Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated

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:

Example The experimental phase of this study was performed between 13 May 2008 and 04 November 2009.

Reliability:

1 (reliable without restriction)

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 methodological 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

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:

SULFOCLOR LF 1013

IUPAC Name:

SULFOCLOR LF 1013

Constituent 2

Reference substance name:

Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated

EC Number:

269-145-7

EC Name:

Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated

Cas Number:

68188-19-2

Molecular formula:

CnHxCly(SO2Cl)z n= 14 - 17 or n=17 - 30 ; x=2n+2-y-z

IUPAC Name:

Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated

Details on test material:

Sponsor's identification: SULFOCLOR LF 1013
CAS Number : 68188-19-2
Description : pale yellow slightly viscous liquid
Purity : > 99%
Batch number: not supplied
Label : Sulfoclor LF 1013 TANQUE 25.09.2009
Date received: 05 February 2010
Expiry date : not supplied
Storage conditions: room temperature in the dark

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in house. Acetone was therefore selected as the vehicle

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix

Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) - Experiment 1

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

METHOD OF APPLICATION: in agar (plate incorporation) - Experiment 2

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): 20 minutes at 37 degress C

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:A precipitate (greasy in appearance) was noted to the naked eye at and above 1500 µg/plate. Additionally a dark, circular precipitate was also noted under an inverted microscope at 5000 µg/plate. These observations did not prevent the scoring of revertant colonies

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS

Preliminary Toxicity Test

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA^-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	71	79	66	68	90	82	73	68	62	78P	71P
+	TA100	78	74	80	66	70	74	76	76	77	81P	80P
-	WP2uvrA^-	22	15	13	18	14	15	25	21	21	16P	18P
+	WP2uvrA^-	30	21	18	31	22	23	24	16	24	18P	19P

Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table1and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Tables 2 -5 (see attached Background material)

A history profile of vehicle and positive control values is presented in Appendix 2 (see attached Background material)

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1               Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
103		18		23		8		8
104	(102)	13	(14)	24	(26)	25	(19)	13	(11)
100		11		30		24		13

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
89		15		14		19		10
90	(82)	18	(14)	25	(19)	23	(22)	11	(12)
67		10		19		24		16

Please also see

Attachments: "Results (Tables 2 - 5)".

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

SULFOCLOR LF 1013 was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction. 

The study was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system. The method used follows that described in the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA^-were treated with SULFOCLOR LF 1013 using both the Ames plate incorporation and pre-incubation methods at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using a dose range 15 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test material formulations. An additional dose level was included in the second experiment to allow for potential toxicity following the change in test methodology.

Results.

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.

SULFOCLOR LF 1013 caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate (greasy in appearance) was noted to the naked eye at and above 1500 µg/plate. Additionally a dark, circular precipitate was also noted under an inverted microscope at 5000 µg/plate. These observations did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Conclusion. SULFOCLOR LF 1013 was considered to be non-mutagenic under the conditions of this test.