NT	0 V	NT	NT
5000 µg	NT	NT	0 VP	NT	NT
N-ethyl-N'-nitro-N-nitrosoguanidine	721	3264	96
4-Nitroquinoline-1-oxide				151
9-Aminoacridine					465
Experiment II	with S9
	TA100	TA1535	WP2 uvrA	TA98	TA1537
Vehicle (acetone)	89	13	25	16	8
0.5 µg	76	14	NT	NT	NT
1.5 µg	89	12	22	20	11
5 µg	77	10	25	23	9
15 µg	78	12	23	21	10
50 µg	86	12	27	26**	11
150 µg	81	10	26	18	8
500 µg	0 V	0 V	26	14 S	11
1500 µg	0 V	0 V	24	0 V	0 V
5000 µg	NT	NT	22 P	0 VP	0 TP
2-aminoanthracene	1314	197	260		210
Benzo(a)pyrene				64

P = precipitation; S = sparse bacterial background law, V = very weak bacterial background lawn, T = toxic, no bacterial background lawn, NT = not tested

** p<0.01

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative