Benzenesulfonic acid, 4-(1-methylethyl)-, ammonium salt (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: Read across from category (Hydrotropes)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

Permanent stocks of these strains are kept at -80°C in ERBC facilities.

Metabolic activation:

with and without

Metabolic activation system:

One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics:
Species Rat
Strain Sprague Dawley
Tissue Liver
Inducing Agents Phenobarbital – 5,6-Benzoflavone
Producer MOLTOX,Molecular Toxicology, Inc.
Batch Number 4086

The production and quality control certificate can be found in Addendum 2 of this report. The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL):
S9 tissue fraction 1.0mL
NADP (100 mM) 0.4mL
G-6-P (100 mM) 0.5mL
KCl (330 mM) 1.0mL
MgCl2 (100 mM) 0.8mL
Phosphate buffer (pH 7.4, 200 mM) 5.0mL
Distilled Water 1.3mL

Test concentrations with justification for top dose:

Toxicity test: 5, 1.58, 0.500, 0.158 and 5.05 µL/plate. A slightly opaque solution without any precipitation, suitable for treatment and serial
dilutions, was obtained at 126 µL/mL corresponding to 50.0 µL/mL (expressed as active
ingredient). This result permitted a maximum concentration of 5.00 µL/plate to be used in
the toxicity test
I and II Main assay: 5.00, 2.50, 1.25, 0.625 and 0.313 µL/plate (no effect obsterved in the first main test and the same doses were used for the second main test)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 and TA100 wihtout metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E.Coli WP2 uvrA wihtout metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains with metabolic activation

Details on test system and experimental conditions:

Solubility of the test item was evaluated in a preliminary trial using sterile water for injection. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity.
A slightly opaque solution without any precipitation, suitable for treatment and serial dilutions, was obtained at 126 µL/mL corresponding to 50.0 µL/mL (expressed as active ingredient). This result permitted a maximum concentration of 5.00 µL/plate to be used in the toxicity test.

Two Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
The Main Assay I was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.

The overlay mixture was composed as follows:
Overlay agar (held at 45°C) 2.0mL
Test or control item solution 0.1mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
Bacterial suspension 0.1mL

The Main Assay II was performed using a pre-incubation method. The components were added in turn to an empty test-tube:
Bacterial suspension 0.1mL
Test item solution 0.1mL
or control item solution 0.05mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL

The incubate was vortexed and placed at 37°C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were immediately scored by counting the number of revertant colonies on each plate and evaluating the condition of the bacterial backgroundlawn. In the preliminary toxicity test, plates were held at approximately 4°C for 24 hours before scoring.

Evaluation criteria:

Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study.
TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens. In addition to amutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall and increases permeability to certain classes of chemicals. All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens. TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system.
Tester strain WP2 uvrA is reverted fromtryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic.

The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

as per OECD 471 guideline

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
No precipitation of the test item was observed at the end of the incubation period at any concentration tested, in the absence or presence of S9 metabolic activation. Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

Main assay: No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment.
Neither toxicity, nor relevant increase in the number of revertant colonies was observed in the pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

Controls: Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data.

Controls of S9:
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Conclusions:

Nut mitagenic in bacteria

Executive summary:

The test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, for all the tested strains under the reported experimental conditions.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: Read across from category (Hydrotropes)

Adequacy of study:

supporting study

Study period:

April 20-23, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study; GLP compliance, Full documentation

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA98 and TA100 strains contain the pKM101 plasmid (carrying the R-factor)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

100, 330, 1000, 3333 and 5000 micrograms/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle:the relatively high solubility of the test substance

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

other: vehicle was water

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-nitrofluorene, sodium azide, 9-aminoacridine

Remarks:

positive controls varied with test strain

Details on test system and experimental conditions:

On the day of use, all tester strain cultures were checked for the correct genotype. The presence of the rfa wall mutation was confirmed by demonstration of sensitivity to crystal violet. The deletion in the uvrB gene was confirmed by demonstration of sensitivity to ultraviolet light. The presence of the pKM101 plasmid was confirmed by demonstration of resistance to ampicillin. Spontaneous reversion frequencies in the vehicle controls were demonstrated by plating 100 microliter aliquots of the culture along with the appropriate vehicle on selective media.

To determine the sterility of the test substance, the highest dose (5000 microliters/plate) was plated on selective agar with an aliquot volume equal to that used in the assay. To determine the sterility of the S9 or Sham mix, a 0.5 mL aliquot of S9 or Sham mix was plated on selective agar.

Test substance dilutions wer prepared immediately before use. 0.5 mL of S9 or Sham mix, 100 microliter of tester strain and 50 microliter of vehicle or test substance were added to 2 mL of agar at 45 degrees C. The positive control was substituted for the test substance. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. The plates were inverted and incubated for 48-72 hours at 37 +/- 2 degrees C. Plates were read immediately or else were stored at 4 degrees C until read.

The dosing solutions reflected the active ingredient in the test substance.

Condition of the bacterial background lawm was evaluated for evidence of test substance toxicity using a dissecting microscope.. Toxicity and precipitate were scored relative to the vehicle control plate. Revertant colonites were counted either entirely by automated colongy counter or entirely by hand (the later in the case of excess precipitate)

All dose levels of test substance, vehicle controls and positive controls were plated in triplicate. For each replicate plating, the mean and standard deviation of the number of revertants per plate was calculated.

Evaluation criteria:

Validity criteria include: the presece of the deep rough mutation (rfa), the deletion in hte uvrB gene and the characteristic mean number of spontaneous revertants in the vehicle control of 10-50 for TA98, 80-240 for TA100, 5-45 for TA1535, 3-21 for TA1537, and 5-35 for TA1538. In addition, TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. Strain culture titers must be greater than or equal to 0.3 x 10-9 cells/mL. The mean of each positive control must be at least a 3X increase in revertants compared with respective vehicle control. A minimum of three non-toxic dose levels are required. A dose level is considered toxic if >50% reduction in revertants versus its control and/or a reduction in the background lawn.

For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one strain with a minimum of two increasing concentrations of the test substance. 2X increases are needed for TA98 and TA100. 3X increases are needed for TA1535, TA1537 and TA 1538.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Historical control data are provided in Appendix I

A range finding study was conducted to a maximum dose of 5000 micrograms active ingredient per plate. Neither precipitate nor appreciable toxicity was observed.

In the definitive assay, neither precipitate nor appreciable toxicity was observed. No positive responses were observed with any of the strains in the presence and absence of S9 activation. All criteria for a valid study were met. The data are presented in Tables 2-11 and summarized in Table 12

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test substance, SAR 33-55, is not genotoxic based on the results of the Ames Test

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: Read across from category (Hydrotropes)

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Protocols and results reviewed and accepted by the National Toxicology Program's Board of Scientific Counselor's Technical Reports Review Subcommittee, USA National Institutes of Health

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

not specified

GLP compliance:

yes

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:

Aroclor 1254-induced male Sprague-Dawley rat liver S9

Test concentrations with justification for top dose:

2513, 3750 and 5000 micrograms active ingredient per milliliter

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: with S9 and mitomycin-C without S9

Details on test system and experimental conditions:

Without S9, cells were incubated in McCoy's 5A medium with the test substance for 18 hours. colcemid was then added and incubation continued for additional 2 hours. The cells were harvested by mitotic shake-off, fixed and stained with Giemsa. The cells with S9 activation were treated with the test substance for 2 hours at which time the treatment medium was removed and the cells were incubated for 10 hours in fresh medium with Colcemid present for the last 2 hours. Harvesting was the same.

Evaluation criteria:

Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 +/- 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each test level. Aberrations were classified as "simple" (breaks and terminal deletions), "complex" (rearrangements and translocations) and "other" (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Statistics:

Analyses were conducted on both the dose response curve and individual dose points. For a single trial, a significant (P

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: chromosome aberration

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Test substance was not genotoxic in this assay

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

other: Read across from category (Hydrotropes)

Adequacy of study:

supporting study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Protocols and results reviewed and accepted by the National Toxicology Program's Board of Scientific Counselor's Technical Reports Review Subcommittee, USA National Institutes of Health

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

mouse lymphoma cells - TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

125, 250, 500, 1000 and 2500 microgram active ingredient per milliliter

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: without S9 and methlcholanthrene with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in Fischer medium

DURATION
Normal cycling time for L5178Y cells was 10 hours. Treated cultures contained 6 x 10-6 cells in 10mL medium.
Incubation with test substance continued for 4 hours, at which time the medium plus test substance was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was maintained at log phase growth. After the 48-hr expression period, cells were plated in medium and soft agar supplemented with TFT (trifluorothymidine) for selection of TFT-resistant cells. Cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 degrees C in 5% CO2 for 10-12 days.


NUMBER OF REPLICATIONS: each treatment level was replicated, including positive and solvent controls


OTHER:

Evaluation criteria:

Minimum criteria for accepting an experiment as valid are presented in Caspary et al 1988

Statistics:

All data were evaluated statistically for trend and peak responses. Both responses had to be significant (P<=0.05) for the test substance to be considered positive; i.e., capable of inducing TFT resistance. A single significant response led to a "questionable" conclusion, and absence of both a trend and a peak response resulted in a "negative" conclusion.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Negative in one replicate series and positive in other replicate series with metabolic activation; leads to "equivocal" conclusion with metabolic activation

Remarks on result:

other: strain/cell type: L5178Y

Remarks:

Migrated from field 'Test system'.

Conclusions:

Not mutagenic without metabolic activation, equivocal with metabolic activation

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

other: Read across from category (Hydrotropes)

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Protocols and results reviewed and accepted by the National Toxicology Program's Board of Scientific Counselor's Technical Reports Review Subcommittee, USA National Institutes of Health

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5900 - In vitro Sister Chromatid Exchange Assay

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver S9

Test concentrations with justification for top dose:

500, 1667, 2513, 3750 and 5000 micrograms active substance per milliliter

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: with S9 and mitomycin-C without S9

Details on test system and experimental conditions:

Testing was performed as reported by Galloway et al 1987.

In the test without S9, CHO cells were incubated for 25.5 hours with the test substance in McCoyy's supplemented 5A medium. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 25.5 hours, fresh medium was provided to include BrdU and Colcemid and further incubated for 2 hours. Cells were harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa.

In the SCE test with S9, cells were incubated with the test substance, serum-free medium and S9 for 2 hours. Then the medium was replaced with medium containing serum and BrdU and no test substance and incubated for 25.5 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as cells treated without S9.

Evaluation criteria:

All slides were scored blind and those from a single test were read by the same person. fifty second-division metaphase cells were scored for frequency of SCEs/cell from each dose level.

Statistics:

Conducted on the slopes of the dose-response curves and the individual data points. An SCE frequency 20% above the concurrent solent control was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose is less than 0.01 and at two doses is less than 0.001. An increase of 20% or greater at any single dose was considered weak evidence of activity; increases at two or more doses was considered a positive response. A statistically significant trend in the absence of any responses reaching 20% above background was considered equivocal.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with metabolic activation

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Clastogenic without metabolic activation at doses >1667 ug/mL. Not clastogenic with metabolic activation up to 5000 ug/mL. Cell cycle delay was apparent at concentrations >2513 ug/mL; incubation time was increased.

Remarks on result:

other: other: sister chromatid exchange

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation positive without metabolic activation

Test substance not genotoxic with metabolic activation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

April 14-17, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline test; GLP compliance; Full documentation

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5385 (In Vivo Mammalian Cytogenetic Tests: Bone Marrow Chromosomal Analysis)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc. in Maryland
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males 27.6-34.3g; females 20.0-27.2g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: in a AAALAC-accredited facility at up to 5 mice of same sex per plastic autoclavable cage with filter tops. Heat-treated hardwood chips were used for bedding.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: minimum of 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 74+/-6
- Humidity (%): 50+/-20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: To:

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: relatively high solubility of the test substance

Details on exposure:

13 experimental groups of 5 males and 5 females in each group based on computer generated assignments taking into account body weight. Vehicle control was water. Positive control was cyclophosphamide dissolved in deionized water at a concentration of 2 mg/mL Test substance concentrations were corrected for purity which was 31.2% active ingredient. The high dose level was calculated to be approximately 80% of the LD50/3

Duration of treatment / exposure:

one IP injection at a constant volume of 20 mL/kg body weight

Frequency of treatment:

one IP injection at a constant volume of 20 mL/kg body weight

Post exposure period:

72 hours

Remarks:

Doses / Concentrations:
145, 290 and 580 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: single IP injection
- Doses / concentrations: 2mg/mL

Tissues and cell types examined:

femur bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: highest dose was approximately 80% of LD50/3


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 5 animals were sacrificed at each dose and control at 24, 48 and 72 hours following the IP injection.


DETAILS OF SLIDE PREPARATION: Femur bone marrow cells were transferred to a capped centrifuge tube with 1 mL fetal bone serum. Cells were pelleted by centrifugation at 100X for 5 minutes. The cells were resuspended by aspiration with a capillary pipet and a small drop spread onto a glass slide. Two to four slides were prepared for each animal. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.


METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were scored for the presence of micronuclei. The number of micronucleated normocytes in the filed of 1000 polychomatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes counted was also recorded per 1000 erythrocytes.


OTHER:

Evaluation criteria:

The test substance was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrrent vehicle control was observed at any sampling time. The test substance was considered positive if a dose-responsive increase in micronucleated polychromatic erythorcytes was observed and one or more doses were statistically elevated relative to the vehicle control at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, teh assay was considerd a suspect or unconfirmed positive.

THe criteria for determination of a valid test is - The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence in the positive control must be significantly increased relative to the vehicle control (p

Statistics:

Kastenbaum-Bowman tables based on the binomial distribution. All analysies were performed separately for each sex and for each treatment group.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

lethargy in both sexes at 580 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A 36% reduction in the ratio of polychromatic erythrocytes to total erythrocytes was observed in male mice sacrificed at 72 hours with 580 mg/kg relative to their respective vehicle controls. The presence of lethargy and reduction in the frequency of polychromatic erythrocytes in the bone marrow demonstrate that suitable dose levels were used. No significant increases in micronucleated polychromatic erythrocytes were observed in either sex at any time period relative to their respective vehicle controls. The positive control induced a significant increase in micronucleated polychromatic erythrocytes in both sexes. The vehicle (negative) and positive controls fulfilled the requirements for determination of a valid test.

Conclusions:

Interpretation of results (migrated information): negative
The test substance was found to be not genotoxic in the mouse micronucleus assay

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: Read across from category (Hydrotropes)

Adequacy of study:

key study

Study period:

June to July 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented study according to OECD test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: BOR:NMRI (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: young adults
- Weight at study initiation: 30.4 +/- 1.7 g (males); 24.3 +/- 1.5 g (females)
- Assigned to test groups randomly: no data
- Fasting period before study: no
- Housing: 5 animals per cage, gender separated, macrolone cages Type III
- Diet : Ssniff R10 exclusive diet for rats (ad libitum); supplied by Ssniff Spezialfutter GmbH, Soest, Germany
- Water: drinking water (ad libitum)
- Acclimation period: one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: July 9, 1991 To: July 18, 1991

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 40 %
- Amount of vehicle: 16.8 ml/kg bw

Frequency of treatment:

single oral application

Post exposure period:

24, 48 and 72 hours

Remarks:

Doses / Concentrations:
4467 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: oral by gavage
- Dose: 100 mg/kg bw
- Vehicle: water
- Total application volume: 10 ml/kg bw
- post exposure period: 24 hours

Tissues and cell types examined:

bone marrow; polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The maximum tolerable dose (MTD)* was selected as dose.

The MTD was determined in a dose range finding study :
Phase 1: Limit test with 5000 mg/kg bw
Phase 2: Determination of the TMTD range with reduced animal number
Phase 3: Determination of the MTD with 5 animals/sex/dose

*MTD is defined as dose with no mortality but clear clinical symptoms within 3 days after application

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
24, 48 and 72 hours after treatment the animals were killed by cervical dislocation and tissue was sampled.

DETAILS OF SLIDE PREPARATION:
The femora were removed and the bone marrow was suspended in fetal calf serum. The cell suspensions were centrifuged with 160 x g for 5 minutes and the supernatand discarded. The serum was resuspended and the suspension purified using a cellulose chromatographic column. The eluate was centrifuged at 800 x g for 10 minutes and the pellet in fetal calf serum /25 mM EDTA suspended. From this suspension 3-4 smears per animals were prepared on slides which were dried for at least 24 hours and stained with May-Grünwald/Giemsa solution.

METHOD OF ANALYSIS:
The cell analysis was performed by means of a Zeiss miscroscope at a 1000fold magnification (oil immersion). At least 1000 polychromatic erythrocytes (PCE) per animal were examined to determine the frequency of micronucleated cells. The ratio of PCE to normochromatic cells (NCO) was determined for a sample of 1000 erythrocytes. The number of micronucleated cells in counted NCE was determined.

Evaluation criteria:

For the identification of micronuclei the following criteria were considered:
a) roundish and clear contour by the nuclear membrane
b) diameter of about 1/20 of the size of the polychromatic erythrocyte
c) lays in the same focus layer as the observed erythrocyte

The micronucleus test is regarded as positive (test substance induces micronuclei in polychromatic erythrocytes) if the frequency of mucronucleated polychromatic erythrocytes of at least one tretament group is statistically significantly increased compared to the negative control and the increase is biologically relevant.

Statistics:

Mean values and standard deviations were calculated for the following parameters:
a) number of polychromatic erythrocytes (PCE) containing micronuclei
b) ratio of PCE/NCE

Comparison of treatment groups with different post exposure periods with negative controls of respective post exposure periods. After control of the relative frequency of micronuclei in the treatment groups on homogeneity with the mean relative frequency a statistical analysis of micronucleus frequency using a 2 x 2 contingency table with chi² test and continuity table according to Yates was performed (see [1]).
The differences of miconucleus frequencies in the positive control were reassessed in the two-sided t-test. This test was also used for the statistical analysis of the PCE/NCE-ratio.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range:
Phase 1: males and females: 5000 mg/kg bw (limit test);
Phase 2: males: 1585, 1995, 2512, 3162 and 3981 mg/kg bw; females: 3162, 3548, 3981, 4467 and 5000 mg/kg bw.
Phase 3: males and females: 3981, 4467 and 5000 mg/kg bw.

- Clinical signs of toxicity in test animals:
Phase 1: 4/5 male and 1/5 female animals died. The males died within 4 hours after application, the female died within 24 hours after application of the test substance. Clinical signs as hunched posture, ruffled fur, closed eyes and diarrhea were observed.
Phase 2: 1/5 female at 3981 mg/kg bw died. At high doses clinicals signs as hunched posture, ruffled fur, closed eyes and diarrhea were observed.
Phase 3: 2/5 male and 2/5 female animals died at 500 mg/kg bw. At 4467 mg/kg bw no mortality occured but severe toxic effects.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals:
Hunched posture, ruffled fur, closed eyes and diarrhea were observed. One day after application the effects subsided and two days after application all animals were free of clinical symptoms. One male and one female animal died 24 and 5.5 hours after application, respectively. These animals were replaced with mice of a concurrent satellite group also treated with the test substance. One male of the satellite group died 5 hours after application.

- Induction of micronuclei:
Positive control: Significantly increased number of polychromatic erythrocytes (PCE) with miconuclei both in male and female animals.
Test substance: No significant increase in the number of PCE with micronuclei after 24 and 48 hours in both males and females and after 72 hours in males. After 72 hours a statistically significant increase of the number of micronuclei in PCEs in females were observed. If the results of males and females were combined no significant difference in the frequency of micronuclei between treatment group and combined vehicle control group is observed. (Tables 1, 2 and 3)

- Ratio of PCE/NCE: Neither in the positive control group nor in the treated males a significant difference of PCE/NCE ratio compared to the control group is observed. In the females of the treatment groups no difference was observed 24 and 48 hours after treatment. After 72 hours the PCE/NCE ratio was decreased compared to vehicle control group but still was above the typically observed ratio of 1.0. (Tabels 1, 2 and 3)
- Appropriateness of dose levels and route: The selected dose level investigated as the maximum tolerable dose (MTD) induced clear toxic symptoms and was lethal in two animals. According to the guideline the recommended criteria for the dose level were fulfilled. The route of administration was appropriate as systemic availability could be demonstrated by clinical signs.

The statistically significant increase in frequency of micronuclei is considered to be of no biological relevance for the following reasons:

- The frequency of micronuclei at this sampling time point (0.18%) is not above the frequency of micronuclei of controls generally observed in this test laboratory (0.07 - 0.22%). The statistical significance in this case is caused by the low frequency of micronuclei in the control group (0.02%) which deviates downwards from the so far observed frequencies for controls. After addition of male and female animals of sampling time point 72 h into one group there is no more a statistically significant difference.

- A delayed effect based on slow excretion is improbable for sodium cumenesulphonate as sulphonic acids in general are readily absorbed and do not show any tendency for accumulation. But this kind of detention is regarded as prerequisite to explain based on the kinetic of erythrocyte maturation an impact on micronuclei frequency at sampling time point of 72 hours.

The results of the positive control affirm the sensitivity of the mouse strain to mutagenic substances.The frequency of micronuclei in polychromatic erythrocytes was considerably increased compared to the control group.

Table 1: Results of in vivo micronucleus test for male animals (mean ± standard deviation)

		Neg. control			test substance 4467 mg/kg bw			Pos. control
sampling time		24 h	48 h	72 h	24 h	48 h	72 h	24 h
micronuclei in 1000 PCE		0.8  ± 0.8	1.8  ± 0.8	0.4 ± 0.5	0.6 ± 0.5	0.8  ± 0,8	0.2  ± 0.4	48.0* ± 19.6
% PCE with micronuclei		0.08	0.18	0.04	0.06	0.08	0.02	4.8
PCE / NCE		1.0 ± 0.2	1.4 ± 0.3	1.6 ± 0.3	1.0 ± 0.3	1.1 ± 0.4	1.9 ± 0.9	0.8  ± 0.1

*p < 0.05

Table 2: Results of in vivo micronucleus test for female animal (mean ± standard deviation)

		Neg. control			test substance 4467 mg/kg bw			Pos. control
sampling time		24 h	48 h	72 h	24 h	48 h	72 h	24 h
micronuclei in 1000 PCE		1.4  ± 1.7	1.8  ± 1.1	0.2 ± 0.4	2.2 ± 1.1	1.2  ± 0,8	1.8*  ± 1.5	36.8* ± 9.8
% PCE with micronuclei		0.14	0.18	0.02	0.22	0.12	0.18	3.6
PCE / NCE		1.2 ± 0.2	1.4 ± 0.2	2.4 ± 0.6	1.0 ± 0.2	1.3 ± 0.3	1.3 ± 0.5	1.0  ± 0.1

*p< 0.05

Table 3: Results of in vivo micronucleus test for male + female animals (mean ± standard deviation)

		Neg. control			test substance 4467 mg/kg bw			Pos. control
sampling time		24 h	48 h	72 h	24 h	48 h	72 h	24 h
micronuclei in 1000 PCE		1.1  ± 1.3	1.8  ± 0.9	0.3 ± 0.5	1.4 ± 1.2	1.0  ± 0.8	1.0  ± 1.3	42.4* ± 15.7
% PCE with micronuclei		0.11	0.18	0.03	0.14	0.10	0.10	4.24
PCE / NCE		1.1 ± 0.2	1.4 ± 0.3	2.0 ± 0.6	1.0 ± 0.2	1.2 ± 0.4	1.6 ± 0.8	0.9  ± 0.1

* p < 0.05

Conclusions:

Interpretation of results (migrated information): negative
All male mice treated with the test substance showed no statistically significant increase in micronucleus frequency at any sampling time compared to control animals. For the female mice treated with the test substance at sampling times 24 and 48 hours after treatment also no statistically significant increase in micronucleus frequency was observed. Only at sampling time point 72 hours a statistically significant increase of polychromatic erythrocytes with micronuclei compared to control animals was observed. This effect was regarded as biologically not relevant as this increase ís based on the exceptional low micronucleus frequency of vehicle control group.
Sodium cumenesulphonate under these test conditions is regarded as not mutagenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are 5 different in vitro genetic toxicity assays and 2 different in vivo genetic toxicity assays conducted with hydrotrope substances, principally sodium xylene sulphonate. The key studies are as follows:

 

An in vitro bacterial cell gene mutation study (Ames Test) on sodium xylene sulphonate with four strains of Salmonella, all with and without S9 metabolic activation, positive and negative controls, and 5 exposure concentrations of the test substance. The genotoxicity results were negative for all conditions.

An in vitro mammalian cell gene mutation study (Mouse Lymphoma) on sodium xylene sulphonate with and without S9 metabolic activation, positive and negative controls, a DMSO vehicle, and 5 exposure concentrations of the test substance. The genotoxicity results were negative without activation and equivocal with activation (one replicate was positive and the 2nd replicate was negative). The study conclusion was "negative genotoxicity".

A mammalian cell DNA damage assay (Sister Chromatid Exchange in Chinese Hamster Ovary) on sodium xylene sulphonate with and without Aroclor 1254 activation, positive and negative controls, using a DMSO vehicle, and 5 exposure concentrations of the test substance. The genotoxicity results were negative with activation and positive without activation (clastogenic at doses of 2513 micrograms active per milliliter and higher). The study conclusion was not genotoxic with metabolic activation.

A mammalian cell chromosome aberration study on sodium xylene sulphonate in Chinese Hamster Ovary cells with and without Aroclor 1254 metabolic activation, positive and negative control, tested up to limit concentrations. The test material was not genotoxic in this assay.

An in vivo micronucleus study on sodium cumene sulphonate where mice were dosed by oral gavage at 4467 mg/kg bw. The study gave a negative result.

 

The other supporting studies also gave negative results.

Short description of key information:

There are 5 different in vitro genetic toxicity assays and 2 different in vivo genetic toxicity assays conducted with hydrotrope category substances, principally sodium xylene sulphonate. The assays are Ames Test, mouse lymphoma mammalian gene mutation, Chinese hamster sister chromatid exchange, Chinese hamster ovary mammalian chromosome aberration, in vivo mouse micronucleus bone marrow chromosome analysis, and in vivo mammalian erythrocyte micronucleus. All are guideline studies, conducted according to GLP requirements and fully documented. The conclusion from the battery of 7 different validated assays is the hydrotrope substances are not mutagenic or genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

On the basis of the negative results obtained across a range of in vitro and in vivo assays, there is no justification for classification.