Sulfuric acid, C16-C18 (even numbered) alkyl esters, sodium salts and C16-18 (even numbered) alcohols

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative results for bacterial mutagenicity were available based on literature data for the long chain alcohols (LCHO) C14 and C18. Negative results for bacterial and in vitro mammalian mutagenicity were available based on literature data for the alkyl sulfates (AS) C12-C16. Negative results for bacterial mutagenicity were available based on literature data for sodium sulfate.

An in vitro micronucleus test with the registered substance tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

OTHER SPECIFICS:
C12 > 98 %
97 % active substance
Trade name: Texapon K 12.

Target gene:

Histidine

Species / strain / cell type:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Test concentrations with justification for top dose:

1st test: 8, 40, 200, 1000 or 5000 µg/plate
2nd test: 5, 10, 20, 40 or 80 µg/plate (-S9) or 2.5, 10, 40, 160 or 640 µg/plate (+S9)

Untreated negative controls:

yes

Remarks:

suspension medium bidist. water and untreated fresh cell suspensions

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S-9: TA 100, TA 1535

Untreated negative controls:

yes

Remarks:

suspension medium bidist. water and untreated fresh cell suspensions

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S-9: TA 1537

Untreated negative controls:

yes

Remarks:

suspension medium bidist. water and untreated fresh cell suspensions

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S-9: TA 98, TA 1538

Untreated negative controls:

yes

Remarks:

suspension medium bidist. water and untreated fresh cell suspensions

Positive controls:

yes

Positive control substance:

other: 2 aminoanthracene

Remarks:

with S-9: all strains

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 80 µg/plate

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 80 µg/plate

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 80 µg/plate

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 80 µg/plate

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 80 µg/plate

Conclusions:

C12-Alkylsulfate (97% active substance) tested negative in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation at doses of 80 µg/plate (-S9) and 640 µg/plate (+S9). Cytotoxic concentration was >= 80 µg/plate.

Executive summary:

The IUCLID dataset of C12-Alkylsulfate (CAS 151-21-3) in the OECD SIDS Initial Assessment Profile on Sodium dodecyl sulfate (SDS) describes the following Klimisch 1 study according to OECD Guideline 471 in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538.

Sodium dodecyl sulfate was tested at the following doses:

1st test: 8, 40, 200, 1000 or 5000 µg/plate.

2nd test: 5, 10, 20, 40 or 80 µg/plate (-S9) or 2.5, 10, 40, 160 or 640 ug/plate (+S9).

The second test was performed with and without metabolic activation. Negative controls (suspension medium bidist. water and untreated fresh cell suspensions) and positive controls sodium azide, 9-aminoacridine, 4-nitro-o-phenylendiamine and 2 aminoanthracene were also tested.

C12-Alkylsulfate (97% active substance) tested negative in this Ames test in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation at doses of 80 µg/plate (-S9) and 640 µg/plate (+S9). Cytotoxic concentration was >= 80 µg/plate.

 

Reference: Henkel KGaA (1988) Texapon K 12. Pruefung auf Mutagenitaet im Amest-Test (Unpublished Report No. 880075). Henkel KGaA, Duesseldorf, 35 pp.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

according to guideline

Guideline:

other: not specified

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

tk+/tk-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Test concentrations with justification for top dose:

3.125 - 100 µg/mL (4 hrs)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 70 µg/mL

Conclusions:

C12-Alkylsulfate tested negative in mouse lymphoma L1578Ytk+/tk- cells with and without metabolic activation with cytotoxic concentration >=70µg/mL.

Executive summary:

The IUCLID dataset of C12-Alkylsulfate (CAS 151-21-3) in the OECD SIDS Initial Assessment Profile on Sodium dodecyl sulfate (SDS) describes the following Klimisch 2 study in Mouse lymphoma L5178Y tk+/tk- cells.

The assay was done with and without metabolic activation at concentrations between 3.125 and 100 µg/mL. The genotoxicity was negative for C12-Alkylsulfate with cytotoxic concentration >= 70 µg/mL.

 

Reference: McGregor DB, Brown A, Cattanach P, Edwards I, McBride D, Riach C & Caspary WJ (1988) Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay: III. 72 coded chemicals. Environ Mol Mutagen 12, 85 - 154

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

according to guideline

Guideline:

other: P&G standard procedure #28

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 (rat liver)

Test concentrations with justification for top dose:

0.25 - 8 µL/mL

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without S9: TA 1535, TA100

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9: TA 1537

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9: TA 1538, TA98

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2AA

Remarks:

With S9: TA 1535, TA 100

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 8AQ

Remarks:

With S9: TA 1537

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2AAF

Remarks:

With S9: TA 1538, TA 98

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: B[a]p

Remarks:

With S9: TA1538, TA 98

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 4 µL/mL

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 4 µL/mL

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 4 µL/mL

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 4 µL/mL

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 4 µL/mL

Conclusions:

C14-Alkylsulfate tested negative in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation at doses of 0.25 - 8 µL/mL. Cytotoxic concentration was >= 4 µL/mL.

Executive summary:

The IUCLID dataset of C14-Alkylsulfate (CAS 1191 -50 -0) in the OECD SIDS Initial Assessment Profile on Sodium tetradecyl sulfate describes the following Klimisch 2 study according to P&G standard procedure #28 in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538.

The assay was done with and without metabolic activation (S9 rat liver) at doses from 0.25 - 8 µL/mL.

C14-Alkylsulfate tested negative in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation at doses of 0.25 - 8 µL/mL. Cytotoxic concentration was >= 4 µL/mL.

 

Reference: Procter & Gamble Co. (1979) Evaluation of various surfactants for compatibility with the Salmonella/Microsome test (Unpublished Report; P&G accession # 22411).Procter & Gamble Co., Cincinnati, Ohio, 60 pp.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Remarks:

no data

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

tk+/tk-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cells were maintained and cleansed of spontaneous TK+/+ mutants biweekly as decscribed by Clive et al. (1979) Mutat Res 59, 61 - 108.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

0.06 - 1 µL/mL

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2AAF

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Testing was performed with a 3-day expression period as described in Coppinger et al. (1979) P&G Internal Report

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 0.13 µL/mL

Conclusions:

C14-Alkylsulfate tested negative in mouse lymphoma L1578Ytk+/tk- cells with and without metabolic activation with cytotoxic concentration >=0.13µg/mL.

Executive summary:

The IUCLID dataset of C14-Alkylsulfate (CAS 1191 -50 -0) in the OECD SIDS Initial Assessment Profile on Sodium tetradecyl sulfate sulfate describes the following Klimisch 2 study in mouse lymphoma L5178Y tk+/tk- cells.

The assay was done with and without metabolic activation at concentrations between 0.06 and 1 µL/mL. The genotoxicity was negative for C14-Alkylsulfate with cytotoxic concentration >=0.13 µL/mL.

 

Reference: Procter & Gamble Co. (1981) Testing of surfactants for mutagenic potential in the L5178 TK+/- mouse lymphoma assay (Unpublished Report; P&G accession # 26142).Procter & Gamble Co., Cincinnati, Ohio, 24 pp

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

according to guideline

Guideline:

other: P&G standard procedure #28

GLP compliance:

not specified

Remarks:

no data

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 (rat liver)

Test concentrations with justification for top dose:

0.25 - 8 µL/mL

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: EMS

Remarks:

Without S9: TA 1535, TA 100

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 7AA

Remarks:

Without S9: TA 1537

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2NF

Remarks:

Without S9: TA1538, TA 98

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2AA

Remarks:

With S9: TA1535, TA 100

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 8AQ

Remarks:

With S9: TA 1537

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2AAF

Remarks:

With S9: TA1538, TA 98

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: B[a]p

Remarks:

With S9: TA1538, TA 98

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 2 µL/mL

Conclusions:

C16-Alkylsulfate tested negative in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation at doses of 0.25 - 8 µL/mL. Cytotoxic concentration was >= 2 µL/mL.

Executive summary:

The IUCLID dataset of C16-Alkylsulfate (CAS 1120 -01 -0) in the OECD SIDS Initial Assessment Profile on Hexadecyl sulfate sodium salt describes the following Klimisch 2 study according to P&G standard procedure #28 in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538.

The assay was done with and without metabolic activation at concentrations between 0.25 and 8 μL/mL.

The genotoxicity was negative for C16-Alkylsulfate with cytotoxic concentration >= 2 μL/mL.

 

Reference: Procter & Gamble Co. (1979) Evaluation of various surfactants for compatibility with the Salmonella/Microsome test (Unpublished Report; P&G accession # 22411).Procter & Gamble Co., Cincinnati, Ohio, 60 pp.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

according to guideline

Guideline:

other: specified in freetext not provided in SIDS dossier

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cells were maintained and cleansed of spontaneous TK+/+ mutants biweekly as decscribed by Clive et al. (1979) Mutat Res 59, 61 - 108.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

0.01 - 1 µL/mL

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2AAF

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Testing was performed with a 3-day expression period as described in Coppinger et al. (1979) P&G Internal Report

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 0.09 uµ/mL

Conclusions:

C16-Alkylsulfate tested negative in mouse lymphoma L1578Ytk+/tk- cells with and without metabolic activation with cytotoxic concentration >= 0.09 µL/mL.

Executive summary:

The IUCLID dataset of C16-Alkylsulfate (CAS 1120-01-0) in the OECD SIDS Initial Assessment Profile on Hexadecyl sulfate sodium salt describes the following Klimisch 2 study in mouse lymphoma L5178Y tk+/tk- cells.

The assay was done with and without metabolic activation at concentrations between 0.01 and 1 µL/mL. The genotoxicity was negative for C16-Alkylsulfate with cytotoxic concentration >= 0.09 µL/mL.

Reference: Procter & Gamble Co. (1981) Testing of surfactants for mutagenic potential in the L5178 TK+/- mouse lymphoma assay (Unpublished Report; P&G accession # 26142).Procter & Gamble Co., Cincinnati, Ohio, 24 pp

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Remarks:

no data

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 (rat liver)

Test concentrations with justification for top dose:

62.5 - 2000 µg/plate (- S9)
15.6 - 500 µg/plate (+ S9)

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

no data

Conclusions:

C16-Alkylsulfate tested negative in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation at doses of 62.5 - 2000 µg/plate (- S9) and 15.6 - 500 µg/plate (+ S9). There were no data on cytotoxic concentration.

Executive summary:

The IUCLID dataset of C16-Alkylsulfate (CAS 1120 -01 -0) in the OECD SIDS Initial Assessment Profile on Hexadecyl sulfate sodium salt describes the following Klimisch 2 study in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538.

The assay was done with and without metabolic activation at following doses:

62.5 - 2000 µg/plate (- S9)

15.6 - 500 µg/plate (+ S9)

The genotoxicity was negative for C16-Alkylsulfate.

 

Reference: Procter & Gamble Co. (1976) Testing for mutagenic activity according to the IRI proposal no. 406481 - Ames test (Unpublished Report; P&G accession # 19060). Inveresk Research Int., 5 pp

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

OTHER SPECIFICS: Tradename Kalcol 4098

Target gene:

Histidine

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA98, and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 Arocholor 1254 induced

Test concentrations with justification for top dose:

Test 1: 15 (-S9 only), 50, 150, 500, 1500 and 5000 µg/plate.
Test 2: 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s): DMSO.- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitrosoguanidine

Remarks:

without S9: 3 µg/plate (TA100), 5 µg/plate (TA1535)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9: 80 µg/plate (TA1537)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene diamine

Remarks:

without S9: 5 µg/plate (TA 1538)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide

Remarks:

without S9: 0.2 μg/plate (TA98)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (0.5, 1 or 2 ug/plate)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9: 0.5, 1 or 2 μg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).

DURATION
- Preincubation period: No
- Exposure duration: 48 hours at 37°C
- Selection time (if incubation with a selection agent): 48 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate

Evaluation criteria:

A dose related and statistically significant increase in reverse mutation rate in one or more bacterial strains at sub-toxic dose levels. For a negative result the numbers of induced revertants should be less than two fold compared to controls.

Statistics:

Dunnetts test was used and showed no statistically significant differences between test and control plates.

Species / strain:

other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increase in reverse mutation rate in any strain at dose levels up to 5000 µg/plate.

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Slight cytotoxicity was indicated in a preliminary toxicity screen with TA100 at dose levels ≥ 500 µg/plate without metabolic activation.

Remarks:

In the actual mutation study there was no evidence of cytotoxicity up to 5000 µg/plate with or without S9.

Conclusions:

The C14 alcohol Kahlcol 4098 did not increase the reverse mutation rate in histidine dependent bacterial strains of Salmonella typhimurium in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate.

Executive summary:

A bacterial reverse mutation assay was performed according to OECD TG 471 in Salmonella typimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 with and without metabolic activation (rat liver S9, Arochlor 1254 induced).

The test item was dissolved in DMSO and the assay was done by plate incorporation at doses of:

Test 1: 15 (-S9 only), 50, 150, 500, 1500 and 5000 µg/plate.

Test 2: 50, 150, 500, 1500 and 5000 µg/plate.

Tests were performed in triplicate. Slight cytotoxicity was indicated in a preliminary toxicity screen with TA100 at dose levels ≥ 500 µg/plate without metabolic activation. In the actual mutation study there was no evidence of cytotoxicity up to 5000 µg/plate with or without S9. Positive and negative controls gave appropriate responses.

(Reference: Thompson, P.W.1996b.Kalcohl 4098: Reverse mutation assay "Ames test" using Salmonella typhimurium. SPL Project Number 140/597)

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

OTHER SPECIFICS: Tradename Kalcol 6098

Target gene:

histidine

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 Arochlor 1254 induced

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate for both tests.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitrosoguanidine

Remarks:

without S9: 3 µg/plate (TA100), 5 µg/plate (TA 1535)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9: 80 µg/plate (TA1537)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene diamine

Remarks:

without S9: 5 µg/plate (TA 1538)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide

Remarks:

without S9: 0.2 µg/plate (TA98)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9: 0.5, 1 or 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No
- Exposure duration: 48 hours at 37°C
- Selection time (if incubation with a selection agent): 48 hours at 37°C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: Duplicate tests each performed in triplicate

Evaluation criteria:

A dose related and statistically significant increase in reverse mutation rate in one or more bacterial strains at sub-toxic dose levels.
For a negative result the numbers of induced revertants should be less than two fold compared to controls.

Statistics:

Dunnetts test was used and showed no statistically significant differences between test and control plates.

Species / strain:

other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increase in reverse mutation rate in any strain at dose levels up to 5000 µg/plate.

Cytotoxicity / choice of top concentrations:

other: There was no evidence of cytotoxicity up to 5000 µg/plate with or without S9.

Remarks:

Precipitation concentration at 5000 µg/plate did not interfere with counting revertant colonies.

Untreated negative controls validity:

valid

Remarks:

Negative controls gave appropriate responses.

Positive controls validity:

valid

Remarks:

Positive controls gave appropriate responses.

Conclusions:

The C16 alcohol Kahlcol 6098 did not increase the reverse mutation rate in histidine dependent bacterial strains of Salmonella typhimurium in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate.

Executive summary:

A bacterial reverse mutation assay was performed according to OECD TG 471 in Salmonella typimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 with and without metabolic activation (rat liver S9, Arochlor 1254 induced). The test item was dissolved in acetone and the assay was done by plate incorporation at doses of 50, 150, 500, 1500 and 5000 µg/plate. Duplicate tests each were performed in triplicate.

There was no evidence of cytotoxicity up to 5000 µg/plate with or without S9. At 5000 µg/plate precipitation occurred but this did not interfere with counting revertant colonies. No increase in reverse mutation rate in any strain with and without metabolic activation at dose levels up to 5000 µg/plate. Positive and negative controls gave appropriate responses.

(Reference: Thompson, P.W. 1996c. Kalcohl 6098: Reverse mutation assay "Ames test" using Salmonella typhimurium. SPL Project Number 140/499.)

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

OTHER SPECIFICS: Tradename Kalcol 8098

Target gene:

Histidine

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA98, and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 Arocholor 1254 induced

Test concentrations with justification for top dose:

Dosing: 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitrosoguanidine

Remarks:

without S9: 3 µg/plate (TA100), 5 µg/plate (TA1535)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9: 80 µg/plate (TA1537)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene diamine

Remarks:

without S9: 5 µg/plate (TA 1538)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide

Remarks:

without S9: 0.2 μg/plate (TA98)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9: 0.5, 1 or 2 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).

DURATION
- Preincubation period: No
- Exposure duration: 48 hours at 37°C
- Selection time (if incubation with a selection agent): 48 hours at 37°C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: Duplicate tests each performed in triplicate

Evaluation criteria:

A dose related and statistically significant increase in reverse mutation rate in one or more bacterial strains at sub-toxic dose levels. For a negative result the numbers of induced revertants should be less than two fold compared to controls.

Statistics:

Dunnetts test was used and showed no statistically significant differences between test and control plates.

Species / strain:

other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increase in reverse mutation rate in any strain at dose levels up to 5000 µg/plate.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

There was no evidence of cytotoxicity up to 5000 µg/plate with or without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: ≥ 500 µg/plate but this did not interfere with scoring of the plate, plates were counted manually at 5000 µg/plate.

Conclusions:

The C18 alcohol Kahlcol 8098 did not increase the reverse mutation rate in histidine dependent bacterial strains of Salmonella typhimurium in the presence or absence of metabolic activation at dose levels up to 5000 µg/plate. This dose level was not cytotoxic.

Executive summary:

A bacterial reverse mutation assay was performed according to OECD TG 471 in Salmonella typimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 with and without metabolic activation (rat liver S9, Arochlor 1254 induced).

The test item, C18 alcohol Kahlcol 8098, was dissolved in ethanol and the assay was done by plate incorporation at doses of 50, 150, 500, 1500 and 5000 μg/plate. Duplicate tests each were performed in triplicate. There was no evidence of cytotoxicity up to 5000 μg/plate with or without S9. At 5000 μg/plate precipitation occurred but this did not interfere with counting revertant colonies. No increase in reverse mutation rate in any strain with and without metabolic activation at dose levels up to 5000 μg/plate. Positive and negative controls gave appropriate responses.

(Reference: Thompson, P.W.1996d.Kalcohl 809 +8: Reverse mutation assay "Ames test" using Salmonella typhimurium. SPL Project Number 140/505).

Reference 11

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted July 29, 2016.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: OE70403001
- Expiration date of the lot/batch: April 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C

Species / strain / cell type:

lymphocytes:

Remarks:

cultured human peripheral lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human peripheral blood was obtained by venipuncture and collected in heparinised vessels.
- Sex, age and number of blood donors if applicable: young, healthy, non-smoking individuals with
no known recent exposures to genotoxic chemicals or radiation.
- Methods for maintenance in cell culture if applicable: Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin
* Ham’s F10 supplemented with 10% fetal calf serum (FCS)
* Chromosome complete medium with 5 μg/mL Cytochalasin B.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

CytoB used in a concentration 5 μg/mL

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver, Aroclor 1254 induced)

Test concentrations with justification for top dose:

-Preliminary experiment (without and with metabolic activation): 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/mL medium.
-Main Test:
4 h exposure with and without S9: 18.75, 37.5, 75, 150 and 300 μg/mL medium.
24h exposure without S9: 6.25, 12.5, 25, 50 and 100 µg/mL medium.
In the preliminary test cytotoxicity was noted starting at a concentration of 100 µg/mL medium in the experiment without metabolic activation (24-hour exposure). Test item precipitation and cytotoxicity were noted at concentrations of 316 and 1000 µg/mL medium in both experiments (24-hour or 4-hour exposure).
Hence, 300 µg/mL medium were employed as the top concentration for the genotoxicity tests without and with metabolic activation with a 4-hour exposure and 100 µg/mL medium for the 24-hour exposure experiment.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 was completely dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg/mL

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

mitomycin C

Remarks:

0.1 µg/mL and 0.2 µg/mL without S9

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

cyclophosphamide

Remarks:

10 µg/mL and 20 µg/mL with S9

Positive controls:

yes

Remarks:

aneugen

Positive control substance:

other: colchicine

Remarks:

0.01 µg/mL and 0.02 µg/mL without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
Without S9: 4 hours and 24 hours.
With S9: 4 hours
- Exposure duration: 20 h incubation with spindle inhibitor Cytochalasin B
- After exposure: 20 h incubation with spindle inhibitor Cytochalasin B

STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: duplicate (main study), one culture per concentration in the preliminary test

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Each culture was harvested and processed separately. After the test item incubation, mitotic activity was arrested by the addition of CytoB to each culture at a final concentration of 5 μg/mL. Afteran additional incubation of 20 hours the cultures were centrifuged for 10 minutes at 800 rpm, the supernatant was discarded and the cells resuspended in KCl (0.56%). After incubation for 17 minutes at 37°C, the cell suspensions were centrifuged for 10 minutes at 800 rpm. The supernatant was discarded and 5 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added. The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm. The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette. Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide. The slides were then stained using 10% Giemsa and left to air-dry at room temperature.

NUMBER OF CELLS EVALUATED:
-At least 500 cells per replicate cell culture were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity.
-The micronucleus frequencies were analysed in at least 2000 binucleate cells per concentration (at least 1000 binucleate cells per culture; two cultures per concentration). If substantially fewer than 1000 binucleate cells per culture are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test would be repeated using more cells, or at less toxic concentrations, whichever is appropriate

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.

DETERMINATION OF CYTOTOXICITY
- Method: other:
The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI). The CBPI indicates the average number of nuclei per cell during the period of exposure to CytoB, and is used to calculate cell proliferation. The RI indicates the relative number of cell cycles in treated cultures compared to control cultures and can be used to calculate the % cytostasis:
CBPI =((No. mononucleate cells)+(2×No. binucleate cells)+(3×No. multinucleate cells)) / (Total num ber of cells).
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.
Cytostasis = 100 - RI
RI = [((No. binucleate cells)+(2×No. multinucleate cells))÷(Total number of cells)T / ((No. binucleate cells)+(2×No. multinucleate cells))÷(Total number of cells)C ] × 100
T= treated cultures
C= control cultures
Thus, an RI of 53% means that, compared to the numbers of cells that have divided to form binucleate and multinucleate cells in the control culture, only 53% of this number divided in the treated culture, i.e. 47% cytostasis.
- Any supplementary information relevant to cytotoxicity: Treatment of cultures with CytoB, and measurement of the relative frequencies of mononucleate, binucleate, and multi-nucleate cells in the culture, provides an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment and ensures that only cells that divided during or after treatment are scored.

Evaluation criteria:

Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction.Concurrent measures of cytotoxicity and/or cytostasis for all treated and vehicle control cultures were determined.Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if: at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control; the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test; any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits).Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if: none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control; there is no concentration-related increase when evaluated with an appropriate trend test; all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).Equivocal results may be clarified by analysis of another 1000 cells from all the cultures to avoid loss of blinding.If this approach does not resolve the result, further testing would be necessary.Modification of study parameters over an extended or narrowed range of conditions, as appropriate, would be considered in follow-up experiments.Study parameters that might be modified include the test concentration spacing, the timing of treatment and cell harvest, and/or the metabolic activation conditions.Although most experiments give clearly positive or negative results, in some cases the data set would preclude making a definite judgement about the activity of the test item.These equivocal or questionable responses may occur regardless of the number of times the experiment is repeated

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Complete cytotoxicity and test item precipitation (macroscopically) were noted in the 4-hour exposure experiments with metabolic activation at concentrations of 300 µg/mL medium.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

vehicle control = negative control

Positive controls validity:

valid

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was noted starting at concentrations of 75 µg/mL medium. Complete cytotoxicity and test item precipitation (macroscopically) were noted at concentrations of 150 µg/mL medium (4 h-exposure without S9).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

vehicle control = negative control

Positive controls validity:

valid

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was noted starting at concentrations of 25 µg/mL medium in the experiments without metabolic activation (24-hour exposure).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

vehicle control = negative control

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant changes in pH of the test item formulations compared to the negative control were noted up to the top concentration of 1000 μg/mL medium. Digital pH meter type SevenCompact S210 Mettler-Toledo GmbH, 35396 Gießen, Germany was used.
- Effects of osmolality: No relevant changes in osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 1000 μg/mL medium. Semi-micro osmometer Typ ML A0299 Knauer GmbH, 14163 Berlin, Germany was used.
- Precipitation of the test item was checked before and after each experiment. Evaluation of precipitation was done by light microscopy at the beginning and end of treatment.
- Definition of acceptable cells for analysis: Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. S
coring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
For the last 27 studies (most recent background data of the years 2015 to 2017, not audited by the QAU-department):
WITHOUT METABOLIC ACTIVATION
*Vehicle controls 4 hour exposure
Mean: 5.4
SD: 2.6
Range:1.0 - 16
95% Confidence interval: 4.8 - 6.1
*Vehicle controls 24 hour exposure
Mean: 5.2
SD: 2.4
Range: 1.0 - 13
95% Confidence interval: 4.6 – 5.8
WITH METABOLIC ACTIVATION
*Vehicle control
Mean: 4.7
SD: 2.1
Range: 1 - 9
95% Confidence interval: 4.2 – 5.4
- Positive historical control data: For the last 27 studies (most recent background data of the years 2015 to 2017, not audited by the QAU-department):
*Mitomycin C
Mean: 26.0
SD: 10.3
Range: 12 - 61
*Colchicine
Mean : 31.2
SD: 20.6
Range:12 - 125
*Cyclophosphamide
Mean : 22.5
SD: 9.1
Range: 12 - 55
- Negative (solvent/vehicle) historical control data: The results for the vehicle controls were within the historical control range.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: . The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).

Remarks on result:

other: 4-hour exposure

Conclusions:

Under the present test conditions, Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.
The results for the vehicle controls were within the historical control range.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Executive summary:

Test samples of Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 was completely dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg/mL. The test item was soluble only by warming up to 37°C for 30 minutes and by carefully stirring after addition to the treatment medium to a final concentration of 1000 µg/mL medium. 1000 µg/mL medium was the maximum feasible concentration.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 µg Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18/mL medium were employed. Cytotoxicity was noted starting at a concentration of 100 µg/mL medium in the experiment without metabolic activation (24-hour exposure). Test item precipitation and cytotoxicity were noted at concentrations of 316 and 1000 µg/mL medium in both experiments (24-hour or 4-hour exposure). No changes in pH or osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 1000 µg/mL medium.

Hence, 300 µg/mL medium were employed as the top concentration for the genotoxicity tests without and with metabolic activation with a 4-hour exposure and 100 µg/mL medium for the 24-hour exposure experiment.

In the main study cytotoxicity was noted starting at concentrations of 75 or 25 µg Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18/mL medium in the experiments without metabolic activation (4-hour or 24-hour exposure, respectively). Complete cytotoxicity and test item precipitation (macroscopically) were noted in the 4-hour exposure experiments without and with metabolic activation at concentrations of 150 and 300 µg/mL medium.

Mitomycin C (at 0.2 µg/mL) and colchicine (at 0.02 µg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 µg/mL) in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The mean micronucleus frequencies of cultures treated with the concentrations of 18.75, 37.5, 75, 150 and 300 or 6.25, 12.5, 25, 50 and 100 µg Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 3.5 to 6.5 micronucleate cells per 1000 binucleate cells. There was no dose-related increase in micronuclei up to the top concentration of 75 or 25 µg/mL medium (4- and 24-hour exposure, respectively). No binucleated cells could be evaluated at the higher concentrations due to cytotoxicity and/or test item precipitation. The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test mean frequencies of 5.5 or 4.0 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively, were observed. The vehicle result was within the historical control ranges.

In the positive control cultures the mean micronucleus frequencies were increased to 17.5 or 18.5 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively.This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure)

The mean micronucleus frequencies of cultures treated with the concentrations of 18.75, 37.5, 75, 150 and 300 µg Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18/mL medium (4-h exposure) in the presence of metabolic activation ranged from 2.0 to 3.5 micronucleate cells per 1000 binucleate cells. There was no dose-related increase in micronuclei up to the top concentration of 75 µg/mL medium. No binucleate cells could be evaluated at the higher concentrations due to cytotoxicity and/or test item precipitation. The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test a mean frequency of 4.5 micronucleate cells per 1000 binucleate cells was observed. The vehicle result was within the historical control ranges.

In the positive control culture the mean micronucleus frequency was increased to 23.5 micronucleate cells per 1000 binucleate cells for the 4-hour exposure. This demonstrated that cyclophosphamide induced significant chromosomal damage.

Conclusion

Under the present test conditions, Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.

The results for the vehicle controls were within the historical control range.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Reference 12

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

LT2 system

Test concentrations with justification for top dose:

4 concentrations:
312.5-625-1250-2500-5000 mg/L and 8-40-200-1000-5000 mg/L
(312.5 to 5000 μg per plate with 4 dilutions)

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Positive control substance:

other: nitrofurantoin

Positive control substance:

other: 4-nitro-1, 2-phenylene diamine

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 4 per test

Evaluation criteria:

Based on Maron and Ames (1983).

Statistics:

n.a.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no toxicity observed up to 5000 mg/L

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no toxicity observed up to 5000 mg/L

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no toxicity observed up to 5000 mg/L

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no toxicity observed up to 5000 mg/L

Conclusions:

Na2SO4 tested negative in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation at doses of 312.5-625-1250-2500-5000 mg/L. No cytotoxicity was observed up to 5000 mg/L.

Executive summary:

The IUCLID data set of Na2SO4 (CAS 7757 -82 -6) in the OECD SIDS INITIAL ASSESSMENT PROFILE of Sodium sulfate describes the following Klimisch 1 study in Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537.

The assay was done with and without metabolic activation at following doses: 312.5-625-1250-2500-5000 mg/L and 8-40-200-1000-5000 mg/L. No toxicity was observed up to 5000mg/L. The genotoxicity was negative for Na2SO4 under the present test conditions.

Reference: Bayer A.G. (1988). Salmonella/Microsome test to evaluate for point mutagenic effects. Unpublished report. Study no. T9027163, Report no. 16839, Wuppertal, Germany

Reference 13

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks on result:

other: not tested with Na2SO4

Conclusions:

The registered substance does not have to be classified for genotoxicity.

Executive summary:

LCOH 14 (Alfol 14; 1-Tetradecanol; CAS 112-72-1) did not increase the reverse mutation rate inSalmonella typhimuriumstrains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate (SIDS, 2006; Thompson, 1996b).

LCOH 16 (1-Hexadecanol; CAS 36653-82-4) did not increase the reverse mutation rate inSalmonella typhimuriumstrains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate (SIDS, 2006; Thompson, 1996c).

LCOH 18 (1-Octadecanol; CAS 112-925) did not increase the reverse mutation rate inSalmonella typhimuriumstrains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate (SIDS, 2006; Thompson, 1996d).

C12-Alkylsulfate (CAS 151-21-3) did not increase the reverse mutation rate inSalmonella typhimuriumstrains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate; as cytotoxicity was noted above 80 µg/plate, a second test was performed at concentrations of 5, 10, 20, 40 or 80 µg/plate (-S9) or 2.5, 10, 40, 160 or 640 µg/plate (+S9) (SIDS, 2008; Henkel, 1988).

C14-Alkylsulfate (CAS 1191-50-0) did not increase the reverse mutation rate inSalmonella typhimuriumstrains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation up to cytotoxic concentration up 4 µL/mL (SIDS, 2008; Procter & Gamble Co., 1979).

C16-Alkylsulfate (CAS 1120-01-0) did not increase the reverse mutation rate inSalmonella typhimuriumstrains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation up to cytotoxic concentration up 2 µL/mL in one study and up to 2000 µg/plate (-S9) and 500 µg/plate (+S9) in another study (SIDS, 2008; Procter & Gamble Co., 1979).

For sodium sulfate (CAS 7757 -82 -6) a reverse mutation study was conducted inSalmonella typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation up to 5000 µg/mL. No toxicity observed and mutagenicity was negative up to highest concentration (SIDS, 2005; Bayer ,1988).

Based on these results and according to CLP (No. 1272/2008 of 16 December 2008), the registered substance does not have to be classified and has no obligatory labelling requirement for genotoxicity.

Reference 14

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Conclusions:

The registered substance does not have to be classified for genotoxicity.

Executive summary:

C12-Alkylsulfate (CAS 151-21-3): A Mouse Lymphoma assay in L5178Ytk+/- cells was negative for mammalian gene mutation both with and without metabolic activation up to cytotoxic concentration of 70 µg/mL (SIDS, 2007; Henkel, 1988). 

C14-Alkylsulfate (CAS 1191-50-0): A Mouse Lymphoma assay in L5178Ytk+/- cells was negative for mammalian gene mutation both with and without metabolic activation up to cytotoxic concentration of 0.13 µL/mL (SIDS, 2007; Procter & Gamble Co., 1981). 

C16-Alkylsulfate (CAS 1120-01-0): A Mouse Lymphoma assay in L5178Ytk+/- cells was negative for mammalian gene mutation both with and without metabolic activation up to cytotoxic concentration of 0.0.9 µL/mL (SIDS, 2007; Procter & Gamble Co., 1981). 

Based on these results and according to CLP (No. 1272/2008 of 16 December 2008), the registered substance does not have to be classified and has no obligatory labelling requirement for genotoxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Limited extra testing was performed for the registered substance as literature data were available as weight-of-evidence for the various components of the registered substances, except for a Micronucleus study. A selection of most reliable sources (Klimisch 1-2) was made.

In vitro:

Limited literature data were available, i.e. only for bacterial mutagenicity, for the long chain alcohols (LCHO) with chain length between C14 and C18, as summarised below.

-      LCOH 14 (Alfol 14; 1-Tetradecanol; CAS 112-72-1) did not increase the reverse mutation rate in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate (SIDS, 2006; Thompson, 1996b).

-      LCOH 16 (1-Hexadecanol; CAS 36653-82-4) did not increase the reverse mutation rate in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate (SIDS, 2006; Thompson, 1996c).

-      LCOH 18 (1-Octadecanol; CAS 112-925) did not increase the reverse mutation rate in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate (SIDS, 2006; Thompson, 1996d).

For the alkyl sulfates (AS), there were more data for the C12-C16 chain lengths, both for the bacterial and gene mutation assays.

-      C12-Alkylsulfate (CAS 151-21-3) did not increase the reverse mutation rate in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate; as cytotoxicity was noted above 80 µg/plate, a second test was performed at concentrations of 5, 10, 20, 40 or 80 µg/plate (-S9) or 2.5, 10, 40, 160 or 640 µg/plate (+S9) (SIDS, 2008; Henkel, 1988). A Mouse Lymphoma assay in L5178Ytk+/- cells was negative for mammalian gene mutation both with and without metabolic activation up to cytotoxic concentration of 70µg/mL (SIDS, 2007; Henkel, 1988). 

-      C14-Alkylsulfate (CAS 1191-50-0) did not increase the reverse mutation rate in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation up to cytotoxic concentration up 4 µL/mL (SIDS, 2008; Procter & Gamble Co., 1979). A Mouse Lymphoma assay in L5178Ytk+/- cells was negative for mammalian gene mutation both with and without metabolic activation up to cytotoxic concentration of 0.13µL/mL (SIDS, 2007; Procter & Gamble Co., 1981). 

-      C16-Alkylsulfate (CAS 1120-01-0) did not increase the reverse mutation rate inSalmonella typhimuriumstrains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 in the presence or absence of metabolic activation up to cytotoxic concentration up 2 µL/mL in one study and up to 2000 µg/plate (-S9) and 500 µg/plate (+S9) in another study (SIDS, 2008; Procter & Gamble Co., 1979). A Mouse Lymphoma assay in L5178Ytk+/- cells was negative for mammalian gene mutation both with and without metabolic activation up to cytotoxic concentration of 0.0.9µL/mL (SIDS, 2007; Procter & Gamble Co., 1981). 

For the sodium sulfate, literature data were available:

-      For sodium sulfate (CAS 7757 -82 -6) a reverse mutation study was conducted inSalmonella typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation up to5000 µg/mL. No toxicity observed and mutagenicity was negative up to highest concentration (SIDS, 2005; Bayer ,1988).

A key in vitro Micronuclues study was performed for the registered substance using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The concentrations employed were chosen based on the results of a cytotoxicity study. Cytotoxicity was noted starting at a concentration of 100 µg/mL medium in the experiment without metabolic activation (24-hour exposure). Test item precipitation and cytotoxicity were noted at concentrations of 316 and 1000 µg/mL medium in both experiments (24-hour or 4-hour exposure). Hence, 300 µg/mL medium were employed as the top concentration for the genotoxicity tests without and with metabolic activation with a 4-hour exposure and 100 µg/mL medium for the 24-hour exposure experiment.

In the main study cytotoxicity was noted starting at concentrations of 75 or 25 µg Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18/mL medium in the experiments without metabolic activation (4-hour or 24-hour exposure, respectively). Complete cytotoxicity and test item precipitation (macroscopically) were noted in the 4-hour exposure experiments without and with metabolic activation at concentrations of 150 and 300 µg/mL medium.

Tests without metabolic activation (4- and 24-hour exposure): no dose-related increase in micronuclei up to the top concentration of 75 or 25 µg/mL medium (4- and 24-hour exposure, respectively). No binucleated cells could be evaluated at the higher concentratoins. concentrations due to cytotoxicity and/or test item precipitation.

Test with metabolic activation (4-hour exposure) : no dose-related increase in micronuclei upto the top concentration of 75 µg/mL medium. No binucleate cells could be evaluated at the higher concentrations due to cytotoxicity and/or test item precipitation.

In the positive control culture the mean micronucleus frequency was increased to 23.5 micronucleate cells per 1000 binucleate cells for the 4-hour exposure.This demonstratedthat cyclophosphamideinduced significant chromosomal damage.

Conclusion

Under the present test conditions, Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.

The results for the vehicle controls were within the historical control range.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Justification for classification or non-classification

Based on these results and according to CLP (No. 1272/2008 of 16 December 2008), the registered substance does not have to be classified and has no obligatory labelling requirement for genotoxicity.