Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs.-, compd. with 1-aminopropane-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A series of in vitro studies were conducted to assess the potential mutagenicity of the read across substance Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts. In the first study (Schoeberl 1993), a bacterial mutagenicity study (Ames test) was conducted 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.

In the second test (Anon. 1995), the potential of Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts to cause mutations in mammalian cells was examined. 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 cytotoxic 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. Therefore, results show that the substance was not mutagenic to CHO cells both in the presence and absence of S9.

The third study (Murie and Innes 1997) examined the potential of Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts to cause chromosomal aberrations in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 2.5, 5, 10, 15, 20, 26, 33, and 39 µg/ml with S9, and 20, 39, 58, 78, 104, 130, and 156 µg/ml without S9. No biologically significant results were seen in treated cultures in the absence of metabolic activation. In the presence of metabolic activation the results were more equivocal. In the first of three tests, no cytotoxicity, and no increase in chromosome aberrations were observed at doses of 10 or 20 μg/ml and 100% cytotoxicity was observed at 39 μg/ml. In the second test, a steep cytotoxicity curve was observed between 10 and 20 μg/ml with a cell count of 68/90 % at the 10 μg/ml dose and no living cells remaining at the 20 μg/ml dose. An increased in aberration frequency could be observed at the 10 μg/ml dose. No increase in aberration frequency has been observed at lower doses which also did not show any cytotoxicity. To gain clarity on the positive result an additional test was conducted. Here, no cytotoxicity and no increase in chromosomal aberration frequency have been observed at the 10 μg/ml dose. At the 15 μg/ml dose the cell number was reduced to 25 % which is why this dose group cannot be evaluated due to excessive cytotoxicity. These results indicate that the substance is weakly clastogenic at cytotoxic concentrations but negative at concentrations below cytotoxic concentrations in this in vitro assay.

For the second read across substance MIPA, the substance was tested in the Ames reverse mutation assay using S. typhimurium strains TA98, TA100, TA1535 and TA1537 up to cytotoxic concentrations, with and without metabolic activation. The substance was not mutagenic in strains TA98, TA100 and TA1537. However, with S9 -activation the TA 1535 strain presented some weak positive results. This result was considered ambiguous (Zeiger et al., 1987). In contrast, in another Ames test MIPA was found not mutagenic in S. typhimurium strains TA98, TA100, TA 1535, TA1537 and TA 1538, and E. coli WP2 uvr A, incubated with 1 to 5000 µg MIPA/plate in the presence and absence of metabolic activation. Cytotoxicity was observed at the highest dose only (Shimizu et al., 1985).The substance purity in the 1st Ames test with ambiguous result was 95% whereas substance purity in the second Ames-test with negative result was 97%. Therefore impurities are likely to be responsible for the ambiguous result of the test-substance with the lower purity. Taken together, MIPA was not considered to be mutagenic.

Induction of gene mutations in mammalian cells by MIPA was investigated in an HGPRT assay using Chinese hamster ovary (CHO) cells at 156 to 2500 µg/mL, with and without metabolic activation. The results indicate that MIPA does not induce gene mutations in this assay. No cytotoxicity was observed (Dow, 1995).

MIPA tested at 83 - 1500 µg/mL (-S9) and 250 - 2500 µg/mL (+S9) did not induce significant increases in chromosomal aberrations using rat lymphocytes with and without metabolic activation. Cytotoxicity was observed without metabolic activation only, at 833 µg/mL. MIPA was judged as non-clastogenic (Dow, 1995).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report which meets basic scientific principles.

Principles of method if other than guideline:

Method: other: according to Haworth et al., Environ. Mutagen., 5 (Suppl. 1), 3-142

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Strain Target Gene:
TA1537 hisC3076
TA1535 hisG46
TA100 hisG46
TA98 hisD3052

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

The S9 fractions were prepared from the liver of Aroclor 1254-induced male Sprague-Dawley rats and male Syrian hamsters.

Test concentrations with justification for top dose:

33 - 4000 ug/plate

Vehicle / solvent:

water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: see remarks

Remarks:

For strains tested in the absence of S9: TA98, 2-nitrofluorene, TA100 and TA1535, sodium azide, TA1537, 9-aminoacridine. For strains tested with S9: All strains, 2-aminoanthracene.

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

Evaluation criteria:

According to Haworth et al. (1983).

Species / strain:

S. typhimurium, other: TA98, TA100, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slightly toxic at highest dose in the 3 strains.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slightly toxic at highest dose

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slightly toxic at highest dose

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Strain TA100

Dose	No Activation (Negative)		10% HLI (Negative)		10% RLI (Negative)
Dose units	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	135	2	141	6.8	139	5.6
33	152	4	144	8.7	132	14.4
100	141	3.8	140	12.2	138	5.8
333	148	4.2	140	5.5	149	11.1
1000	155	0.9	128	6.4	124	4.3
3333	130S	7.8	159S	7	156S	6.5
Positive Control	1298	63.2	1452	83.5	947	33.3

Strain TA98

Dose	No Activation (Negative)		10% HLI (Negative)		10% RLI (Negative)
Dose units	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	18	2	30	0.6	24	1.2
33	19	2.6	36	1.5	25	4.4
100	25	2.9	43	1.9	30	6.3
333	17	2.3	25	2.1	33	0.7
1000	21	3.3	31	4.5	26	4.1
3333	8T	3.5	20S	4.3	20S	1.8
Positive Control	1475	103.9	935	33.9	637	42.8

Strain TA1537

Dose	No Activation (Negative)		10% HLI (Negative)		10% RLI (Negative)
Dose units	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	8	1.7	10	1.7	8	0.9
33	7	2.9	12	1.5	11	2.9
100	7	0.3	13	1.7	9	0.9
333	6	1.8	7	1.2	7	1
1000	6	0.6	7	1.3	8	0.3
3333	4S	1.2	4S	0.3	5S	0.3
Positive Control	87	12.3	124	6.1	97	2.9

Strain TA1535:

Dose	No Activation (Negative)		No Activation (Negative)		No Activation (Negative)		No Activation (Negative)		10% HLI (Equivocal)		10% HLI (Weak Positive)		10% HLI (Negative)		10% HLI (Positive)		10% RLI (Negative)		10% RLI (Positive)		10% RLI (Negative)		10% RLI (Positive)
Dose units	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	27	5	54	6.4	7	1.2	36	3.6	9	0.9	11	3.2	9	2.5	5	1	9	1.5	9	0.7	7	1.5	11	0
33	30	1.5							11	2.2							7	2.6
100	30	4.4	51	4.2	7	0.9	34	0.9	11	1	9	1	8	1.2	10	2.3	11	1.2	10	0.6	7	1.9	11	1.3
333	27	4.4	45	2.2	6	1.5	32	5	14	0.9	14	1.2	6	0.7	11	2	9	0.9	14	2.8	6	0.7	7	0.3
1000	20	1.5	47	3.8	4	0.7	23	4.8	10	1.2	9	0.3	7	1.3	11	4.2	7	1.8	14	0.7	5	1.5	9	0.7
1800			34	3.8	5	1.3	28	1.3			24	1.9	10	1.3	29	0.7			15	1.9	5	1.7	13	3.2
2800			25S	0.9	4S	1	23S	2			30S	2.9	5	0.6	28	6.6			33S	4.3	8	2	32	2.1
3333	12S	3.8	25S	4.5	4S	0.9	16S	3.2	27S	1.7	20S	1.8	8S	2.1	24S	2.7	17S	2.5	32S	2.6	6S	2.2	32S	2.2
3500			16S	0.6	3S	0.6	21S	0.3			18S	2.7	6S	1.5	25S	4.7			20S	1.5	6S	1	26S	2.2
4000			13S	1.7	2S	0.6	17S	2.1			21S	0.9	3S	0.3	15S	2.4			26S	5.1	4S	1.7	23S	3.3
Positive Control	1005	61	1484	111	869	23.8	1124	41.3	124	3.5	99	2.3	85	2.3	231	16.1	98	6.4	95	11.4	83	4.7	103	13.6

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination

Conclusions:

Interpretation of results: ambiguous with metabolic activation

Under the conditions tested the test substance was not mutagenic in the strains TA98, TA100 and TA1537.
However, with S9-activation the TA1535 strain presented some weak positive results. Therefore the test substance was rated as ambiguous.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication which meets basic scientific principles.

Principles of method if other than guideline:

Method: other: Ames et al., Mut Res 31, 347-364, 1975

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 98, TA100, TA1535, TA1537, TA1538

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from the liver of male Sprague-Dawley rats, pretreated with polychlorinated biphenyl (KC 500) at a dose of 500 mg/kg body weight five days before sacrifice.

Test concentrations with justification for top dose:

1 - 5000 ug/plate

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other:

Remarks:

positive controls used: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitroquinoline- 1-oxide, benzo(a)pyren, aminoanthracene, 2-nitrofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Dose (µg/plate	TA100		TA1535		WP2 uvrA		TA98		TA1537		TA1535
	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
1	177	183	38	12	34	30	21	43	7	10	18	28
5	160	187	41	14	35	34	18	36	8	14	18	24
10	164	181	38	14	41	44	19	45	9	13	20	29
50	167	183	36	18	33	37	22	37	9	13	23	28
100	175	165	41	12	31	32	21	34	10	10	24	30
500	144	189	38	18	34	37	16	34	8	19	21	24
1000	163	183	44	19	35	42	24	31	9	8	22	30
5000	94*	106*	17*	18	36	37	15	26*	4*	12	19	30
H20	149±17.1	161±16.2	28±6.9	15±3.6	32±7.3	33±10.3	29±6.2	39±8.6	16±6.4	21±8.1	21±5.5	28±7.0
AF2	501 ± 84.7	-	-	-	1082 ± 293.7	-	278 ± 64.8	-	-	-	-	-
ENNG	-	-	1101 ± 683.1	-	-	-	-	-	-	-	-	-
9AC	-	-	-	-	-	-	-	-	889 ± 275.7	-	-	-
4NQO	-	-	-	-	-	-	-	-	-	-	270 ± 66.2	-
B(a)P	-	1084  ± 236.3	-	-	-	-	-	809 ± 108.4	-	313 ± 48.6	-	354 ± 89.4
2AA	-	-	-	440 ± 198.6	-	359 ± 127.0	-	-	-	-	-	-

 * growth inhibition was observed

Conclusions:

Interpretation of results (migrated information):
negative

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read across to Kilimisch 1 GLP, Guideline study (OECD Test Guideline 473)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: rat lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver.

Test concentrations with justification for top dose:

83.3-1500 ug/mL (-S9) and 250-2500 ug/mL (+S9)
Analytical method: GC/HPLC

Vehicle / solvent:

water

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

- Without metabolic activation: Mitomycin C; With metabolic activation: Cyclophosphamide

Details on test system and experimental conditions:

ADMINISTRATION:
-Treatment time/harvest time:
-S9-mix: 24/24 hours and 48/48 hours (second test)
+S9-mix: 4/24 hours and 4/48 hours (second test)
- Dosing;
-S9-mix: 2.5, 8.33, 25.0, 83.3, 250, 833, 2500 ug/mL
(first test)
-S9-mix: 83.3, 250, 833, 1500 ug/mL (second test)
+S9-mix: 2.5, 8.33, 25.0, 83.3, 250, 833, 2500 ug/mL
(first test)
+S9-mix: 83.3, 250, 833, 2500 ug/mL (second test)
-Doses used for evaluation
-S9-mix: 83.3, 250, 833 ug/mL (first test)
-S9-mix: 250, 833, 1500 ug/mL (24 h harvest)
1500 ug/ml (48 h harvest) (second test)
+S9-mix: 250, 833, 2500 ug/mL (first test)
+S9-mix: 250, 833, 2500 ug/mL (24 h harvest)
2500 ug/mL (48 h harvest) (second test)
- Number of replicates: 2

Mitotic index: based on 1000 cells.
No. of metaphases analyzed: 100 metaphases/replicate (200/dose).

Evaluation criteria:

- Statistically significant increase in the aberrant cells frequency, with or without S9-mix

Statistics:

At each dose level, data from the replicates were pooled. A 2-way contingency table was constucted to analyze the frequencies of cytogenetic abnormalities. An overall Chi-square statistic, based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the two global hypotheses of (1) no differences in average number of cells with aberrations among the dose groups, and (2) no linear trend of increasing number of cells with aberrations with increasing dose. An ordinal metric was used for the doses in the statistical evaluation. If either statistic was found to be significant at α=0.01 versus a one-sided increasing alternative, pairwise tests (i.e., control vs treatment) were performed at each dose level and evaluated at α=0.01 again versus a one-sided alternative. For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the negative controls, the aberration frequency in the negative control should be within reasonable limits of the laboratory historical values. A test chemical is considered positive in this assay if it induces a significant dose-related, and reproducible increase in the frequency of cells with aberrations.

Species / strain:

lymphocytes: primary cultures from the rat

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Without metabolic activation: 833 ug/mL; With metabolic activation: no toxicity up to 2500 ug/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS:
- with metabolic activation: no effects
- without metabolic activation: no effects
MITOTIC INDEX:
- Without metabolic activation
- 1500 ug/mL (24/24): 78% of the control
- 1500 ug/mL (24/48): 86% of the control
- With metabolic activation
- 2500 ug/mL (4/24 and 4/48): no significant reduction.

TEST-SPECIFIC CONFOUNDING FACTORS:
Highest dose was based on changes in pH/osmolality. pH at 2500 ug/mL was too high and was lowered to approximately 7.4. Osmolality at 2500 ug/mL was at an acceptable level (55-57 mOsmol/kg water above the control). Higher concentrations needed more HCl to adjust the pH, revealing an unacceptably high osmolality.

Conclusions:

Interpretation of results (migrated information):
negative

Non-clastogenic

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read across to Kilimisch 1 GLP, Guideline study (OECD Test Guideline 476)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

other: CHO -K1-BH4 cells

Details on mammalian cell type (if applicable):

Proficiences: low spontaneous mutation frequency at the HGPRT locus.

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254-induced rat liver.

Test concentrations with justification for top dose:

156.3-2500 µg/ml
Number of replicates: 2
Analytical method: GC/HPLC

Vehicle / solvent:

water

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

- S9: Ethyl methanesulfonate; +S9: 20-Methylcholanthrene

Details on test system and experimental conditions:

HGPRT assay
- Application: Cells were treated for approximately 4 hours. Then, cells were subcultured into petri dishes (1x10E6/dish). This was repeated every 2-3 days up to 7-8 days. Then the cells were trypsinised and plated in selection medium (2x10E5 cells/dish). Mutant frequency was determined 8-10 days later.

Evaluation criteria:

Based on statistical evaluation.

DESCRIPTION OF FOLLOW UP REPEAT STUDY: repeat study was similar to first study.

Statistics:

The frequency of mutants per 10E6 clonable cells was evaluated using ANOVA; weighrs are derived from the inverse of the mutation frequency variance. The actual plate counts are assumed to follow a poisson distribution therefore, the mean plate count is used as an estimate of variance. A linear trend test and lack of fit test are employed (α=0.05) as an omnibus test to compare treated groups to the negative control. If there is a significant increasing trend or a significant lack of fit, a Dunnett’s t-test is conducted, comparing each treated group and the positive control to the negative control (α=0.05, one-sided). An additional comparison of the positive control to the negative control (α=0.05) is counducted using a linear contrast statement. For an assay to be acceptable, the mutation frequency in positive controls should be significantly higher than the negative controls, and the negative controls should be within reasonable limits of laboratory historical controls and literature values. The chemical is considered positive if it induces a statistically significant, dose related, reproducible increase in mutation frequency. The final interpretation of the data also takes into consideration such factors as the mutation frequency and cloning efficiencies in the negative controls.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: highest concentration showed no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
Highest dose was based on changes in pH/osmolality. pH at 2500 ug/mL was 9.19. This was lowered to 7.40 (pH of control was 7.05). Osmolality 307 mOsmol/kg water versus 257 in the control. Higher concentrations needed more HCl to adjust the pH, revealing an unacceptably high osmolality.

Conclusions:

Interpretation of results (migrated information):
negative

Not mutagenic .

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

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)

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 TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

also TA 1538

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 ug/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 TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with and without activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TA 1538 also tested negative. During the pre-incubation test, signs of toxicity were noted at concentrations as low as 125 ug/plate. 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 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

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

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:

All concentrations in micrograms/ml

Test 1 with S9: 0.32, 0.63, 1.25, 2.5, 5, 10, 20, 39, 78
Test 1 without S9: 1.25, 2.5, 5, 10, 20, 39, 58,78, 156

Test 2 with S9: 2.5, 5, 10, 20, 26, 33, 39
Test 2 without S9: 20, 39, 58, 78, 130, 156

An additional test was done with S9 at the following dose levels:
2.5, 5, 7.5, 10, 15, 20, 25, and 30 ug/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 and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 15 microgram/ml with S9, >=58 microgram/ml without S9

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 ug/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 micrograms/ml and above. In Test 2, in the absence of S9, cytoxicity was seen at concentrations of 58 micrograms/ml and above.

In Test 1, in the presence of S9, no positive results were seen at concentrations of up to 20 micrograms/ml. Metaphases could not be analyzed due to severe cytotoxicity at the 39 and 78 microgram/ml concentrations. In Test 2, in the presence of S9, one of the cultures at the 5 microgram/ml concentration gave a suspicious result, and both cultures at the 10 microgram/ml concentrations gave positive responses. Mild cytotoxity was also seen at the 10 microgram/ml concentration. At concentrations at and above 20 micrograms/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 microgram/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 micrograms/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 micrograms/ml in the presence of S9, with negative results.

Abbreviations used in tables:

T - Toxicity evident from morphological changes

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

TTT- Too toxic for metaphase assessment

Concentration (micrograms/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 micrograms/ml)	0.14	8	4	-
Cyclophosphamide (30 micrograms/ml)	0.06	4	4	-
Cyclophosphamide (40 micrograms/ml)	0.33	20	19	-

Test 1 - Without S9 Mix, 24 hr Harvest

Concentration (micrograms/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 micrograms/ml)	0.03	3	1	-
Cyclophosphamide (20 micrograms/ml)	0.16	14	10	-

Test 2 - With S9 Mix, 24 hr Harvest

Concentration (micrograms/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 micrograms/ml)	0.38	20	17	-
Cyclophosphamide (50 micrograms/ml)	0.31	18	11	-

Test 2 - With S9 Mix, 48 hr Harvest

Concentration (micrograms/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 micrograms/ml)	0.03	3	2	-
Cyclophosphamide (50 micrograms/ml)	0.10	8	7	-

Test 2 ¿ Without S9 Mix, 24 hr Harvest

Concentration (micrograms/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 micrograms/ml)	0.30	21	14	-
Methyl methane-sulphonate (20 micrograms/ml)	0.71	33	28	-

Test 2 - Without S9 Mix, 48 hr Harvest

Concentration (micrograms/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 micrograms/ml)	0.21	11	8	-
Methyl methane- sulphonate (40 micrograms/ml)	3.20	60	60	-

Test 3 - With S9 Mix, 24 hr Harvest

Concentration (micrograms/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 micrograms/ml)	0.24	14	12	-
Cyclophosphamide (40 micrograms/ml)	0.32	17	11	-

Test 3 - see tables below

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
positive with metabolic activation at cytotoxic concentrations or above

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 Marlon A 350 to cause chromosomal aberrations in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 0.32 to 78 ug/ml with S9, and 1.25 to 156 ug/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 LAS is weakly clastogenic at cytotoxic concentrations but negative at concentrations below cytotoxic concentrations

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

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:

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:

mammalian cell gene mutation assay

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 ug/ml without S9
0, 6, 10, 18, 30, 60 ug/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:

preliminary test showed cytotoxicity at >= 50 ug/ml without S9, and >= 100 ug/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 (ug/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 (ug/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 (ug/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 (ug/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:

Interpretation of results (migrated information):
negative

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 ug/ml without S9, and 0, 6, 10, 18, 30, and 60 ug/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. 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A robust in vivo mammalian micronucleus study is available on the structurally related substance Benzenesulfonic acid, C10-13-sec alkyl derivatives . In this study (Fedtke 1991), 40 male and 40 female mice were given a single oral dose by gavage of 1122 mg/kg test substance and evaluated for chromosome aberrations. Only a single dose has been evaluated which was in the range of the acute oral LD50 value for Benzenesulfonic acid, C10-13-sec alkyl derivatives 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.

In an in vivo mammalian chromosome aberration study on the read across substance Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts by Inoue et al. 1976, groups of male mice were given doses of 200, 400, or 800 mg/kg of test substance. This is about half the acute oral LD50 of 1655 mg/kg, as cited by the authors of the study. Mice were sacrificed at 6, 24, and 48 hrs, three of the mice from each dosage group were sacrificed, and bone marrow cells from the femurs 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 commercial detergents containing 19% or 17.1% of Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts. None of the treatment groups showed any significant increase in chromosome aberrations as compared to negative controls while the positive control, Mitomycin C, clearly showed an increase in chromosomal aberration frequency. Therefore, the substance was not considered clastogenic in this assay.

A second chromosomal aberration study was conducted by Masubuchi et al. 1976. In this study, groups of five male mice or five male rats were fed diets containing 0.9% of the read across substance Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts for 9 months (1125 mg/kg bw/d for mice, 405 mg/kg bw/d for rats). 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. The substance was not clastogenic in this assay.

These same authors (Masubuchi et al. 1976) also conducted a dominant lethal study in mice. A group of seven male mice were fed a diet containing 0.6% of the read across substance Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts for 9 months (750 mg/kg bw/d). At the end of this period they were each mated with two untreated females. Females were sacrificed on day 13 of gestation for examination of ovaries and uteri. No evidence of dominant lethal mutations was observed as compared to the controls.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP laboratory study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

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 degrees 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

Remarks:

Doses / Concentrations:
1122 mg/kg
Basis:
actual ingested

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:

Interpretation of results: negative

Executive summary:

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

Reference 2

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:

other: Well-documented publication.

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 commerical detergents containing 19% or 17.1% of the test substance. Mitomycin C was used as a positive control.

GLP compliance:

no

Type of assay:

chromosome aberration assay

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(s)/solvent(s) 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

Remarks:

Doses / Concentrations:
200, 400, 800 mg/kg
Basis:
nominal conc.
test substance

Remarks:

Doses / Concentrations:
800, 1600, 3200 mg/kg
Basis:
nominal conc.
commercial detergent containing 19% test substance

Remarks:

Doses / Concentrations:
1000, 2000, 4000 mg/kg
Basis:
nominal conc.
commercial detergent containing 17.1% test substance

No. of animals per sex per dose:

9

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C1

- Route of administration: intraperitoneally
- Doses / concentrations: 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:

Interpretation of results: negative
The test substance is 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 3

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:

other: Well-documented journal article.

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 germ cell cytogenetic 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 (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

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 (Type of food): feed powder CE-2

Duration of treatment / exposure:

9 months

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0.9%, 1170 mg/kg bw d
Basis:
nominal 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 degree 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.

Sex:

male

Genotoxicity:

negative

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:

Interpretation of results: negative
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 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:

other: Well-documented journal article.

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 germ cell cytogenetic assay

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 (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

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 (Type of food): feed powder CE-2

Duration of treatment / exposure:

9 months

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0.9%, 450 mg/kg bw d
Basis:
nominal 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 degree 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.

Sex:

male

Genotoxicity:

negative

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:

Interpretation of results: negative
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 5

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:

other: Well-documented journal article.

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 (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): 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 (Type of food): feed powder CE-2

Duration of treatment / exposure:

9 months

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0.6%, 780 mg/kg bw d
Basis:
nominal in diet

No. of animals per sex per dose:

7

Control animals:

yes

Statistics:

Rohrborn's method.

Sex:

male

Genotoxicity:

negative

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:

Interpretation of results: negative
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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The read across substance Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts was consistently found not to cause induction of gene mutations in the Ames bacterial reverse mutation assay as well as in the OECD 476 in vitro mammalian cell gene mutation test. Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts was found to be positive at cytotoxic concentrations with metabolic activation, but negative at non-cytotoxic concentrations with metabolic activation and negative without metabolic activation, when tested in an in vitro chromosome aberration assay in CHO cells. When tested in a battery of in vivo genotoxicity studies, Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts was consistently found not to cause any mutagenic or clastogenic responses. An additional study conducted on Benzenesulfonic acid, C10-13-sec alkyl derivatives further supports that this groups of substances are not expected to be mutagenic or clastogenic. The positive result in the in vitro chromosome aberration study using a rodent cell line (CHO cells) derived from cancer tissues that is lacking proper cell cycle control has to be seen in the context of the extensive in vivo data. In vivo studies do assess genotoxicity under more realistic conditions, including eADME and metabolic activation.

In addition, the read across substance MIPA did not cause gene mutations in Salmonella typhimurium (Ames test) or in Chinese hamster ovary cells (CHO/HGPRT), nor were chromosomal aberrations induced in rat lymphocytes. All studies were performed in the absence and presence of metabolic activation. Thus, MIPA is not considered to be genotoxic.

Finally, in a supporting study, the read across substance Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 1-aminopropane-2-ol was tested in the Ames reverse mutation assay using S. typhimurium and E. coli strains up to cytotoxic concentrations, both with and without metabolic activation. No significant revertant colonies were observed.

Justification for classification or non-classification

Based on all of the in vitro and in vivo experimental studies available for read across substances, Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs.-, compd. with 1-aminopropane-2-ol is not classified for genetic toxicity.