Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro reverse gene mutation assay in bacteria: Negative; OECD 471; Dreher, 2017

In vitro cytogenicity/micronucleus in mammalian cells: Waiver

In vitro gene mutation in mammalian cells: Waiver

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

Study period:

29 March 2017 to 07 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial forward mutation assay

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stable at room temperature
- Stability under test conditions: Not required
- Solubility and stability of the test substance in the solvent/vehicle: Not required
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not required

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test article stock solutions were prepared by the test article under subdued lighting in purified water with the aid of vortex mixing, ultrasonication and warming at 37 °C, to give the maximum required treatment concentration. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32 mm/CR filter, 0.2 µm pore size) and subsequent dilutions were made using purified water. The test article solutions were protected from light and used within approximately 2 hours of initial formulation.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 50, 16, 5, 1.6, 0.5, 0.16 and 0.05 mg/mL treatment solutions prepared.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as an aqueous formulation.

OTHER SPECIFICS: n/a

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Experiment 1 (S9 ±): 5, 16, 50, 160, 500, 1600, 5000 µg/plate
Experiment 2 (S9 -): 0.5, 1.6, 5, 16, 50, 160, 500 µg/plate
Experimetn 2 (S9 +): 15, 30, 60, 125, 250, 500, 1000 µg/plate

A maximum concentration of 5000 µg/plate was selected for Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997). For Experiment 2 the maximum concentration tested was selected on the basis of toxicity seen in the assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Test item is completely soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Mutation Experiment

Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45 ± 1 °C:
• 0.1 mL bacterial culture
• 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
• 0.5 mL 10% S-9 mix or buffer solution
This was followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37 ± 1 °C protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony Enumeration Section 4.4).
As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step in order to increase the sensitivity of the test system. Quantities of test article, vehicle control solution or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37 ± 1 °C, with shaking, before the addition of 2 mL molten agar at 45 ± 1 °C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure.

Toxicity Assessment

The background lawns of the plates were examined for thinning as sign of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Colony Enumeration

Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or splits in the agar affected the accuracy of the automated counter.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:

1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. The positive trend/effects described above were reproducible.

The test article was considered positive in this assay if both of the above criteria were met.

The test article was considered negative in this assay if neither of the above criteria were met.

Statistics:

Statistical analysis using Dunnett’s test was used to aid evaluation of a concentration response, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate). However, adequate interpretation of biological relevance was of critical importance.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 1600 µg/plate. Slightly thinned bacterial lawn observed at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 500 µg/plate. Slight thinning of bacterial lawn at 160 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 1600 µg/plate. Slight thinning of bacterial lawn observed at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 500 µg/plate. Slight thinning of bacterial lawn observed at 160 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 1600 µg/plate. Slight thinning of bacterial lawn observed at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 500 µg/plate. Slight thinning of the bacterial lawn observed at 160 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 1600 µg/plate. Slight thinning of bacterial lawn observed at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 5000 µg/plate. Very thin bacterial lawn present at 1600 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: test item readily soluble at highest tested concentration
- Precipitation: not obsered
- Definition of acceptable cells for analysis: The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline).
- Other confounding effects: n/a

RANGE-FINDING/SCREENING STUDIES: no

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: n/a
- Indication whether binucleate or mononucleate where appropriate: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: visual inspection of bacterial lawn
- Other observations when applicable: n/a

Table 2:       Mean number of revertants per plate (experiment 1 – pre-incubation not conducted)

Conc. (µg/plate)	TA98			TA100			TA1535			TA1537			TA102
	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)
Neg. control	27.3	49.3	- / -	124.3	89.3	- / -	18.7	15.3	- / -	12.7	8.0	- / -	260.0	353.3	- / -
5	21.7	33.0	- / -	104.7	91.7	- / -	12.7	16.0	- / -	12.3	13.0	- / -	279.0	344.0	- / -
16	25.7	29.3	- / -	95.7	107.3	- / -	19.7	12.0	- / -	7.5	14.0	- / -	252.3	316.7	- / -
50	29.3	39.0	- / -	94.0	109.3	- / -	16.3	12.3	- / -	10.0	14.3	- / -	254.3	362.7	- / -
160	22.7	22.7	- / -	62.3	95.3	S / -	18.0	20.3	S / -	8.7	9.3	S / -	185.7	422.0	- / -
500	9.0	25.0	S / S	0	80.0	T / S	0	15.0	T / S	0	12.7	T / S	0	342.0	T / -
1600	0	0	T / T	0	0	T / T	0	0	T / T	0	0	T / T	0	116.0	T / V
5000	0	0	T / T	0	0	T / T	0	0	T / T	0	0	T / T	0	0	T / T
Pos. control	545.3	102.0	- / -	528.0	785.3	- / -	631.7	224.0	- / -	321.7	426.3	- / -	904.3	3078.3	- / -

T= cytotoxic, no sign of revertant colonies

- = no sign of cytotoxicity

S= slight reduction in bacterial lawn

V= very thin bacterial lawn

^*= p<0.05

^**= p<0.01

 

Table 3:       Mean number of revertants per plate (experiment 2 – pre-incubation conducted)

Conc. (µg/plate)	TA98		TA100		TA1535		TA1537		TA102
	S9 -	Toxicity	S9 -	Toxicity	S9 -	Toxicity	S9 -	Toxicity	S9 -	Toxicity
Neg. control	22.7	-	100.0	-	13.3	-	10.0	-	259.7	-
0.5	25.7	-	90.0	-	14.7	-	8.0	-	325.3	-
1.6	23.7	-	78.0	-	11.7	-	14.7	-	299.0	-
5	25.7	-	83.0	-	11.3	-	9.3	-	305.7	-
16	27.7	-	98.7	-	11.0	-	6.0	-	294.0	-
50	30.3	-	90.3	-	21.0	-	7.7	-	260.7	-
160	28.0	-	75.3	-	12.3	-	6.3	-	201.0	-
500	22.3	-	13.0	V	10.7	-	1.3	-	126.7	S
Pos. control	591.0	-	885.3	-	642.0	-	227.7	-	722.0	-

 

Conc. (µg/plate)	TA98		TA100		TA1535		TA1537		TA102
	S9 +	Toxicity	S9 +	Toxicity	S9 +	Toxicity	S9 +	Toxicity	S9 +	Toxicity
Neg. control	33.3	-	101.0	-	18.0	-	17.7	-	298.3	-
15	45.7^*	-	109.7	-	15.0	-	16.0	-	372.3^*	-
30	33.7	-	117.0	-	13.0	-	11.0	-	390.3^**	-
60	36.7	-	88.3	-	14.3	-	10.3	-	340.7	-
125	31.0	-	91.3	-	13.7	-	9.3	-	360.0	-
250	39.7	-	82.3	-	15.0	-	11.0	-	349.7	-
500	29.7	-	96.7	-	17.7	-	8.7	-	244.7	-
1000	19.0	-	24.7	-	14.3	-	7.3	-	218.7	-
Pos. control	303.0	-	1296.3	-	177.3	-	427.0	-	1552.0	-

- = no sign of cytotoxicity

S= slight reduction in bacterial lawn

V= very thin background bacterial lawn

T= cytotoxic, no revertant colonies

^*= p<0.05

^**= p<0.01

Conclusions:

It was concluded that under the condition of this study, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines).

Executive summary:

OECD 471 (2017) - In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA98, TA100, TA1535, TA1537 and TA102) were exposed to Benzenesulfonic acid, 4 -C10 -13 -sec-alkyl derivs., ammonium salts, formulated in purified water at concentration range of 0.5 – 5000 µg/plate in the presence and absence of mammalian metabolic activation S9-mix.

The test item was tested up to 5000 µg/plate (or its cytotoxic limit, where relevant), the maximum recommended concentration according to OECD 471.  The positive controls induced the appropriate responses in the corresponding strains.  There was no evidence (or a concentration related positive response) of induced mutant colonies over background in the test item treated plates.

This study is classified acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo mammalian bone marrow chromosome aberration test: Negative; Mammalian somatic cell cytogenetic assay (no guideline followed); Masubuchi, 1976 (read across from benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. - data on LAS as part of category approach)

In vivo rodent dominant lethal assay: Negative; Rodent dominant lethal assay (no guideline followed); Masubchi, 1976 (read across from benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. - data on LAS as part of category approach)

In vivo chromosome aberration assay: Negative; Chromosome aberration assay (no guideline followed); Anon., 1976 (read across from benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. - data on LAS-Na salt as part of category approach)

In vivo mammalian bone marrow chromosome aberration test: Negative: Mammalian somatic cell cytogenetic assay (no guideline followed); Masubuchi, 1976 (read across from benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. - data on LAS as part of category approach)

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Well-documented journal article.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

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

Remarks:

Study pre-dates GLP

Type of assay:

mammalian germ cell 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 (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

Dose / conc.:

1 170 mg/kg bw/day (nominal)

Remarks:

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

Remarks on result:

other: No increase in chromosome aberrations was noted.

Additional information on results:

No increase in chromosome aberrations was noted.

Chromosome Aberrations

	1170 mg/kg bw/d in diet	Control
Number of cells with chromatid breaks	1	2
Number of cells with isochromatid breaks	1	0
Number of cells with chromatid gaps	4	5
Number of cells with isochromatic gaps	0	0
Number 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 2

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:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Negative controls validity:

valid

Remarks on result:

other: REPORTING FORMAT FOR THE ANALOGUE APPROACH Refer to Section 13.2 for read-across justification document.

Conclusions:

Interpretation of results: negative
The test substance is not clastogenic.

Executive summary:

In a one-to-one read-across approach, the substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (data on LAS as part of category approach) is considered appropriate for direct read-across (one-to-one) to benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts (target substance) for the endpoint Genetic toxicity in vivo.  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.

In conclusion, The test substance in not clastogenic.  A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 3

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Well-documented journal article.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

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

Remarks:

Study pre-dates GLP

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 (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

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

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

Remarks on result:

other: No increase in chromosome aberrations was noted.

Chromosome Aberrations

	450 mg/kg diet - Wistar Rats	450 mg/kg diet - SD Rats	Control	Control
Number of cells with chromatid breaks	0	0	1	0
Number of cells with isochromatid breaks	0	0	0	0
Number of cells with chromatid gaps	3	4	3	4
Number of cells with isochromatid gaps	0	0	0	0
Number of cells with other aberrations	0	0	0	0

Conclusions:

Interpretation of results (migrated information): 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 4

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Negative controls validity:

valid

Remarks on result:

other: No increase in chromosome aberrations was noted.

Conclusions:

Interpretation of results (migrated information): negative
The test substance is not clastogenic.

Executive summary:

In a one-to-one read-across approach, the substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (data on LAS as part of category approach) is considered appropriate for direct read-across (one-to-one) to benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts (target substance) for the endpoint Genetic toxocity in vivo. In conclusion, no increase in chromosome aberrations was seen as compared to controls following exposure of 5 male rats fed with a diet containing 0.9% test substance for 9 months. The test substance is not clastogenic.  A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 5

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Well-documented publication.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

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

Remarks:

Study pre-dates GLP

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(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:

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

Remarks:

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

Remarks:

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

Remarks on result:

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

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)	-	-
mitomycon 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 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:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

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

Conclusions:

Interpretation of results: negative
The test substance is not clastogenic.

Executive summary:

In a one-to-one read-across approach, the substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (data on LAS-Na salt as part of category approach) is considered appropriate for direct read-across (one-to-one) to benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts (target substance) for the endpoint Genetic toxicity in vivo.  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.

In conclusion, The test substance in not clastogenic.  A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 7

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Well-documented journal article.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

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

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

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

Remarks on result:

other: 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 (300 mg/kg bw/day)	Control
Number of mating females	14	18
Number pregnant	11	12
Number with dead embryos	6	10
Dead embryos per pregnant female	54.6 %	83.3 %
Number of corpora lutea	156	161
Corpora lutea per pregnant female	14.2	13.4
Number of implants	148	156
Implants per pregnant female	13.5	13.0
Implants per corpora lutea	94.9	96.9
Number 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
Number of early dead fetuses	4	12
Number 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.

Reference 8

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Negative controls validity:

valid

Remarks on result:

other: 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.

Conclusions:

Interpretation of results: negative
The test substance did not cause genetic disorders in mice.

Executive summary:

In a one-to-one read-across approach, the substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (data on LAS as part of category approach) is considered appropriate for direct read-across (one-to-one) to benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts (target substance) for the endpoint Genetic toxicity in vivo. In conclusion, no increase in dominant lethal induction was seen as compared to controls following 9 months dietary exposure to males at 300 mg/kg bw/d, followed by mating with untreated females.  On day 13 of pregnancy, the females were sacrificed, and the ovaries and uteri were examined. The test substance does not cause genetic disorders.  A full justification for the read-across approach is presented in IUCLID Section 13.2.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not applicable, no adverse effects observed.

Additional information

Genetic Toxicity - in vitro

OECD 471 (2017) - In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA98, TA100, TA1535, TA1537 and TA102) were exposed to Benzenesulfonic acid, 4 -C10 -13 -sec-alkyl derivs., ammonium salts, formulated in purified water at concentration range of 0.5 – 5000 µg/plate in the presence and absence of mammalian metabolic activation S9-mix. The test item was tested up to 5000 µg/plate (or its cytotoxic limit, where relevant), the maximum recommended concentration according to OECD 471.  The positive controls induced the appropriate responses in the corresponding strains.  There was no evidence (or a concentration related positive response) of induced mutant colonies over background in the test item treated plates.

Genetic Toxicity - in vivo

Masubuchi (1976) CAT - In a one-to-one read-across approach, the substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (data on LAS as part of category approach) is considered appropriate for direct read-across (one-to-one) to benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts (target substance) for the endpoint Genetic toxocity in vivo. In conclusion, no increase in chromosome aberrations was seen as compared to controls following exposure of 5 male rats fed with a diet containing 0.9% test substance for 9 months. The test substance is not clastogenic.

Masubuchi (1976) RDLA - In a one-to-one read-across approach, the substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (data on LAS as part of category approach) is considered appropriate for direct read-across (one-to-one) to benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts (target substance) for the endpoint Genetic toxicity in vivo. In conclusion, no increase in dominant lethal induction was seen as compared to controls following 9 months dietary exposure to males at 300 mg/kg bw/d, followed by mating with untreated females.  On day 13 of pregnancy, the females were sacrificed, and the ovaries and uteri were examined.  The test substance does not cause genetic disorders.

Anon. (1976) CAT - In a one-to-one read-across approach, the substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (data on LAS-Na salt as part of category approach) is considered appropriate for direct read-across (one-to-one) to benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts (target substance) for the endpoint Genetic toxicity in vivo.  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.

In conclusion, The test substance in not clastogenic.

Masubuchi (1976) CAT - In a one-to-one read-across approach, the substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (data on LAS as part of category approach) is considered appropriate for direct read-across (one-to-one) to benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts (target substance) for the endpoint Genetic toxicity in vivo.  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.

In conclusion, The test substance in not clastogenic.

Justification for classification or non-classification

The substance does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008.