Sulfuric acid, mono-C16-20-alkyl esters, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data are available for the target substance Sulfuric acid, mono-C16-20 (even numbered)-alkyl esters, sodium salts (CAS 91648-55-4). Therefore, read-across from structural analogue substances has been applied.

Bacterial reverse mutation assay (Ames test / OECD 471): negative

Read-across from source substances Sulfuric acid, mono-C16-18-alkyl esters, sodium salts (CAS 68955-20-4) and C12-18-alkyl esters, sodium salts (CAS 68955-19-1)

In vitro mammalian cell gene mutation assay (MLA / OECD 476): negative

Read-across from source substance sodium dodecyl sulfate (CAS 151-21-3)

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No E.coli WP2 or S. typhimurium TA102 strain tested.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1. test: 8, 40, 200, 1000, 5000 µg/plate
2. test: 11.1, 33.3, 100, 300, 600 µg/plate

Vehicle / solvent:

water

Untreated negative controls:

yes

Remarks:

untreated cells

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, 4-nitroquinoline-N-oxide, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Evaluation criteria:

According to Guideline.

Statistics:

no

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:

cytotoxicity

Remarks:

At 200 µg/plate and higher.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 200 µg/plate and higher.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1st test: 8, 40, 200, 1000 or 5000 µg/plate
2nd test (and repitition of 2nd test): 6.25, 25, 100, 400 or 1600 µg/plate

Vehicle / solvent:

bidist. water

Untreated negative controls:

yes

Remarks:

solvent medium bidist. water and untreated fresh cell suspensions

Positive controls:

yes

Positive control substance:

other: -S-9: sodium azide (TA 100 / TA 1535); 9-aminoacridine (TA 1537); 4-nitro-o-phenylendiamine (TA 98 / TA 1538); +S-9: 2 aminoanthracene (all strains)

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: >= 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

lack of details on test substance

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus (tk)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fisher’s medium supplemented with 2 mM L-glutamine, sodium pyruvate, 110 µg/mL 0.05% pluronic F68, antbiotics and 10% heat-inactivated donor horse serum (v/v)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Fischer 344 rats, intraperitoneally induced with Arochlor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Experiments 1-5: -S9: 3.125, 6.25, 10, 12.5, 20, 25, 30, 40, 50, 55, 60,65, 70, 80 and 100 µg/mL
Experiments 6-8: +S9: 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 µg/mL

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

-S9: methylmethanesulfonate (MMS), 15 µg/mL; +S9: 3-methylcholanthrene (3-MCA), 2.5 µg/mL

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h with and without S9 mix
- Expression time: 2 days
- Selection time: 11-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-16 days

SELECTION AGENT: 3 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: four cultures for vehicle control; two cultures for positive controls and each test substance concentration

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:

Four response categories for evaluation of results were defined (see below).

Response Categories for Experiments:
Positive response (+): The dose-related trend and the response at one of the three highest acceptable doses were statistically significant.
Negative response (-) Two categories were used. In both there was
a) no dose-related trend,
b) no statistically significant response at any dose,
c) an acceptable positive control response.
Nontoxic, negative response ( = )
There was an RTG among the acceptable doses of >30% (approximately), higher toxicities being unattainable due to intrinsic properties of either the compound or the system.
Toxic, negative response (-)
There was either an RTG of <30% (approximately) at the maximum acceptable dose, or the lethal concentration was no greater than 1.5 x a lower concentration at which the RTG was >30%.
Inconclusive (i)
There was
a) no dose-related trend and a statistically significant dose was any other than one of the highest three doses,
b) a response which would have been negative, but the lowest RTG acceptable doses was >35%,
c) a response which would have been negative, but there were no acceptable positive controls.
Questionable (?)
There was either
a) no dose-related trend, but a statistically significant response occurred at one of the highest three doses, or
b) a statistically significant dose-related trend, but none of the acceptable doses was statistically significant on its own.

Primary judgments were made at the level of individual experiments, but judgment on the mutagenic potential of a chemical was made on a basis of consensus of all valid experimental results (see "Any other information on materials and methods inlc. tables").

Statistics:

The statistical analysis was based upon the mathematical model proposed for this system and consisted of a dose-trend test and a variance analysis of pair-wise comparisons of each dose against the vehicle control. Significant differences from concurrent vehicle control values are indicated at the 5% level.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9: 70, 80 and 90 µg/mL; +S9: 95 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

RESULTS OF EXPERIMENTS 1-8

Eight acceptable experiments were conducted, five in the absence of S9 mix.

In the first of these, statistically significant increases in mutant fraction were observed at three dose levels: 6.25, 25, and 50 µg/mL; 100 µg/mL was a lethal concentration in cells (see Table 1). Over the nonlethal range, there were generally elevated mutant fractors, the highest being 1.9-fold the control level at 25 µg/mL. Although these increases in mutant fraction were significant, the lack of an obvious dose-related response with a relatively soluble chemical over a dose range which was not toxic encouraged speculation that the increases were not due to treatment with the test material.

 

Table 1. Experiment 1 - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency	Relative Total Growth	Mutants per 1E+06 surviving cells	Mutation factor	Average Mutation factor
DMSO (NC)	62	95	56	30	43
	68	98	90	44
	65	95	66	34
	80	112	153	64
3.125	77	106	84	36	61
	59	97	151	85
6.25	71	119	176	83	78*
	73	107	160	74
12.5	90	145	133	49	65
	67	133	160	80
25	84	90	247	99	83*
	65	88	130	67
50	86	82	192	75	69*
	76	98	145	64
100	lethal	lethal	n.a.	n.a.	n.a.
			n.a.	n.a.
MMS (15 µg/mL) PC	27	21	135	167	232*
	25	23	219	298

MMS = methylmethanesulfonate; NC = negative control; PC = positive control; *p < 0.05; n.a. = not applicable

 

In the second experiment without S9 mix, there was a clearly significant response at 60 µg/mL, but at no other concentration. The RTG was about 22%

Table 2. Experiment II - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency	Relative Total Growth	Mutants per 1E+06 surviving cells	Mutation factor	Average Mutation factor
DMSO (NC)	80	100	115	48	48
	74	108	119	53
	86	106	115	45
	61	87	83	45
10	75	112	107	47	58
	66	94	137	69
20	67	89	119	59	52
	53	76	70	44
30	71	70	98	46	60
	64	86	144	75
40	77	77	126	54	n.a.
50	94	61	191	68	74
	68	56	163	80
60	81	27	365	150	203*
	77	16	595	256
70	lethal	lethal	n.a.	n.a.	n.a.
			n.a.	n.a.
MMS (15 µg/mL) PC	34	30	683	666	664*
	29	27	573	662

MMS = methylmethanesulfonate; NC = negative control; PC = positive control; *p < 0.05; n.a. = not applicable

Experiment 3 gave a statistically significant response (1.7-fold increase) at 60 µg/mL, but not at the next higher concentration of 65 µg/mL. The mutant fraction at 70 µg/ml was only 44/106 survivors, so this single culture result supported the view that the statistically significant result at the lower dose level was a chance event. Thus, this experiment was judged to be questionable.

 

Table 3. Experiment 3 - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency	Relative Total Growth	Mutants per 1E+06 surviving cells	Mutation factor	Average Mutation factor
DMSO (NC)	76	103	84	37	34
	71	104	73	34
	82	97	98	40
	74	96	58	26
50	60	71	65	36	36
	75	75	82	36
55	68	44	68	33	38
	53	71	69	44
60	72	63	107	50	56*
	72	74	134	62
65	64	71	87	45	42
	79	68	93	39
70	80	83	107	44	n.a.
MMS (15 µg/mL) PC	36	26	127	119	158*
	26	23	156	197

MMS = methylmethanesulfonate; NC = negative control; PC = positive control; *p < 0.05; n.a. = not applicable

 

However, the succeeding experiments 4 and 5 without S9 mix were unambiguously negative; therefore the test substance was considered to be non-mutagenic in the absence of S9 mix.

Two experiments (6 and 7) were performed in the presence of S9 mix, showing unambiguously negative results. The last experiment with S9 mix was inconclusive because the cloning efficiency at 80 µg/mL was about 86% and there was no indication of a mutagenic response. However, based on the two experiments with S9 mix showing clearly negative results, the test substance was considered to be not mutagenic in the presence of S9 mix.

Conclusions:

Interpretation of results: negative

Executive summary:

No enhanced mutation rate in the S9 treated or untreated cells was observed in this mouse lymphoma assay. Therefore, the test substance was not considered to be mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No data are available for the target substance Sulfuric acid, mono-C16-20 (even numbered)-alkyl esters, sodium salts (CAS 91648-55-4). Therefore, read-across from structural analogue substances has been applied.

Mammalian Erythrocyte Micronucleus Test (MNT / OECD 474): negative

Read-across from source substance Sulfuric acid, mono-C16-18-alkyl esters, sodium salts (CAS 68955-20-4)

Mammalian Bone Marrow Chromosome Aberration Test (CA / OECD 475): negative

Read-across from source substance Sulfuric acid, mono-C12-15-alkyl esters, sodium salts (CAS 68890-70-0)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

: lack of details on test substance

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

According to guideline.

Route of administration:

oral: feed

Vehicle:

water

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Post exposure period:

none

Dose / conc.:

1.13 other: % nominal in diet

No. of animals per sex per dose:

6

Control animals:

yes, plain diet

Positive control(s):

Yes.

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

SAMPLING TIMES:
after 90 days

Evaluation criteria:

According to guideline.

Statistics:

No.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

: lack of details on test substance

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: CFW 1

Sex:

male/female

Details on test animals or test system and environmental conditions:

According to guideline.

Route of administration:

oral: gavage

Vehicle:

water

Duration of treatment / exposure:

24 h (400, 2000, 4000 mg/kg bw)
48, 72 h (4000 mg/kg bw)

Frequency of treatment:

Single

Post exposure period:

n.a.

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

4 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes.

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

SAMPLING TIMES:
24, 48 and 72 hours after application

Evaluation criteria:

According to guideline.

Statistics:

Yes.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The lack of mutagenic activity for the alkyl sulfates category is predictable based on structural and mechanistic considerations. Mutagens are chemicals that either 1) contain highly reactive electrophilic centers capable of interacting with nucleophilic sites on DNA (direct acting agents) or 2) can be metabolized to highly reactive electrophiles. The chemical structures represented by this chemical class do not contain electrophilic functional groups or functional groups capable of being metabolized to electrophiles. Alkyl sulfates with fully saturated carbon chains are not metabolized to reactive electrophiles. The consistent lack of mutagenic activity with alkyl sulfates is consistent with these mechanistic predictions.

There is no study regarding genotoxicity available for C16-20 AS Na (CAS 91648-55-4). Therefore, this endpoint is covered by read across to structurally related alkyl sulfates (AS). The possibility of a read-across to other alkyl sulfates in accordance with Regulation (EC) No. 1907/2006 Annex XI 1.5 “Grouping of substances and read-across approach” was assessed. In Annex XI 1.5 it is given that a read-across approach is possible for substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity. The AS reported within the AS category show structural similarity. The most important common structural feature of the category members is the presence of a predominantly linear aliphatic hydrocarbon chain with a polar sulfate group, neutralized with a counter ion. This structural feature confers the surfactant properties of the alkyl sulfates. The surfactant property of the members of the AS category in turn represent the predominant attribute in mediating effects on mammalian health. Therefore, the AS of the AS category have similar physicochemical, environmental and toxicological properties, validating the read across approach within the category. The approach of grouping different AS for the evaluation of their effects on human health and the environment was also made by the OECD in the SIDS initial assessment profile [1] and by a voluntary industry program carrying out Human and Environmental Risk Assessments (HERA [2]), further supporting the read across approach between structurally related AS.

 

Mutagenicity in bacteria

In the first study, performed according to OECD Guideline 471, Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 1538 and TA 100 were treated with C16-18 AS Na (CAS 68955-20-4) in presence and absence of metabolic activation. The tester strains TA 102 or E.coli were not used during the conduct of the study (BASF, 1992). In this study the dose range was 8, 40, 200, 1000 and 5000 µg/plate in the first experiment and 6.25, 25, 100, 400 and 1600 µg/plate in a second experiment (and the repetition of the second experiment). Results achieved with negative control (untreated and medium), vehicle (water) and positive controls were valid. Cytotoxicity was observed in presence and absence of metabolic activation at and above 200 µg/plate. No genotoxicity was observed.

 

In the second study, performed according to OECD Guideline 471, Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 1538 and TA 100 were treated with C12-18 AS Na (CAS 68955-19-1, analytical purity 39.4%) in presence and absence of metabolic activation according to the plate incorporation method. The tester strains TA 102 or E.coli were not used during the conduct of the study (BASF, 1991). In this study the dose range was 8, 40, 200, 1000, 5000 µg/plate in the first experiment and 11.1, 33.3, 100, 300, 600 µg/plate in a second experiment. Results achieved with negative control (untreated), vehicle (water) and positive controls were valid. Cytotoxicity was observed in presence and absence of metabolic activation at and above 200 µg/plate. No genotoxicity was observed.

 

Mutagenicity in mammalian cells

The mutagenicity of C12 AS Na (CAS 151-21-3) in a mammalian cell line was investigated similar to OECD guideline 476 using the mouse lymphoma L5178Y cells with and without metabolic activation (McGregor, 1988). The test concentrations were 3.125, 6.25, 10, 12.5, 20, 25, 30, 40, 50, 55, 60, 65, 70, 80 and 100 µg/mL without and 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 µg/mL with metabolic activation. Results achieved with the negative (untreated), vehicle (DMSO) and positive controls were valid. Cytotoxicity was observed in presence and absence of metabolic activation while no genotoxicity was observed under both circumstances for C12AS Na (CAS 151-21-3).

 

Genotoxicity in vivo

The potential of C12-15 AS Na (CAS 68890-70-0, analytical purity approx. 30%) to induce in vivo chromosomal aberration was assessed in a study conducted similar to OECD guideline 475 in rats (Unilever, 1976c). The test substance was administered via feed at a dose of 1.13% for a period of 90 days to 6 animals per sex and dose and bone marrow was sampled thereafter. Results achieved with the vehicle (DMSO) and positive controls were valid. No signs of toxicity were noted. As no enhanced chromosome aberrations were observed in this micronucleus test the test substance was considered to be not clastogenic.

 

The potential of C16-18 AS Na (CAS 68955-20-4, analytical purity 55%) to induce micronuclei in vivo was assessed in a study conducted according to OECD guideline 474 with CFW-1 mouse (BASF, 1986d). The test substance was administered via gavage at doses of 400, 2000 and 4000 mg/kg bw to 7 animals per sex and dose. Bone marrow was sampled 24 h (400 and 2000 mg/kg bw) and 24, 48 and 72 h (4000 mg/kg bw) after gavage. Results achieved with the vehicle (water) and positive controls were valid. No signs of toxicity were noted. As no enhanced chromosome aberrations were observed in this micronucleus test the test substance was considered to be not clastogenic.

 

In conclusion, the substance did not show any genotoxic potential. This is supported by the conclusions of the HERA Draft report “AS are not genotoxic, mutagenic or carcinogenic…” and the conclusions of the SIDS initial assessment profile “Alkyl sulfates of different chain length and with different counter ions were not mutagenic in standard bacterial and mammalian cell systems [...]. There was also no indication for a genotoxic potential of alkyl sulfates in various in vivo studies on mice […].”

 

[1] SIDS initial assessment profile, (2007); http://www.aciscience.org/docs/Alkyl_Sulfates_Final_SIAP.pdf

[2] (HERA Draft report, 2002); http://www.heraproject.com/files/3-HH-04-%20HERA%20AS%20HH%20web%20wd.pdf  

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.