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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-C14-16 (even numbered)-alkyl esters, sodium salts (CAS 91648-54-3). 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-C12-16-alkyl esters, sodium salts (CAS 73296-89-6) 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

Referenceopen allclose all

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
Remarks:
Lack of details on the test substance.
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:
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
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 strains 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: 2.2, 6.6, 20, 60, 180 µg/plate
Vehicle / solvent:
water
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
Remarks:
medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine, sodium azide, 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 180 µg/plate and higher.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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 180 µg/plate and higher.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results:
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No data regarding genetic toxicity in vitro are available for the target substance. 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

Referenceopen allclose all

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
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: 72 h
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 C14-16 AS Na (CAS 91648-54-3). Therefore, these endpoints are 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 read-across approach show structural similarity. The most important common structural feature of the source and target substances 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 source and target substances in turn represent the predominant attribute in mediating effects on mammalian health. Therefore, the AS have similar physicochemical, environmental and toxicological properties. 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 programme carrying out Human and Environmental Risk Assessments (HERA [2]), further supporting the read across approach between structurally related AS.

Mutagenicity in bacteria

In a first study, the mutagenicity in bacteria was assessed in a study performed according to OECD Guideline 471. However, tester strains TA 102 or E.coli were missing (BASF, 1992). In this study with C12-16 AS Na (CAS 73296-89-6), Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 1538 and TA 100 were treated with and without addition of a rat liver S9-mix. The dose range was 8, 40, 200, 1000, 5000 µg/plate with and without S9-mix in the first experiment and 2.2, 6.6, 20, 60, 180 µg/plate with and without metabolic activation in the second experiment. Results achieved with vehicle (medium), untreated cells and positive controls were valid. Cytotoxicity was observed in presence and absence of metabolic activation at concentrations of 180 µg/plate and higher while 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 C12 AS 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, 1986). 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 read across substances 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.