Reaction products resulting from the esterification of Sorbitol with C8 – 18 (even) and C18 unsaturated fatty acids in the ratio of 1:1

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

09 Oct - 23 Oct 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sorbitan, octanoate (2:3)

EC Number:

295-118-4

EC Name:

Sorbitan, octanoate (2:3)

Cas Number:

91844-53-0

Molecular formula:

Molecular formula cannot be given as substance is a mixture.

IUPAC Name:

91844-53-0

Method

Target gene:

his operon

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital i.p. and ß-Naphthoflavone p.o.

Test concentrations with justification for top dose:

first experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation
second experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen because of it´s solubility properties and its relative non-toxicity to the bacteria

Controls

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls were performed.

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent vehicle controls were performed.

True negative controls:

no

Positive controls:

yes

Remarks:

sodium azide (NaN3), 4-nitro-o-phenylene-diamine (4-NOPD), methyl-methane-sulfonate (MMS) and 2-aminoanthracene (2-AA)

Positive control substance:

other: without S9 mix: NaN3, 10 µg/plate in TA 1535 and TA 100; 4-NOPD, 10 µg/plate in TA 98, 50 µg/plate in TA 1537; MMS, 3.0 µL/plate in TA 102; with S9 mix: 2-AA, 2.5 µg/plate in TA 1535, TA 1537, TA 98, TA 100 and 10 µg/plate in TA 102

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: three replicates in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative growth

The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to reduced background growth some plates were counted manually.

Evaluation criteria:

A test item was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control was observed. A dose dependent increase was considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase is not considered biologically relevant

Statistics:

Mean values and standard deviation were calculated.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Based on the toxic effects observed in the range finding study, eight concentrations were tested in main experiment and 5,000 µg/plate were chosen as maximal concentration.

ADDITIONAL INFORMATION ON CYTOTOXICITY: see tables 3 and 4

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Summary of results Pre-experiment/Experiment I

Metabolic activation	Test group	Dose Level (µg/plate)	Revertant Colony Counts (Mean±SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	DMSO		23±11	13±0	36±3	146±7	520±46
	Untreated		21±8	9±4	26±2	138±24	530±38
	Test substance	3	19±0	12±7	34±13	149±11	513±48
		10	20±3	7±2	33±6	153±27	508±10
		33	14±2	10±3	31±6	146±18	525±12
		100	25±3	14±3	37±12	130±14	500±3
		333	23±6	10±5	36±3	116±15	412±12
		1000	13±3	7±2	19±3	86±6	354±52
		2500	9±1	1±1	16±2	47±10	250±26
		5000	4±2	0±1	11±3	27±2	132±9
	NaN3	10	1980±52			2043±204
	4-NOPD	10			390±24
	4-NOPD	50		106±17
	MMS	3					2736±155
With activation	DMSO		21±6	17±6	48±6	156±26	628±69
	Untreated		26±4	17±4	39±9	154±4	671±76
	Test substance	3	24±3	18±11	43±6	173±6	584±41
		10	26±2	15±7	37±1	174±17	643±33
		33	24±4	10±4	41±6	158±11	677±16
		100	24±1	12±3	38±7	164±1	662±31
		333	26±5	12±3	35±6	176±8	570±49
		1000	23±5	18±6	45±6	153±5	630±82
		2500	21±6	11±3	39±10	143±13	575±110
		5000	11±2	4±2	14±6	93±8	348±30
	2-AA	2.5	400±29	701±76	3163±334	4283±196
	2-AA	10					2495±221

DMSO = dimethylsulfoxide

NaN3 = sodium azide

4-NOPD = 4 -nitro-o-phenylene-diamine

MMS = methyl-methane-sulfonate

2-AA = 2 -aminoanthracene

Table 2. Summary of results Experiment II

Metabolic activation	Test group	Dose Level (µg/plate)	Revertant Colony Counts (Mean±SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	DMSO		19±5	13±0	34±7	116±18	404±16
	Untreated		20±11	11±2	32±6	150±2	444±10
	Test substance	3	14±2	13±3	28±5	149±136	391±17
		10	19±5	12±6	22±4	106±8	388±17
		33	22±2	10±4	24±3	118±11	315±5
		100	15±2	6±3	25±8	82±5	262±20
		333	17±3	14±2	23±6	69±13	243±9
		1000	8±1	1±1	14±2	39±17	83±19
		2500	0±1	0±0	7±2	11±3	7±7
		5000	0±0	0±0	0±0	0±0	0±0
	NaN3	10	1672±35			1717±137
	4-NOPD	10			524±14
	4-NOPD	50		119±13
	MMS	3					1718±205
With activation	DMSO		28±6	11±4	42±6	123±8	433±19
	Untreated		37±15	17±3	43±6	153±12	549±46
	Test substance	3	31±5	18±3	39±6	150±3	485±38
		10	32±11	17±5	43±14	129±12	435±56
		33	39±11	10±2	54±18	113±26	406±79
		100	32±15	12±4	34±1	120±19	392±29
		333	25±3	10±2	42±6	128±18	194±64
		1000	20±3	8±5	28±3	72±6	58±10
		2500	9±4	10±4	22±7	46±10	39±11
		5000	7±2	0±0	12±3	15±5	14±3
	2-AA	2.5	298±15	263±12	2097±6	2118±6
	2-AA	10					1383±148

DMSO = dimethylsulfoxide

NaN3 = sodium azide

4-NOPD = 4 -nitro-o-phenylene-diamine

MMS = methyl-methane-sulfonate

2-AA = 2 -aminoanthracene

Table 3. Reduced backround growth were observed at the following plates:

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	5000	5000	1000-5000	5000
TA 1537	2500-5000	5000	1000-5000	5000
TA 98	2500sodium azide (NaN3), 4-nitro-o-phenylene-diamine (4-NOPD), methyl-methane-sulfonate (MMS) and 2-aminoanthracene (2-AA)-5000	5000	1000-5000	5000
TA 100	5000	5000	1000-5000	5000
TA 102	2500-5000	5000	1000-5000	1000-5000

Table 4. Toxic effects as a reduction in number of revertants were observed at the following plates:

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	2500-5000	-	1000-5000	2500-5000
TA 1537	2500-5000	5000	1000-5000	5000
TA 98	2500-5000	5000	1000-5000	5000
TA 100	2500-5000	-	1000-5000	2500-5000
TA 102	5000	-	1000-5000	333-5000

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies. Negative and positive control values were within the range of historical control data (see Table 5).

Table 5. These data represent the laboratory's historical control data from May 2005 until June 2006 representing approx. 200 experiments (TA 102 the historical control data are based on approx. 100 experiments).

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent Control	20.8	4.7	9	35	24.7	5.9	7	43
	Negative Control	20.4	4.4	11	31	24.2	5.5	10	38
	Positive Control	1422.0	464.7	781	1900	332.0	95.3	107	695
TA 1537	Solvent Control	11.2	3.7	5	28	16.2	5.0	6	36
	Negative Control	11.6	4.0	4	28	17.1	5.4	7	34
	Positive Control	99.8	32.5	53	425	276.8	132.6	59	746
TA 98	Solvent Control	28.1	6.1	15	49	37.9	7.4	20	57
	Negative Control	30.2	6.6	16	60	39.0	7.5	18	64
	Positive Control	439.0	155.2	176	1818	1839.4	898.6	407	4891
TA 100	Solvent Control	130.7	20.8	87	197	147.0	25.5	84	255
	Negative Control	138.2	21.6	86	216	150.1	24.2	96	214
	Positive Control	2083.1	281.3	616	2872	2372.9	958.4	417	5230
TA 102	Solvent Control	407.1	78.3	129	530	501.2	106.5	192	674
	Negative Control	401.5	78.4	197	550	508.0	109.5	160	682
	Positive Control	3432.0	1516.2	1183	6415	2278.9	608.0	872	3370

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative