Reaction mass of 1-phenyloctadecane-1,3-dione and phenylicosane-1,3-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 31 May 2011 to 14 October 2011

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-phenyloctadecane-1,3-dione and phenylicosane-1,3-dione

EC Number:

915-316-2

Molecular formula:

C50H80O4

IUPAC Name:

Reaction mass of 1-phenyloctadecane-1,3-dione and phenylicosane-1,3-dione

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

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 from the liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Concentration tested in Experiments 1 and 2 (plate incorporation, with and without metabolic activation): 12.3, 37, 111.1, 333.3 and 1000 µg/plate (A strong precipitate was observed in the Petri plates at dose levels > or = at 1000 µg/plate).
Due to a severe toxicity observed with S9 mix using the pre-incubation method, a third experiment was undertaken using a lower range of dose levels. The selected dose-levels for this third experiment were:
- 0.137, 0.412, 1.23, 3.7, 11.1 and 33.3 µg/plate for the TA 102 strain,
- 0.412, 1.23, 3.7, 11.1, 33.3 and 100 µg/plate for the TA 1537 strain,
- 0.8, 2.5, 7.4, 22.2, 66.7 and 200 µg/plate for the TA 98 strain,
- 1.23, 3.7, 11.1, 33.3, 100 and 300 µg/plate for the TA 100 strain.

Vehicle / solvent:

- Solvent used: Tetrahydrofuran (THF)
- Justification for choice of solvent: THF was chosen because of its solubility properties.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see details below

Details on test system and experimental conditions:

Positive controls:
Without S9 mix:
- sodium azide for strains TA 1535 and TA 100 (1 µg/plate),
- 9-Aminoacridine for strain TA 1537 (50 µg/plate),
- 2-Nitrofluorene for strain TA 98 (0.5 µg/plate),
- Mitomycin C for strain TA 102 (0.5 µg/plate).

With S9 mix:
- 2-Anthramine for strains TA 1535, TA 1537, TA 98 (2 µg/plate) and TA102 (10 µg/plate),
- Benzo(a)pyrene for strain TA 100 (5 µg/plate).

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results Experiment I

Direct plate incorporation method

 

Metabolic activation	Test group	Dose level per plate	Mean revertant colony counts
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	THF		15	11	18	108	272
	Rhodiastab 55P	12.3 µg	17	14	23	113	312
		37 µg	23	7	22	112	304
		111.1 µg	31	12	22	115	311
		333.3 µg	39	16	32	111	307
		1000 µg	29	14	27	101	366
	NAN3	1 µg	669			452
	9AA	50 µg		337
	2NF	0.5 µg			163
	MMC	0.5 µg					1872
With activation	THF		10	5	23	147	438
	Rhodiastab 55P	12.3 µg	14	8	28	123	411
		37 µg	10	15	28	114	483
		111.1 µg	14	8	29	116	485
		333.3 µg	16	9	27	164	429
		1000 µg	29	7	23	150	500
	2AM	2 µg	241	57	895
		10 µg					3065
	BAP	5 µg				565

 

Summary of Results Experiment II

Direct plate incorporation method (without S9 mix) and Pre-incubation method (with S9 mix)

 

Metabolic activation	Test group	Dose level per plate	Mean revertant colony counts
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	THF		17	7	24	110	410
	Rhodiastab 55P	12.3 µg	19	10	26	93	370
		37 µg	17	12	26	93	345
		111.1 µg	19	6	26	88	405
		333.3 µg	30	12	19	102	318
		1000 µg	33	2	21	86	347
	NAN3	1 µg	443			516
	9AA	50 µg		132
	2NF	0.5 µg			128
	MMC	0.5 µg					2089
With activation	THF		14	10	32	139	387
	Rhodiastab 55P	12.3 µg	10	6	24	137	215
		37 µg	23	13	31	137	186
		111.1 µg	18	2	26	126	110
		333.3 µg	19	14	22	97	55
		1000 µg	27	13	51	41	37
	2AM	2 µg	170	203	1659
		10 µg					2085
	BAP	5 µg				541

 

Summary of Results Experiment III

With metabolic activation, Pre-incubation method

 

Metabolic activation	Test group	Dose level per plate	Mean revertant colony counts
TA 102	THF		483
	Rhodiastab 55P	0.137 µg	359
		0.412 µg	349
		1.23µg	378
		3.7 µg	319
		11.1 µg	357
		33.3 µg	206
	2AM	10 µg	1570
TA 1537	THF		7
	Rhodiastab 55P	0.412 µg	9
		1.23µg	4
		3.7 µg	2
		11.1 µg	5
		33.3 µg	4
		100 µg	3
	2AM	2 µg	99
TA 98	THF		31
	Rhodiastab 55P	0.8 µg	30
		2.5 µg	29
		7.4 µg	22
		22.2 µg	17
		66.7 µg	21
		200 µg	16
	2AM	2 µg	1572
TA 100	THF		109
	Rhodiastab 55P	1.23µg	141
		3.7 µg	112
		11.1 µg	100
		33.3 µg	120
		100 µg	142
		300 µg	117
	BAP	5 µg	590

 

 Vehicle:

THF: Tetrahydrofuran

 

Positive Controls:

NAN3: sodium azide

9-AA: 9-aminoacridine

2NF: 2-Nitrofluorene

MMC: Mitomycin C

2AM: 2-Anthramine

BAP: Benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Reaction mass of Stearoylbenzoylmethane and Palmitoylbenzoylmethane is considered to be non-mutagenic in Ames test, with and without metabolic activation.

Executive summary:

This study was performed to investigate the potential of the test item, RHODIASTAB 55 P, to induce reverse mutation in Salmonella typhimurium. The study was performed according to OECD guideline no. 471 and EC guideline n° B13/14 and in compliance with the Principles of Good Laboratory Practice. 

 

A preliminary toxicity test was performed to define the dose-levels of RHODIASTAB 55 P to be used for the mutagenicity study. The test item was then tested in three independent experiments, with and/or without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. The negative control was the vehicle (Tetrahydrofuran).

The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second and third experiments with S9 mix were performed according to the preincubation method (60 minutes,37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

Since the test item was found poorly soluble in the preliminary test, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines.

The selected treatment-levels were 12.3, 37, 111.1, 333.3 and 1000 µg/plate for the first and second experiments with and without S9 mix. Due to a severe toxicity observed with S9 mix using the preincubation method, a third experiment was undertaken using a lower range of dose-levels. The selected dose-levels for this third experiment were:

.           0.137, 0.412,1.23,3.7, 11.1 and 33.3 µg/plate for the TA 102 strain,

.           0.412,1.23,3.7, 11.1, 33.3 and 100 µg/plate for the TA 1537 strain,

.           0.8, 2.5, 7.4, 22.2, 66.7 and 200 µg/plate for the TA 98 strain,

.           1.23,3.7, 11.1, 33.3, 100 and 300 µg/plate for the TA 100 strain.

 

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid. 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains and in either experiment.

In conclusion, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, in the absence or in the presence of a rat metabolising system therefore RHODIASTAB 55 P is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).