Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro and in vivo genotoxicity studies were conducted with the test substance C10-13 LAS. Overall, results from these studies indicate no evidence of genotoxic potential of the tested substance.

 

Three in vitro genotoxicity studies are available, all providing evidence that LAS Na is negative for in vitro genotoxicity.

The Ames test gave a negative result (Schoeberl, 1993, K1 CSR). This key study was conducted in 1993 and met all test guideline requirements at the time, including use of the five standard test strains,S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538. Williams et al., 2019 reviewed the sensitivity and selectivity of the bacterial strains specified in OECD TG 471, including S. typhimurium TA102,E. coliWP2 uvrA and E. coli WP2 uvrA (pKM101). The authors conclude that “Of the mutagens detected by the full TG471 strain battery, 93% were detected using only strains TA98 and TA100; consideration of results from in vitro genotoxicity assays that detect clastogenicity increased the mutagens detected to 99%.” In this case, possible clastogenicity is examined in an in vitro and in vivo chromosome aberration study and therefore not considered necessary in the Ames test. 

Moreover, a reliable in vitro study (Murie and Innes, 1997 K3 CSR) is available in which the potential of LAS Na to cause chromosomal aberrations in mammalian cells was examined. These results indicate that LAS Na is weakly clastogenic in vitro at cytotoxic concentrations but negative at concentrations below cytotoxic concentrations.

Furthermore, the additional in vitro test (Anon., 1995 K2 CSR) that measures the potentialin vitrogenotoxicity of LAS Na to cause mutations in mammalian cells shows that LAS Na was not mutagenic to Chinese Hamster Ovary (CHO) cells both in the presence and absence of S9.

 

Williams, R.V., DeMarini, D.M., Stankowski Jr., L.F., Escobar, P.A., Zeigler, E., Howe, J., Elespuru, R. and Cross, K.P. 2019. Are all bacterial strains required by OECD mutagenicity test guideline TG471 needed? Mutat. Res. Gen. Tox. En. 848 (2019) 503081, available at: https://doi.org/10.1016/j.mrgentox.2019.503081

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

198

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Directive 84/449/EEC, B.14 Mutagenicity (Salmonella typhimurium - reverse mutation assay)" 1984; equivalent to OECD 471

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

Arochlor-induced S9 fraction

Test concentrations with justification for top dose:

8, 40, 200, 1000 and 5000 µg/plate

Vehicle / solvent:

Water solution at 50 g/L

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Positive controls:

yes

Remarks:

aminoanthracene

Positive control substance:

other: nitrofluorene, sodium azide and aminoacridine

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with and without activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

During the pre-incubation test, signs of toxicity were noted at concentrations as low as 125 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the product was observed at any concentration tested.

Conclusions:

LAS is not mutagenic in the Ames test.

Executive summary:

A bacterial mutagenicity study (Ames test) was conducted on LAS and was found to be negative for mutagenicity.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 16, 1995-June 30, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 from aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

0, 0.6, 1, 1.8, 3, 6 µg/mL without S9
0, 6, 10, 18, 30, 60 µg/mL with S9

Vehicle / solvent:

none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Remarks:

(H0 medium)

Positive controls:

yes

Positive control substance:

other: ethyl methane sulfonate; 3-(20-)methylcholanthrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 1 week
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 6 days at 37 degree C for cloning efficiency study, 9 days for mutation assay

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

A test substance was considered mutagenic if a statistically significant dose-related increase in mutant frequency was found in concentrations with greater than 20% survival rate. The mean mutant frequency must also be significantly above the maximum spontaneous mutant frequency

Statistics:

Statistical significance was determined by the t-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity preliminary test showed cytotoxicity at >= 50 µg/mL without S9, and >= 100 µg/mL with S9.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: In both the studies with and without S9, the mutant frequencies in the treated groups were statistically significantly higher than in the concurrent negative controls. However, the mutant frequencies in the treated groups were not significantly increased when compared to historical negative controls. There was also no dose-response relationship. The increased mutant frequency in treated groups was therefore not considered to be biologically significant.

Results of Test 1 - Without S9 Mix            

Concentration (µg/mL)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	82	3 ± 2
0.6	86	7 ± 1
1	85	3 ± 2
1.8	78	5 ± 2
3	86	1 ± 1
6	83	0 ± 1
EMS	83	277 ± 17

Results of Test 1 - With S9 Mix     

Concentration (µg/mL)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	90	2 ± 1
6	88	1 ± 1
10	84	9 ± 4
18	78	5 ± 3
30	89	3 ± 2
60	89	7 ± 2
MCA	81	91 ± 9

Results of Test 2 - Without S9 Mix

Concentration (µg/mL)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	96	1 ± 1
0.6	92	2 ± 3
1	95	1 ± 1
1.8	93	5 ± 2
3	90	2 ± 1
6	91	6 ± 6
EMS	90	309 ± 20

Results of Test 2 - With S9 Mix     

Concentration (µg/mL)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	90	2 ± 1
6	92	7 ± 3
10	88	9 ± 2
18	94	2 ± 1
30	93	2 ± 2
60	90	5 ± 1
MCA	95	89 ± 6

Conclusions:

The test substance is not mutagenic in either the presence or absence of metabolic activation.

Executive summary:

This study examined the potential of the test substance to cause mutations in mammalian cells. Chinese Hamster Ovary (CHO) cells were exposed to concentrations of 0, 0.6, 1, 1.8, 3, and 6 µg/mL without S9, and 0, 6, 10, 18, 30, and 60 µg/mL with S9. The cells were then examined for cytogenicity and mutation frequency. Ethyl methane sulfonate and 3-(20-)methylcholanthrene were used as positive control substances. Preliminary tests show the test substance was cytogenic at concentrations of 50 µg/mL or greater with metabolic activation, and 100 µg/mL or above without metabolic activation. There was no biologically significant increase in mutation frequency in the treated groups. The test substance is considered not mutagenic to CHO cells both in the presence and absence of S9.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 25, 1995-November 23, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study does not adequately address the results obtained at mildly cytotoxic concentrations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Test 1 with S9: 0.32, 0.63, 1.25, 2.5, 5, 10, 20, 39, 78 µg/mL
Test 1 without S9: 1.25, 2.5, 5, 10, 20, 39, 58,78, 156 µg/mL
Test 2 with S9: 2.5, 5, 10, 20, 26, 33, 39 µg/mL
Test 2 without S9: 20, 39, 58, 78, 130, 156 µg/mL
An additional test was done with S9 at the following dose levels:
2.5, 5, 7.5, 10, 15, 20, 25, and 30 µg/mL

Vehicle / solvent:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methyl methanesulphonate, cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 hrs with S9, 22 hrs without S9
- Expression time (cells in growth medium): 16-40 hrs with S9, 40 hrs without S9
- Selection time (if incubation with a selection agent): 2 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 24-48 hrs

SELECTION AGENT (mutation assays): Colcemid

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 100 metaphases

DETERMINATION OF CYTOTOXICITY
- Method: number of cells per culture

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A dosage was considered toxic if cell count was less then 60% of cell cultures. A test substance was considered clastogenic if a single dose caused the percentage of aberrant cells to be consistently greater than the 99% confidence limits of negative controls and there was also an increase at another dose level.

Statistics:

95% and 99% confidence limits

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Only at cytotoxic concentrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 15 µg/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=58 µg/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence of S9, only one culture (Test 2, 24 hr harvest, 20 µg/mL) showed a suspicious result. This single result was considered sporadic, as other cultures at this concentration, or at higher concentrations did not show a positive response. In Test 1, in the absence of S9, cytoxicity was seen at 78 µg/mL and above. In Test 2, in the absence of S9, cytoxicity was seen at concentrations of 58 µg/mL and above.

In Test 1, in the presence of S9, no positive results were seen at concentrations of up to 20 µg/mL. Metaphases could not be analyzed due to severe cytotoxicity at 39 and 78 µg/mL. In Test 2, in the presence of S9, one of the cultures at 5 µg/mL gave a suspicious result, and both cultures at 10µg/mL gave positive responses. Mild cytotoxity was also seen at 10 µg/mL. At concentrations at and above 20 µg/mL, metaphases could not be analyzed due to severe cytotoxicity. No positive results were seen in the Test 2, 48 hr harvest cultures grown in the presence of S9, though moderate cytotoxicity was seen in one of the 20 µg/mL cultures, and severe cytotoxicity was seen in all cultures above this concentration.

A third test was done in the presence of S9, which showed positive results at the 15 µg/mL concentration. However, this concentration was also moderately cytotoxic with only 26% of cells survival. However, due to the low survival of cells, these results are not definitive for determining clastogenicity. Higher concentrations were completely cytotoxic. An additional assessment was then performed at 10 µg/mL in the presence of S9, with negative results.

Concentration (µg/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham's F10 medium	0.01	1	0	Nil
Ham's F10 medium	0.02	1	1	Nil
0.32	-	-	-	Nil
0.32	-	-	-	Nil
0.63	-	-	-	Nil
0.63	-	-	-	Nil
1.25	-	-	-	Nil
1.25	-	-	-	Nil
2.5	0.01	1	0	Nil
2.5	0.00	0	0	Nil
5	0.00	0	0	Nil
5	0.05	5	0	Nil
10	0.01	1	0	Nil
10	0.01	1	0	Nil
20	0.00	0	0	Nil
20	0.00	0	0	Nil
39	-	-	-	TTT
39	-	-	-	TTT
78	-	-	-	TTT
78	-	-	-	TTT
Cyclophosphamide (20 µg/mL)	0.14	8	4	-
Cyclophosphamide (30 µg/mL)	0.06	4	4	-
Cyclophosphamide (40 µg/mL)	0.33	20	19	-

Test 1 - Without S9 Mix, 24 hr Harvest

Concentration (µg/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham's F10 medium	0.00	0	0	Nil
Ham's F10 medium	0.00	0	0	Nil
1.25	-	-	-	Nil
1.25	-	-	-	Nil
2.5	-	-	-	Nil
2.5	-	-	-	Nil
5	-	-	-	Nil
5	-	-	-	Nil
10	-	-	-	Nil
10	-	-	-	Nil
20	-	-	-	Nil
20	-	-	-	Nil
39	0.01	1	0	Nil
39	0.00	0	0	Nil
58	0.01	1	0	Nil
58	0.00	0	0	Nil
78	0.00	0	0	T
78	0.00	0	0	T
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (10 µg/mL)	0.03	3	1	-
Cyclophosphamide (20 µg/mL)	0.16	14	10	-

T- Toxicity evident from morphological changes

TT- Toxicity evident from reduced cell count (<60% of vehicle)

TTT- Too toxic for metaphase assessment

Test 2 - With S9 Mix, 24 hr Harvest

Concentration (µg/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham's F-10 medium	0.01	1	0	Nil
Ham's F-10 medium	0.02	2	1	Nil
2.5	0.07	2	1	Nil
2.5	0.04	3	1	Nil
5	0.04	3	2	Nil
5	0.06	6	4	Nil
10	0.12	8	6	T
10	0.19	13	5	T
20	-	-	-	TTT
20	-	-	-	TTT
26	-	-	-	TTT
26	-	-	-	TTT
33	-	-	-	TTT
33	-	-	-	TTT
39	-	-	-	TTT
39	-	-	-	TTT
Cyclophosphamide (40 µg/mL)	0.38	20	17	-
Cyclophosphamide (50 µg/mL)	0.31	18	11	-

Test 2 - With S9 Mix, 48 hr Harvest

Concentration (µg/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham's F-10 medium	0.00	0	0	Nil
Ham's F-10 medium	0.00	0	0	Nil
2.5	0.01	1	0	Nil
2.5	0.01	1	1	Nil
5	0.00	0	0	Nil
5	0.02	2	2	Nil
10	0.03	2	1	Nil
10	0.02	2	1	TT
20	-	-	-	TTT
20	-	-	-	TTT
26	-	-	-	TTT
26	-	-	-	TTT
33	-	-	-	TTT
33	-	-	-	TTT
39	-	-	-	TTT
39	-	-	-	TTT
Cyclophosphamide (40 µg/mL)	0.03	3	2	-
Cyclophosphamide (50 µg/mL)	0.10	8	7	-

Test 2 - Without S9 Mix, 24 hr Harvest

Concentration (µg/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham's F-10 medium	0.02	2	2	Nil
Ham's F-10 medium	0.03	3	0	Nil
20	0.02	2	0	Nil
20	0.05	5	3	Nil
39	0.02	2	1	Nil
39	0.04	4	0	Nil
58	0.01	1	1	Nil
58	0.06	6	1	Nil
78	-	-	-	TTT
78	-	-	-	TTT
104	-	-	-	TTT
104	-	-	-	TTT
130	-	-	-	TTT
130	-	-	-	TTT
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (10 µg/mL)	0.30	21	14	-
Methyl methane-sulphonate (20 µg/mL)	0.71	33	28	-

Test 2 - Without S9 Mix, 48 hr Harvest

Concentration (µg/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham's F-10 medium	0.01	1	1	Nil
Ham's F-10 medium	0.00	0	0	Nil
20	0.00	0	0	Nil
20	0.00	0	0	Nil
39	0.01	1	1	Nil
39	0.00	0	0	Nil
58	0.00	0	0	T
58	0.01	1	0	T
78	-	-	-	TTT
78	-	-	-	TTT
104	-	-	-	TTT
104	-	-	-	TTT
130	-	-	-	TTT
130	-	-	-	TTT
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (20 µg/mL)	0.21	11	8	-
Methyl methane- sulphonate (40 µg/mL)	3.20	60	60	-

Test 3 - With S9 Mix, 24 hr Harvest

Concentration (µg/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytoxicity
Ham's F-10 medium	0.04	4	0	Nil
Ham's F-10 medium	0.04	4	0	Nil
2.5	-	-	-	Nil
2.5	-	-	-	Nil
5	-	-	-	Nil
5	-	-	-	Nil
7.5	-	-	-	Nil
7.5	-	-	-	Nil
10	-	-	-	Nil
10	-	-	-	Nil
15	0.20	12	8	TT
15	0.18	12	6	TT
20	-	-	-	TTT
20	-	-	-	TTT
25	-	-	-	TTT
25	-	-	-	TTT
30	-	-	-	TTT
30	-	-	-	TTT
Cyclophosphamide (30 µg/mL)	0.24	14	12	-
Cyclophosphamide (40 µg/mL)	0.32	17	11	-

Test 3: With S9 Mix, 24 Hr Harvest

Concentration (µg/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytoxicity
Ham's F-10 medium	0.04	4	0	Nil
Ham's F-10 medium	0.04	4	0	Nil
2.5	-	-	-	Nil
2.5	-	-	-	Nil
5	-	-	-	Nil
5	-	-	-	Nil
7.5	-	-	-	Nil
7.5	-	-	-	Nil
15	0.20	12	8	TT
15	0.18	12	6	TT
20	-	-	-	TTT
20	-	-	-	TTT
25	-	-	-	TTT
25	-	-	-	TTT
30	-	-	-	TTT
30	-	-	-	TTT
Cyclophosphamide (30 µg/mL)	0.24	14	12	-
Cyclophosphamide (40 µg/mL)	0.32	17	11	-

Test 3: With S9 Mix, 24 Hr Harvest: Additional assessment

Concentration (µg/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytoxicity
Ham's F-10 medium	0.02	2	0	Nil
10	0.01	1	0	Nil
10	0.02	2	0	Nil
Cyclophosphamide (30 µg/mL)	0.22	14	10	-

Conclusions:

The test substance is not clastogenic in the absence of metabolic activation. The test substance is also not clastogenic in the presence of metabolic activation at non-cytotoxic concentrations. At cytotoxic concentrations, the test substance is weakly clastogenic.

Executive summary:

This study examined the potential of the test substance to cause chromosomal aberrations in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 0.32 to 78 µg/mL with S9, and 1.25 to 156 µg/mL without S9. Methyl methanesuflphonate and cyclophosphamide were used as positive controls. No biologically significant results were seen in treated cultures in the absence of metabolic activation. Positive responses were seen at cytotoxic concentrations in the presence of S9. Concentrations below the level of cytotoxicty with S9 did not show positive results. The test substance is not clastogenic in the absence of metabolic activation, or with metabolic activation below cytotoxic concentrations. These results indicate that the test substance is weakly clastogenic at cytotoxic concentrations but negative at concentrations below cytotoxic concentrations

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Three key studies to fulfill the information requirement of in vivo genotoxicity are available: a chromosomal aberration assay, in vivo cytogenicity study and dominant lethal test (Masabuchi, 1976 a, b, c; K1, 2, 3) were conducted according to generally accepted procedures at the time the studies were conducted. All studies provide evidence that LAS Na is negative forin vivogenetic toxicity, including chromosomal aberrations.

Furthermore, two supporting studies performed with substances analogous to the test substance are included in the dossier: 1) LAS Acid, the source substance for study (i), in vivo micronucleus assay (Fedtke, 1991; S2 CSR), is identical to the sponsored substance LAS Na as both disassociate in vivo to form the identical substance, the LAS anion, and 2) C10-14 LAS Na, the source substance in study (ii),in vivochromosomal aberration assay (Inoue et al., 1976; S1 CSR) which has very similar composition (slight differences in C10 and C14 content) to the sponsored substance LAS Na. Both of these tests gave negative results, confirming the absence of genotoxic properties in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A group of 7 male mice was fed a diet containing 0.6% test substance for 9 months. At the end of this period, the animals were each mated with two untreated females. On day 13 of pregnancy, the females were sacrificed, and the ovaries and uteri were examined.

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

other: JCL-ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan, Inc.
- Age at study initiation: 4 weeks
- Housing: individually, except during breeding
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 1
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with: feed powder CE-2

Duration of treatment / exposure:

9 months

Frequency of treatment:

daily

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

0.6% in diet

No. of animals per sex per dose:

7

Control animals:

yes

Statistics:

Rohrborn's method.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Negative controls validity:

valid

Additional information on results:

There were no significant differences in fertility, the mortality of ova and embryos, the number of surviving fetuses, or the index of dominant lethal induction between the experimental groups and the control group.

Dominant Lethal Assay Results

	0.6% in Diet	Control
Number of mating females	14	18
Number pregnant	11	12
No. with dead embryos	6	10
Dead embryos per pregnant female	54.6%	83.3%
No. of corpora lutea	156	161
Corpora lutea per pregnant female	14.2	13.4
No. of implants	148	156
Implants per pregnant female	13.5	13.0
Implants per corpora lutea	94.9	96.9
No. of live fetuses	142	143
Live fetuses per pregnant female	12.9	11.9
Live fetuses per corpora lutea	91.0	88.8
Live fetuses per total implants	96.0	91.7
No. of early dead fetuses	4	12
No. of late dead fetuses	2	1
% of dominant lethals	-4.67	-
% of dominant lethals	-8.33	-

Conclusions:

The test substance did not cause genetic disorders in mice.

Executive summary:

A group of 7 male mice was fed a diet containing 0.6% test substance for 9 months. At the end of this period, the animals were each mated with two untreated females. On day 13 of pregnancy, the females were sacrificed, and the ovaries and uteri were examined. No increase in dominant lethal induction was seen as compared to controls. The test substance does not cause genetic disorders.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 5 male rats were fed a diet containing 0.9% test substance for 9 months. At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations.

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

rat

Strain:

other: Wistar and SD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan, Inc.
- Age at study initiation: 4 weeks
- Housing: individually
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 1
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with: feed powder CE-2

Duration of treatment / exposure:

9 months

Frequency of treatment:

Daily

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

0.9% in diet

No. of animals per sex per dose:

10

Control animals:

yes

Tissues and cell types examined:

femur bone marrow cells

Details of tissue and slide preparation:

Animals were sacrificed by administration of 1 ml/kg of 1% colchine solution. Femurs were then removed, and bone marrow cells washed into centrifuge tubes. The cells were then treated with 0.075 M KCl solution at 37 °C for 15 min, and then fixed with an acetic acid 1: ethanol 3 solution. Samples were then flame dried and treated with Giemsa.

Evaluation criteria:

Presence and absence of chromosomal aberrations. 50 metaphases per individual.

Statistics:

Rohrborn's method.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Negative controls validity:

valid

Additional information on results:

No increase in chromosome aberrations was noted.

Chromosome Aberrations

	0.9% in Diet – Wister Rats	0.9% in Diet – SD Rats	Control	Control
No. of cells with chromatid breaks	0	0	1	0
No. of cells with isochromatid breaks	0	0	0	0
No. of cells with chromatid gaps	3	4	3	4
No. of cells with isochromatid gaps	0	0	0	0
No. of cells with other aberrations	0	0	0	0

Conclusions:

The test substance is not clastogenic.

Executive summary:

Groups of 5 male rats were fed a diet containing 0.9% test substance for 9 months. At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations. No increase in chromosome aberrations was seen as compared to controls. The test substance is not clastogenic.

Reference 3

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A group of 5 male mice was fed a diet containing 0.9% test substance for 9 months. At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations.

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

other: JCL-ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan, Inc.
- Age at study initiation: 4 weeks
- Housing: individually, except during breeding
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 1
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with: feed powder CE-2

Duration of treatment / exposure:

9 months

Frequency of treatment:

Daily

Dose / conc.:

1 170 mg/kg bw/day (nominal)

Remarks:

0.9% in diet

No. of animals per sex per dose:

5

Control animals:

yes

Tissues and cell types examined:

femur bone marrow cells

Details of tissue and slide preparation:

Animals were sacrificed by administration of 1 ml/kg of 1% colchine solution. Femurs were then removed, and bone marrow cells washed into centrifuge tubes. The cells were then treated with 0.075 M KCl solution at 37 °C for 15 min, and then fixed with an acetic acid 1: ethanol 3 solution. Samples were then flame dried and treated with Giemsa.

Evaluation criteria:

Presence and absence of chromosomal aberrations. 50 metaphases per individual.

Statistics:

Rohrborn's method.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Negative controls validity:

valid

Additional information on results:

No increase in chromosome aberrations was noted.

Chromosome Aberrations

	0.9% in Diet	Control
No. of cells with chromatid breaks	1	2
No. of cells with isochromatid breaks	1	0
No. of cells with chromatid gaps	4	5
No. of cells with isochromatid gaps	0	0
No. of cells with other aberrations	0	0

Conclusions:

The test substance is not clastogenic.

Executive summary:

A group of 5 male mice was fed a diet containing 0.9% test substance for 9 months. At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations. No increase in chromosome aberrations was seen as compared to controls. The test substance is not clastogenic.

Reference 4

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of male mice were given doses of 200, 400, or 800 mg/kg of Benzenesulfonic acid, C10-14-alkyl derivs., sodium salts. At 6, 24, and 48 hrs, 3 of the mice from each dosage group were sacrificed. The bone marrow cells from the femurs were collected and examined for chromosome aberrations. In addition, one group of mice was exposed daily for 5 consecutive days. Additional groups of mice were exposed to commericial detergents containing 19% or 17.1% of the test substance. Mitomycin C was used as a positive control.

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

other: ICR/JCL

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan Inc.
- Age at study initiation: 9-11 weeks
- Housing: individually in plastic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12 hrs

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Samples were diluted to make a 5 mL/kg dose volume

Duration of treatment / exposure:

Single treatment, except for one group which was given 5 consecutive daily exposures to the test substance

Frequency of treatment:

Once daily

Dose / conc.:

200 mg/kg bw (total dose)

Remarks:

Nominal concentration

Dose / conc.:

400 mg/kg bw (total dose)

Remarks:

Nominal concentration

Dose / conc.:

800 mg/kg bw (total dose)

Remarks:

Nominal concentration

Dose / conc.:

800 mg/kg bw (total dose)

Remarks:

Nominal concentration. Commercial detergent containing 19% test substance

Dose / conc.:

1 600 mg/kg bw (total dose)

Remarks:

Nominal concentration. Commercial detergent containing 19% test substance

Dose / conc.:

3 200 mg/kg bw (total dose)

Remarks:

Nominal concentration. Commercial detergent containing 19% test substance

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Nominal concentration. Commercial detergent containing 17.1% test substance

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

Nominal concentration. Commercial detergent containing 17.1% test substance

Dose / conc.:

4 000 mg/kg bw (total dose)

Remarks:

Nominal concentration. Commercial detergent containing 17.1% test substance

No. of animals per sex per dose:

9

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: mitomycin C1
- Route of administration: intraperitoneally
- Doses: 5 mg/kg

Tissues and cell types examined:

bone marrow cells from femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 3 animals were sacrificed at 6, 24, and 48 hrs after treatment. One group was exposed daily for 5 days prior to sacrifice.

DETAILS OF SLIDE PREPARATION: Cells were placed in Hank's solution, then centrifuged at 1000 rpm for 5 min. Supernatant was discarded, and 5 ml of 0.075 M KCl was added. The mixture then stood for 5 min., then was stirred and centrifuged again. This was repeated several times, before finally placing one or two drops on the slides, and staining with 2% Giemsa solution.

Evaluation criteria:

Number of cells with chromatid and chromosome gaps, number of cells with aberrations.
50 metaphases per animal (150 total) were imaged at each time point.

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No significant differences in the incidence of chromosomal aberrations were observed in any test substance treatment group relative to the controls.

Chromosome Aberrations

	Total number of cells having aberrations and occurrence (%)
	6 hrs	24 hrs	48 hrs	5 days
200 mg/kg	0 (0)	0 (0)	0 (0)	1 (0.7)
400 mg/kg	1 (0.7)	0 (0)	0 (0)	0 (0)
800 mg/kg	0 (0)	0 (0)	0 (0)	0 (0)
800 mg/kg of 17.1% detergent	0 (0)	0 (0)	0 (0)	-
1600 mg/kg of 17.1% detergent	0 (0)	1 (0.7)	0 (0)	-
3200 mg/kg of 17.1% detergent	2 (1.3)	2 (1.3)	0 (0)	-
1000 mg/kg of 19% detergent	-	0 (0)	-	-
2000 mg/kg of 19% detergent	-	0 (0)	-	-
4000 mg/kg of 19% detergent	-	0 (0)	-	-
Mitomycin C	16 (10.7)	53 (353)	13 (8.7)	112 (74.7)
Distilled water	0 (0)	0 (0)	0 (0)	0 (0)
untreated	0 (0)	0 (0)	1 (0.7)	0 (0)

Conclusions:

The test substance in not clastogenic.

Executive summary:

Groups of male mice were given doses of 200, 400, or 800 mg/kg of Benzenesulfonic acid, C10-14-alkyl derivs., sodium salts. At 6, 24, and 48 hrs, 3 of the mice from each dosage group were sacrificed. The bone marrow cells from the femurs were collected and examined for chromosome aberrations. In addition, one group of mice was exposed daily for 5 consecutive days. Additional groups of mice were exposed to commerical detergents containing 19% or 17.1% of the test substance. Mitomycin C was used as a positive control. None of the treatment groups showed any significant increase in chromosome aberrations as compared to negative controls. The test substance in not clastogenic.

Reference 5

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain NMRI. Animals were approximately 22-26 g (male) and 20-25 g (female) and acclimated for 1 week to the test conditions (20±3 °C, 30-70% relative humidity, 12 hour light/dark cycle). Food was given daily and water was ad libitum. All animals were healthy at the time of test initiation.

Route of administration:

oral: gavage

Vehicle:

NaCl

Duration of treatment / exposure:

72 hours

Frequency of treatment:

Single dose

Dose / conc.:

1 122 mg/kg bw (total dose)

No. of animals per sex per dose:

40 males and 40 females per dose

Control animals:

yes

Positive control(s):

Endoxan (cyclophosphamid)

Tissues and cell types examined:

Cells were taken from the thigh.

Details of tissue and slide preparation:

Cells were mixed with cattle serum and suspended, then centrifuged. The sediment was then resuspended. The suspension was seperated in a cellulose chromatography column. This was centrifuged, and mixed with fetal calf serum and EDTA. This was air-dried for 24 hrs and stained with Giemsa.

Evaluation criteria:

number of polychromatid erythrocytes (PCE)
ratio of PCE to normochromatid erythrocytes (NCE)
number of cells with micronucleus

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

No significant increases in the number of polychromatic erythrocytes with micronuclei were observed.

Conclusions:

No significant increases in the number of polychromatic erythrocytes with micronuclei were observed.

Executive summary:

No significant increases in the number of polychromatic erythrocytes with micronuclei were observed.4

Reference 6

Endpoint:

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

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The in vivo genetic toxicity study of C10-13 LAS is based on read-across with C10-14 LAS. The read-across justification is presented in the assessment reports section. A cross-reference is made to that record.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Remarks:

read-across justification

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity, in vitro

Study 1 - AMES - (C10-13 LAS): 

 A bacterial reverse mutation assay was conducted with C10-13 LAS, sodium salt to assess the potential mutagenicity of LAS. The experiment was run using S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, as well as TA1538 at test concentrations of 8, 40, 200, 1000 and 5000 µg/plate. All strains tested negative with and without S9 activation (Schoeberl, 1993).

 

Study 2 - OECD 476 - (C10-13 LAS): 

An in vitro genetic toxicity study was conducted to evaluate the potential of C10 -13 LAS to cause mutations in mammalian cells. Chinese Hamster Ovary (CHO) cells were exposed to concentrations of 0, 0.6, 1, 1.8, 3, and 6 µg/mL without metabolic activation (S9), and 0, 6, 10, 18, 30, and 60 µg/mL with S9. The cells were then examined for cytogenicity and mutation frequency. Ethyl methane sulfonate and 3-(20-)methylcholanthrene were used as positive control substances. Preliminary tests show the test substance was cytogenic at concentrations of 50 ug/ml or greater with metabolic activation, and 100 ug/ml or above without metabolic activation. There was no biologically significant increase in mutation frequency in the treated groups. The test substance is considered not mutagenic to CHO cells both in the presence and absence of S9 (Anon., 1995). 

 

Study 3 - OECD 473 - (C10 -13 LAS):

An in vitro genetic toxicity study was conducted to evaluate the potential of the test substance to cause chromosomal aberrations in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 0.32 to 78 µg/mL with S9, and 1.25 to 156 µg/mL without S9. Methyl methanesuflphonate and cyclophosphamide were used as positive controls. No biologically significant results were seen in treated cultures in the absence of metabolic activation. Positive responses were seen at cytotoxic concentrations in the presence of S9. Concentrations below the level of cytotoxicty with S9 did not show positive results. The test substance is not clastogenic in the absence of metabolic activation, or with metabolic activation below cytotoxic concentrations. These results indicate that the test substance is weakly clastogenic at cytotoxic concentrations but negative at concentrations below cytotoxic concentrations (Murie & Innes, 1997).

Genetic toxicity, in vivo      

Study 1 (C10-13 LAS):  

An in vivo dominant lethal mutation assay was conducted with C10-13 LAS, sodium salt in male mice. A group of 7 male mice was fed a diet containing 0.6% test substance for 9 months. At the end of this period, the animals were each mated with two untreated females. On day 13 of pregnancy, the females were sacrificed, and the ovaries and uteri were examined. No increase in dominant lethal induction was seen as compared to controls. Under the study conditions, LAS was not mutagenic in mice (Masabuchi, 1976a).     

     

Study 2 (C10-13 LAS): 

An in vivo chromosomal aberration assay was conducted with C10-13 LAS, sodium salt in rats. In this study, groups of five male rats were fed diets containing 0.9% LAS for 9 months (equivalent to 405 mg/kg bw/day). At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations. No increase in chromosome aberrations was seen as compared to negative controls in either species but no positive controls have been included in this assay. Under the study conditions, LAS was not clastogenic in this assay (Masabuchi, 1976b).

     

Study 3 (C10 -13 LAS):      

An in vivo chromosomal aberration assay was conducted with C10-13 LAS, sodium salt in male mice. In this study, groups of five male mice were fed diets containing 0.9% LAS for 9 months (equivalent to 1,125 mg/kg bw/day). At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations. No increase in chromosome aberrations was seen as compared to negative controls in either species but no positive controls have been included in this assay. Under the study conditions, LAS was not clastogenic in this assay (Masabuchi, 1976c).

     

Study 4 (C10-14 LAS): 

An in vivo chromosomal aberration assay was conducted with C10-14 LAS, sodium salt in male mice. Groups of male mice were given doses of 200, 400, or 800 mg/kg bw of the test substance. At 6, 24, and 48 h, 3 of the mice from each dosage group were sacrificed. The bone marrow cells from the femurs were collected and examined for chromosome aberrations. In addition, one group of mice was exposed daily for 5 consecutive days. Mitomycin C was used as a positive control. None of the treatment groups showed any significant increase in chromosome aberrations as compared to negative controls. Under the study conditions, LAS was not clastogenic (Inoue, 1979).

 

Study 5 (10-13 LAS):

An in vivo micronucleus was conducted with C10-13 LAS acid in male and female mice (NMRI). Groups of 40 mice per sex were given a single dose of 1122 mg/kg bw test substance via oral gavage and evaluated for chromosome aberrations. Only a single dose has been evaluated which was in the range of the acute oral LD50 value for LAS Acid in rats (LD50 = 1470 mg/kg). Furthermore, slight cytotoxicity has been observed after 48 hours. No statistically significant or biologically relevant increases in the number of polychromatic erythrocytes with micronuclei were observed; therefore the test material is considered negative for cytogenicity (Fedtke, 1991)

 

Justification for classification or non-classification

Based on in vitro and in vivo genotoxicity studies conducted with LAS Na and its dissociation products no classification is warranted for this endpoint according to EU CLP (1272/2008/EC) criteria.