Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters

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:

June - July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters

Molecular formula:

unspecified

IUPAC Name:

Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters

Test material form:

liquid: viscous

Details on test material:

- Name as cited in the report: SAT150011
- Chemical name: Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters
- Batch No.: RP GW 915 MU
- Storage Conditions: room temperature
- Expiration date: March/April 2016
- Description: clear, light amber, viscous liquid
- Molecular weight: approx. 340 Da
- Purity: 73.8 % Mono- and Diester (NMR)

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- PRE-EXPERIMENT:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
- EXPERIMENT I:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (for all tester strains except TA 1537 without metabolic activation)
1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate (TA 1537 without metabolic activation)
- EXPERIMENT II:
1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate (for all tester strains except TA 100 without metabolic activation and TA 102)
0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 µg/plate (TA 100 without metabolic activation)
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 102)

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (TA98, TA1537)

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all strains)

Remarks:

With S9-mix

Details on test system and experimental conditions:

Samples of each tester strain were grown by culturing for 12 h at 37°C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 10E9 cells/mL). The nutrient medium consists per litre:
- 8 g Nutrient Broth
- 5 g NaCI
A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.

The Vogel-Bonner Medium E agar plates with 2 % glucose used in the Ames Test were prepared by Eurofins Munich or provided by an appropriate supplier. Quality controls were performed. Sterilisation was performed for 20 min at 121°C in an autoclave.

The overlay agar contains per litre:
- 7.0 g Agar Agar
- 6.0 g NaCI
- 10.5 mg L-histidine x HCI x H2O
- 12.2 mg biotin
Sterilisation was performed for 20 min at 121°C in an autoclave.

Evaluation criteria:

CYTOTOXICITY:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <= 0.5 in relation to the solvent control.

VALIDITY:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the mean values of the spontaneous reversion frequency of the control plates with and without S9 mix are within the historical control data range
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (exact values).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as folIows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Precipitation of the test item was observed in all tester strains used in experiment l and II at a concentration of 5000 µg/plate (with metabolic activation). In experiment II precipitation of the test item was found at a concentration of 5000 µg/plate (with metabolic activation), if tested.

Toxic effects of the test item were noted in most tester strains evaluated in experiment l and II.

In experiment I toxic effects of the test item were observed in tester strains TA 98 and TA 1535 at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 100 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 10.0 µg/plate and higher (without metabolic activation) and at concentrations of 100 µg/plate and higher (with metabolic activation).

In experiment II toxic effects of the test item were observed in tester strains TA 98 and TA 1535 at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 10.0 µg/plate and higher (without metabolic activation) and at concentrations of 31.6 µg/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 10.0 µg/plate and higher (without metabolic activation) and at concentrations of 100 µg/plate and higher (with metabolic activation). In tester strain TA 102 toxic effects of the test item were observed at a concentration of 5000 µg/plate (without metabolic activation).

No biologieally relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with SAT150011 at any concentration level, neither in the presence nor absence of metabolic activation in experiment l and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

EXPERIMENT I (Plate-incorporation Test)

Mean revertant colonies per plate

	TA 98	TA98	TA 100	TA 100	TA 1535	TA 1535	TA 1537	TA 1537	TA 102	TA 102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
neg. control	21	34	118	112	10	11	7	6	215	298
solvent control	34	43	106	124	5	5	10	11	215	265
test item 1.00 µg	-	-	-	-	-	-	10	-	-	-
test item 3.16 µg	26	32	100	114	13	8	7	7	264	315
test item 10.0 µg	32	28	77	117	7	9	4	13	262	316
test item 31.6 µg	28	35	69	107	9	8	3	12	234	250
test item 100 µg	27	32	58	98	6	5	4	9	186	235
test item 316 µg	17	40	52	81	0	7	4	7	202	223
test item 1000 µg	1	17	33	68	0	1	2	5	227	206
test item 2500 µg	0	0	29	62	0	0	-	6	214	273
test item 5000 µg	0	0	20	50	0	0	-	4	122	241
pos. control	291	2211	1025	2645	575	104	148	305	993	669

EXPERIMENT II (Pre-incubation Test)

Mean revertant colonies per plate

	TA 98	TA98	TA 100	TA 100	TA 1535	TA 1535	TA 1537	TA 1537	TA 102	TA 102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
neg. control	22	27	104	109	11	8	7	8	226	413
solvent control	20	23	78	88	11	6	6	7	221	294
test item 0.100 µg	-	-	82	-	-	-	-	-	-	-
test item 0.316 µg	-	-	79	-	-	-	-	-	-	-
test item 1.00 µg	22	17	76	88	12	11	4	5	-	-
test item 3.16 µg	21	28	76	85	10	6	4	9	-	-
test item 10.0 µg	18	23	55	82	10	9	5	5	283	363
test item 31.6 µg	14	28	49	84	6	8	1	5	293	325
test item 100 µg	10	23	37	68	1	9	2	3	186	289
test item 316 µg	9	22	36	50	2	7	0	6	210	282
test item 1000 µg	1	13	-	50	1	0	0	8	197	243
test item 2500 µg	0	0	-	48	0	0	1	4	209	260
test item 5000 µg	-	-	-	-	-	-	-	-	85	301
pos. control	531	595	571	1201	567	33	109	94	1531	908

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, SAT150011 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, SAT150011 is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of SAT150011 for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:

3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (for all tester strains except TA 1537 without metabolic activation)

1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate (TA 1537 without metabolic activation)

Experiment II:

1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate (for all tester strains except TA 100 without metabolic activation and TA 102)

0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 µg/plate (TA 100 without metabolic activation)

10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 102)

Precipitation was observed in all tester strains used in experiment I and in one tester strain in experiment II (in both experiments: with metabolic activation and only at a concentration of 5000 µg/plate).

Toxic effects of the test item were noted in most tester strains used in experiment l and II:

In experiment I toxic effects of the test item were observed at concentrations of 10.0 µg/plate and higher (without metabolic activation) and at concentrations of 100 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

In experiment 11 toxic effects of the test item were noted at concentrations of 10.0 µg/plate and higher (without metabolic activation) and at concentrations of 31.6 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with SAT150011 at any concentration level, neither in the presence nor absence of metabolic activation in experiment l and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, SAT150011 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, SAT150011 is considered to be non-mutagenic in this bacterial reverse mutation assay.