Alcohols, C18-22, reaction products with maleic anhydride and sodium bisulfite

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 from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from 2012-07-16 to 2012-08-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodoligcal deficiencies, which do not affect the quality of the relevant results. the study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions on a similar substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

Alkenes hydroformylated, sulfosuccinates, sodium salt

IUPAC Name:

Alkenes hydroformylated, sulfosuccinates, sodium salt

Details on test material:

- Name of test material: Alkenes hydroformylated, sulfosuccinates, sodium salt
- Physical state: solid (waxy), yellowish
- Lot no. / Batch no.: Rif. 7018
- Purity : 100 %; active ingredient: 78 %; dose calculation was no adjusted to purity
- Expiration date of the lot/batch: 2013-05-31
- Stability under test conditions: Not indicated by the sponsor
- Storage condition of test material: In the freezer at ≤ - 18°C

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Migrated to IUCLID6: sodium azide, NaN3 used with Strains TA 1535, TA 100 @ 10 µg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD used with Strains: TA 1537, TA 98 @ 10 µg/plate in TA 98, 50 µg/plate in TA 1537

Remarks:

Without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Migrated to IUCLID6: methyl methane sulfonate, MMS used with Strains: WP2 uvrA @ 3 µL/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA used with Strains:TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA @ 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100),10 µg/plate (WP2 uvrA)

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major draw¬back an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

Phenobarbital/b-naphthoflavone induced rat liver S9 will be used as the metabolic activation system. The S9 is prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of b-naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.
The protein concentration in the S9 preparation was 39.5 mg/mL (lot no. R 260412) in both experiments.

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

Evaluation criteria:

A test item is 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 WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

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

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
The test item precipitated in the overlay agar in the test tubes 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation in experiment I. In experiment II, toxic effects were observed in strain TA 1535 at 2500 and 5000 µg/plate in the absence of metabolic activation. No toxic effects were observed in the remaining strain with and without metabolic activation.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results Pre-Experiment and Experiment I

Study Name: 1491203	Study Code: Harlan CCR 1491203
Experiment: 1491203 VV Plate	Date Plated: 16/07/2012
Assay Conditions:	Date Counted: 19/07/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			10 ± 4	11 ± 4	24 ± 2	176 ± 19	51 ± 1
	Untreated			17 ± 4	9 ± 5	28 ± 3	161 ± 13	39 ± 7
	Alkenes,	3 µg		12 ± 2	9 ± 4	27 ± 5	167 ± 27	47 ± 7
	hydroformylated	10 µg		9 ± 1	9 ± 5	29 ± 4	178 ± 14	45 ± 4
	sulfosuccinates,	33 µg		13 ± 4	9 ± 3	22 ± 5	176 ± 1	46 ± 4
	sodium salt	100 µg		9 ± 1	10 ± 3	27 ± 9	185 ± 6	55 ± 8
		333 µg		10 ± 2	13 ± 4	23 ± 5	164 ± 11	45 ± 7
		1000 µg		12 ± 6	9 ± 3	25 ± 4	162 ± 16	56 ± 16
		2500 µg		9 ± 1^P	8 ± 2^P	30 ± 8^P	163 ± 26^P	51 ± 13^P
		5000 µg		16 ± 5^P	12 ± 3^P	30 ± 5^P	173 ± 9^P	55 ± 6^P
	NaN3	10 µg		1835 ± 56			1967 ± 122
	4-NOPD	10 µg				297 ± 19
	4-NOPD	50 µg			67 ± 7
	MMS	3.0 µL						997 ± 58
With Activation	Deionised water			19 ± 8	22 ± 4	40 ± 6	199 ± 17	58 ± 6
	Untreated			19 ± 9	18 ± 1	40 ± 9	171 ± 7	53 ± 9
	Alkenes,	3 µg		22 ± 4	22 ± 4	41 ± 3	177 ± 6	52 ± 5
	hydroformylated	10 µg		21 ± 6	25 ± 3	35 ± 6	196 ± 6	44 ± 1
	sulfosuccinates,	33 µg		19 ± 4	22 ± 8	37 ± 8	201 ± 8	58 ± 24
	sodium salt	100 µg		18 ± 5	21 ± 5	44 ± 3	199 ± 6	52 ± 2
		333 µg		20 ± 6	25 ± 1	46 ± 5	197 ± 13	55 ± 7
		1000 µg		21 ± 4	17 ± 5	34 ± 9	195 ± 16	59 ± 4
		2500 µg		16 ± 8^P	16 ± 6^P	31 ± 8^P	208 ± 19^P	65 ± 3^P
		5000 µg		14 ± 4^P	14 ± 2^P	38 ± 5^P	195 ± 14^P	61 ± 8^P
	2-AA	2.5 µg		347 ± 19	292 ± 10	1749 ± 240	2236 ± 355
	2-AA	10.0 µg						351 ± 47

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P	Precipitate

1.1     Summary of Results Experiment II

Study Name: 1491203	Study Code: Harlan CCR 1491203
Experiment: 1491203 HV2 Pre	Date Plated: 25/07/2012
Assay Conditions:	Date Counted: 01/08/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			21 ± 2	20 ± 6	29 ± 12	166 ± 17	50 ± 6
	Untreated			17 ± 3	21 ± 2	34 ± 10	196 ± 24	53 ± 12
	Alkenes,	33 µg		20 ± 6	21 ± 7	28 ± 7	177 ± 7	50 ± 10
	hydroformylated	100 µg		21 ± 3	19 ± 1	29 ± 5	188 ± 8	52 ± 4
	sulfosuccinates,	333 µg		16 ± 2	18 ± 2	29 ± 5	180 ± 8	55 ± 10
	sodium salt	1000 µg		16 ± 4	16 ± 1	23 ± 4	135 ± 7	45 ± 6
		2500 µg		8 ± 2^P	11 ± 4^P	23 ± 3^P	107 ± 11^P	42 ± 7^P
		5000 µg		5 ± 1^P	9 ± 6^P	18 ± 9^P	82 ± 9^P	44 ± 3^P
	NaN3	10 µg		1614 ± 30			1901 ± 74
	4-NOPD	10 µg				755 ± 21
	4-NOPD	50 µg			106 ± 20
	MMS	3.0 µL						279 ± 39
With Activation	Deionised water			25 ± 5	30 ± 1	46 ± 4	226 ± 6	65 ± 8
	Untreated			28 ± 5	35 ± 3	48 ± 5	202 ± 25	61 ± 6
	Alkenes,	33 µg		25 ± 1	30 ± 4	41 ± 7	204 ± 21	69 ± 14
	hydroformylated	100 µg		28 ± 2	30 ± 2	54 ± 10	224 ± 16	62 ± 4
	sulfosuccinates,	333 µg		25 ± 2	32 ± 4	44 ± 9	217 ± 14	66 ± 8
	sodium salt	1000 µg		24 ± 2	27 ± 2	47 ± 3	215 ± 16	70 ± 1
		2500 µg		24 ± 4^P	22 ± 9^P	46 ± 10^P	187 ± 17^P	66 ± 10^P
		5000 µg		15 ± 5^P	21 ± 5^P	36 ± 4^P	157 ± 10^P	58 ± 5^P
	2-AA	2.5 µg		288 ± 22	252 ± 26	1825 ± 124	1583 ± 114
	2-AA	10.0 µg						222 ± 14

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P	Precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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

Executive summary:

The test item Alkenes hydroformylated, sulfosuccinates, sodium salt was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) and the pre-incubation test (experiment II) usingSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, and TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:          3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                    33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation in experiment I. In experiment II, toxic effects were observed in strain TA 1535 at 2500 and 5000 µg/plate in the absence of metabolic activation. No toxic effects were observed in the remaining strain with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Alkenes hydroformylated, sulfosuccinates, sodium salt at any dose 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 of induced revertant colonies.

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