Dodecylbenzenesulphonic acid, compound with 2-amino-2-methylpropan-1-ol (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For dodecylbenzenesulfonic acid no mutations in the Salmonella typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and Escherichia coli (strain WP2 uvrA) were observed in the presence and absence of metabolic system.

AMP, under the conditions of this study, did not cause a positive response with TA 1535, TA 1537, TA 98 and TA 100 and E. coli (tester strain WP2uvrA) in the presence and absence of Aroclor-induced rat liver S9.

Dodecylbenzenesulfonic acid did not induce chromosomal aberrations at the concentration range tested in cultured CHL cells in the presence and absence of metabolic activation system.

The results of the L5178Y/TK+- Mouse Lymphoma Mutagenesis Assay indicate that, under the conditions of this study, AMP did not cause a positive response in the non-activated and S9-activated systems, and was concluded to be negative.

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

Remarks:

the tested substance is AMP one of the components of the compound

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline, GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Remarks:

Not specified in report

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

uvrA (for WP2 uvrA cultures) and uvrB gene ( for Salmonella cultures), TA-98- his D, TA-100- his G, TA-1535- his G, TA-1537- his C

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli (tester strain WP2uvrA)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used for the metabolic activation system.

Test concentrations with justification for top dose:

100, 333, 1000, 3333, 5000 ug per plate

Vehicle / solvent:

Water was selected as the solvent of choice. The test material was soluble in water at approximately 100ug.mL, the maximum concentration tested.

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: All strains, activated - 2-aminoanthracene ;WP2uvrA, nonactivated - methyl methanesulfonate; TA98, nonactivated - 2-nitrofluorene; TA100, TA1535, nonactivated - sodium azide; TA1537, nonactivated - 9-aminoacridine

Details on test system and experimental conditions:

Salmonella tester strains were recieved directly from Dr. Bruce Ames, University of California, Berkeley, and the E.coli was received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester TA1535 is reverted by mutagens that cause basepair substitutions. Tester TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E.coli is sensitive to basepair substitution mutations, rather than frameshift mutations.To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Each culture wasmonitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 10^9 cells per milliliter.Acoclor 1254-induced rat liver S9 was used for the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single IP injection of Acroclor 1254, 500mg/kg, five days prior to sacrifice. Each batch was assayed for its ability to metabolize.Positive controls, appropriate to the given tester strains, were used as follows:All strains, activated - 2-aminoanthraceneWP2uvrA, nonactivated - methyl methanesulfonateTA98, nonactivated - 2-nitrofluoreneTA100, TA1535, nonactivated - sodium azideTA1537, nonactivated - 9-aminoacridineThe concentration varied according to positive control compound. The sterility of the test article was verified.Preliminary Toxicity AssayTen dose levels of the test article were plated, one plate per dose, with overnight cultures of TA100 and WP2 uvrA on selective minimal agar in the presence and absence of rat liver S9 activation.Mutagenicity AssayAn initial and confirmatory assay was used to evaluate the mutagenic potential of the test material. A minimum of 5 dose levels of the test article, with vehicle and positive controls, were plated with tester strains in the presence and absence of rat liver S9 activation. All dose levels of test article, vehicle controls, and positive controls were plated in triplicate. The test systems were exposed to the test article via the plate incorporation methodology described by Ames, et.al.( (1975) and updated by Maron and Ames (1983). Plates were coded according to the testing laboratory's standard procedures. Plates were incubated for 48-72 hours at 35-39C. Plates not counted immediately following the incubation period were stored at 2-6C until the colonies could be counted.Toxicity and degree of precipation were scored relative to the vehicle control plate using a well-defined 9-point scale. Colonies were counted either by an automated colony counter or by hand unless the assay was preliminary or the plate exhibited toxicity. Plates exhibiting test article precipitate that interferes with an automated colony counter were counted by hand.

Evaluation criteria:

For a positive finding, the test material must cause a dose-related increase in mean revertants per plate of at least one tester strain with a minimum of 2 increasing concentrations of test article. Positive finding for TA1535 and TA1537 is an increase in mean revertants at the peak of dose-response greater than, or equal to, 3x mean control value. Positive finding for TA98, TA100, WP2 uvrA is an increase in mean revertants at the peak of dose-response greater than, or equal to, 2x mean control value.

Statistics:

Mean and standard deviation of the number of revertants per plate were calculated and reported.

Species / strain:

other: S. typhimurium (tester strains TA98, TA100, TA1535, TA1537) and E.coli (tester strain WP2 uvrA)

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:

Preliminary Toxicity AssayThe maximum dose tested was 5000ug/plate, achieved by using a concentration of 100mg/mL and a 50uL plating aliquot. Based on the findings, the maximum dose plated in the mutagenicity assay was 5000ug/plate. Neither precipitate nor appreciable toxicity was observed.Mutagenicity AssayNeither precipitate nor appreciable toxicity was observed in the initial or confirmatory assays. In neither the initial nor confirmatory assay was there a positive response with any of the tester strains at any dose level, in the presence or absence of S9 activation. The mean of each positive control exhibited at least a 3-fold increase in the number of revertants over the mean value of the respective vehicle control, indicating a valid test.

Conclusions:

Interpretation of result: :negativeThe results of this study indicate that, under the conditions of this study, the test material did not cause a positive response with any of the tester strains in the presence and absence of Acrclor-induced rat liver S9.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Remarks:

the tested substance is AMP one of the components of the compound

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline, GLP study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Not specified in report

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

0.5, 1.5, 5.0, 15, 50, 150, 500, 1500, 5000 ug/mL

Vehicle / solvent:

sterile distilled water

Untreated negative controls:

yes

Remarks:

sterile distilled water

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

Positive controls:

yes

Positive control substance:

other: Methyl methanesulfonate and 7,12 Dimethyl benz(a) anthracene

Details on test system and experimental conditions:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. A preliminary toxicity assay was used to establish the optimal dose levels for the mutagenesis assay. L5178Y cells were exposed to the vehicle alone and to concentrations of the test article ranging from 0.5-5000 uL in both the absence and presence of S9-activation. Cell population density was determined at 24 and 48 hours after exposure to the test article. Toxicity was measured as suspension of growth relative to the growth of the solvent controls. The mutagenesis assay, initial and independent repeat, was used to evaluate the mutagenic potential of the test article. L5178Y mouse lymphoma cells were exposed to the vehicle alone and 10 concentrations of the test article in both the absence and presence of S9. Positive controls, with and without S9, were tested concurrently. Methyl methanesulfonate was used as the positive control for the non-activated system and 7,12- Dimethyl-benz(a)anthracene was used as the positive control for the S-9 activated system.Tubes containing L5178Y/TK+- cells with F0P medium or S9 activation mixture, and 100uL of test solution at concentrations of 0.5, 1.5, 5.0, 15, 50, 150, 500, 1500, 5000 ug/mL were prepared. Positive controls treated with MMS and 7,12-DMBA were also prepared. Treatment tubes were gassed with CO2 and incubated in the dark for 4 hours. Cells were washed twice, resuspended, gassed with CO2 in air again, and placed on a roller. Cells were counted, and numbers were adjusted if necessary. For expression of TK+- cells, cells were placed in a cloning meduim of granulated agar, mixed with Trifluorothymidine (TFT) or viable count, prewarmed and filled with cloning media. The tubes were centrifuged, supernatant decanted, and cells resuspended in cloning media. Tests were conducted in duplicate. TFT or Viable Count were added to the flask and placed on a shaker. The suspension was placed in petri dishes, placed in cold storage for 30 minutes, then incubated for 10-14 days.Diameters and numbers of colonies were recorded, and analyzed relative to positive and solvent controls. Previous published data indicates that the large colony mutants received localized damage, likely in the form of a point mutation or small deletion within the TK locus, and small colony mutants received damage to collateral loci concordant with the loss of TK activity.

Evaluation criteria:

The result was considered to be a positive repsonse if a concentration-related increase in mutant frequency was observed and one or more dose levels with 10% or greater total growth exhibit mutant frequencies of > or= 100 mutants per 10exp6 clonable cells over background level. A result was considered equivocal if the mutant frequency in treated cultures was between 55 and 99 mutants per 10exp6 clonable cells over background level. Test articles producing fewer than 55 mutants per 10exp6 clonable cells over the background level were concluded to be negative.

Statistics:

Means and standard deviations

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:

Sterile distilled water was the solvent of choice. The test material was miscible in water at 500 mg/mL, the maximum tested concentration.Preliminary Toxicity AssayThe high dose tested was 5000ug/mL. Osmolality of the solvent control was 300mmol/kg and of the highest dose was 396mmol/kg. Suspension growth relative to the solvent control was 13% at 5000ug/mL without S9 activation, and 31% with S9 activation. Based on these results, the dose levels chosen for the mutagenesis assay ranged from 500-5000ug/mL for both the non-activated and S9-activated cultures.Mutagenesis AssayIn the non-activated system, cultures treated with 1000-500ug/mL were cloned and produced a range in suspension growth of 11-55%. In the S9-activated system, cultures treated with 500-5000ug/mL were cloned and produced a range in suspension growth of 9-98%.No treated cultures exhibited mutant frequencies more than 55 mutants per 10^6 clonable cells over the solvent control. A dose-response trend was not observed in either the activated or non-activated systems. Over the concentration ranges, total growth for the non-activated cultures ranged from 12-60%, and 10-137% for the S9-activated cultures. Results of the independent repeat assay are similar (12-62% and 32-124% for non-activated and S9-activated system suspension growth, respectively; 14-67% and 29-191% for non-activated and S9-activated system total growths, respectively). There was likewise no increase in mutant frequencies over the solvent control. Colony sizing for the positive control yielded the expected increase in small colonies, thereby verifying the methods used to detect small colony mutants.

Conclusions:

Interpretation of results: negative
The results of the L5178Y/TK+- Mouse Lymphoma Mutagenesis Assay indicate that, under the conditions of this study, 2-amino-2-methyl-1-propanol did not cause a positive response in the non-activated and S9-activated systems, and was concluded to be negative.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Remarks:

the tested substance is DDBSA one of the components of the compound

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

- Species and cell type: Rat (Sprague Dawley strain), male, liver - Quantity: 5 % S9 mix induced with Aroclor 1254

Test concentrations with justification for top dose:

- TA 98: 23.44, 46.88, 93.75, 187.5, 375, 750, 1,500 μg/plate (-)
- TA 98: 5.86, 11.72, 23.44, 46.88, 93.75, 187.5, 375, 750, 1,500, 3,000 μg/plate (+)
- TA 100: 5.86, 11.72, 23.44, 46.88, 93.75, 187.5 μg/plate (-)
- TA 100: 23.44, 46.88, 93.75, 187.5, 375, 750 μg/plate (+)
- TA 1535: 5.86, 11.72, 23.44, 46.88, 93.75, 187.5 μg/plate (-)
- TA 1535: 23.44, 46.88, 93.75, 187.5, 375, 750 μg/plate (+)
- TA 1537: 5.86, 11.72, 23.44, 46.88, 93.75, 187.5 μg/plate (-)
- TA 1537: 23.44, 46.88, 93.75, 187.5, 375, 750 μg/plate (+)
- WP2uvrA: 5.86, 11.72, 23.44, 46.88, 93.75, 187.5 μg/plate (-)
- WP2uvrA: 23.44, 46.88, 93.75, 187.5, 375, 750, 1500 μg/plate (+)

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

yes

Remarks:

Dimethylsulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide

Positive controls:

yes

Positive control substance:

other: 2-Nitrofluorene, Sodium azide, 9-Aminoacridine, 4-Nitroquinoline 1-oxide ,2-Aminoanthracene and Benzo(a)pyrene

Details on test system and experimental conditions:

- Number of replicated: 3 plates/dose - Dose range finding experiment was carried out using dose levels of 8, 40, 200, 1,000, 3,000 and 5,000 μg/plate both in the absence and in the presence of metabolic activation system.- Cytotoxicity: A preliminary toxicity test was performed to define the concentrations to be used for the mutagenicity study.

Evaluation criteria:

The assay was considered valid if the number of spontaneous revertant colonies in vehicle control plates falls within the normal range and the positive control chemicals induce significant increases in the number of mutagen-induced revertant colonies compared to vehicle control. It was judged to be positive if there was a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without the metabolic system. In addition, it was judged to have a toxic effect (antibacterial effect) when a clearing or diminution of background lawn, the appearance of micro-colonies, and/or thedecrease more than 50% in the number of colonies compared to that of vehicle control was observed.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥375 μg/plate (+) (TA 100, TA 1535, TA 1537) ≥93.75 μg/plate (-) (TA 100, TA 1535, TA 1537) ≥1500 μg/plate (+) (TA 98) ≥375 μg/plate (-) (TA 98)

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥750 μg/plate (+) (WP2 uvr ) 1500 μg/plate (+) (WP2 uvr )

Bacterial tests

A bacterial reverse mutation test was performed in accordance with [OECD TG 471] using Salmonella typhimurium (strains TA98,TA100, TA1535 and TA 1537) and Escherichia coli (strain WP2uvrA) with and without a metabolic activation system. Test doses were as follows:

	5.86, 11.72, 23.44, 46.88, 93.75, 187.5, 375, 750, 1,500, 3,000μg/plate (+/-)

 

Dimethylsulfoxide (DMSO) was used as a vehicle and Sodium azide (SA), 2-Nitrofluorene (2-NF), 9-Aminoacridine (9-AA), 4-Nitroquinoline 1-oxide (4NQO),2-Aminoanthracene (2-AA)andBenzo(a)pyrene (BP)were used as positive control item.In all strains used, there was no increase in the number of revertant colonies compared to the vehicle control at any concentration of dodecylbenzenesulfonic acid either in the presence or absence of metabolic activation system. However, in the first and second experiment, it was observed a clearing or diminution of background lawn, the appearance of micro-colonies, and/or more than 50% decrease in the number of colonies at more than 375 μg/plate and 93.75 μg/plate in TA100, TA1535 and TA1537 strains in the presence and absence of S9 activation, respectively. Also, in TA98 strain, it was observed a clearing or diminution of background lawn, the appearance of micro-colonies, and/or more than 50% decrease in the number of colonies at more than 1500 μg/plate and 375μg/plate in the presence and absence of S9 activation, respectively. InE. coliWP2uvrA strain, the more than 50% decrease in the number of colonies was observed at more than 750μg/plate and at 1500 μg/plate in the presence of S9 activation in the first and second experiment, respectively. The positive controls induced a significant increase in the numbers of revertant colonies indicating the assay was valid.

 

Conclusions:

Interpretation of results: negative

In the presence and absence of metabolic system, no mutation in the Salmonella typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and Escherichia coli (strain WP2 uvrA) occurred with dodecylbenzenesulfonic acid .

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Remarks:

the tested substance is DDBSA one of the components of the compound

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

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:

mammalian cell line, other: Chinese Hamster Lung (CHL)

Metabolic activation:

with and without

Metabolic activation system:

- Species and cell type: Rat (Sprague Dawley strain), male, liver - Quantity: S9 mix induced with Aroclor-1254

Test concentrations with justification for top dose:

- 6hr (+S): 60, 62, 66, 67, 68 μg/mL- 6hr (-S): 70, 72, 74, 76, 78 μg/mL- 22hr (-S): 60, 70, 72, 73, 74 μg/mL

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

yes

Remarks:

Dimethylsulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide

Positive controls:

yes

Remarks:

Cyclophosphamide monohydrate(CPA), ethylmethanesulfonate (EMS)

Remarks:

Positive controls were prepared in sterile distilled water. 100 fold Stock solutions of CPA and EMS were stored at –20 deg. C

Details on test system and experimental conditions:

- Number of replicated: 2 plates/dose
- Culture establishment; The cells were incubated at 37+/- 1 deg C in 5 % CO2 atmosphere. Cells were subcultured twice weekly. Rapidly growing cell cultures were trypsinized, suspended in culture medium and counted. Three series (series I, II and III) of 25 cm^2 culture flask (Falcon) were seeded with 4x10^4 cells each in 5 mL medium and incubated for 3 days before treatment.
- Number of metaphases analyzed: 100
- Criteria for scoring aberrations: Structural- A hundred metaphases that were well spread and had a chromosome count between 23~27 were evaluated for aberrations. The microscope stage co-ordinates were recorded for each aberrant metaphase. Each type of aberration was recorded, and the number of aberrant metaphases (showing one or more aberrations, including/excluding gaps) and total aberrations (including/excluding gaps) were calculated.Numerical-Regardless of the presence of aberrations, an additional 100 metaphases were examined to determine the frequency of diploid (DP), polyploid (PP, ≥ 37 chromosomes), and endoreduplication (ER).

Evaluation criteria:

The result of the study was judged as positive if there was a concentration-related increase or a clear, reproducible and statistically significant increase in the number of cells with chromosome aberrations.

Statistics:

The statistical analyses, according to Richardson et al. (1989), were done using Statistical Analysis System (SAS) program (version 8.2, SAS Institute Inc., Cary,NC). The number of aberrant metaphases (excluding gaps, according to OECD guideline) and number of (PP+ER) were analyzed. Differences were regarded as statistically significant, if P<0.05.1) The vehicle control and test article-treated groups: After carrying out χ2-test, performed Fisher's exact test, if P<0.05.2) Test for dose-response: Cochran-Armitage trend test.3) The vehicle and positive control groups: Fisher's exact test.

Key result

Species / strain:

mammalian cell line, other: Chinese Hamster Lung (CHL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Additional information on results:

In the presence and absence of metabolic system, no significant increase in the number of aberrant metaphase including structural and numerical aberrations in the Dodecylbenzenesulfonic acid-treated groups.

Conclusions:

Interpretation of results: negative

Dodecylbenzenesulfonic acid did not induce chromosomal aberrations at the concentration range tested in cultured CHL cells under the present experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A single dose of DDBSA was administered to mice by gavage at 1122 mg/kg (LAS Acid) and evaluated for chromosome aberrations. 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.

AMP did not induce a significant increase in the incidence of micro-nucleated polychromatic erythrocytes in bone marrow, and was concluded to be negative in the micronucleus test using male and female ICR mice (single dose at 16, 30 and 60 mg/kg bw).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Remarks:

the tested substance is AMP one of the components of the compound

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline, GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Remarks:

Not specified in report

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

ICR mice from Harlan Sprague-Dawley, INC, were 6-8 weeks old at study initiation. For the pilot, toxicity, and MNT assay, males ranged in weight from 27.4g to 31.9g, and females ranged from 23.0g to 28.0g. After arrival to the lab, the animals were monitored for parasites and infections, and were quarantined a minimum of 5 days. They were observed daily for general health, food and water consumption patterns, and other conditions. The animals were deemed healthy prior to placement on study.Mice were housed in an AALAC-accredited facility with appropriate temperature, humidity, and photocycle for the species. They were housed up to 5 per cage, and given food and water ad libitum. The feed and water contained no contaminants that influenced the study.The mice were randomized and placed into assigned dose groups, and were identified by a uniquely numbered ear tag.

Route of administration:

intraperitoneal

Vehicle:

water

Details on exposure:

In each the Pilot & Toxicity Studies (designed to set appropriate dose levels for the micronucleus assay), and the Micronucleus Assay, mice were weighed immediately prior to dose administration and the dose volume was based on individual body weights. Mice were given a single IP injection of the test article in water, the vehicle alone, or the positive control substance.Pilot Study: 2 male mice were dosed with 1, 10, 100, or 1000 mg test material /kg body weight, and 5 males and 5 females were dosed with 2000 mg/kg. Toxicity Study:5 male and 5 female mice were dosed with 200, 400, 600, 800 mg/kg

Duration of treatment / exposure:

Single IP injection

Frequency of treatment:

Once

Post exposure period:

24 and 48 hours

Remarks:

Doses / Concentrations:200, 400, 600, 800 mg AMP/kgBasis:nominal conc.

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes, cyclophosphamide

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

At the scheduled sacrifice times (24 and 48 hours), up to 5 mice / sex / dose were sacrificed, the femurs exposed, and bone marrow aspirated into a syringe of fetal bovine serum. The bone marrow cells were pelleted by centrifugation and the supernatant drawn off. The cells were resuspended and a small amount of suspension spread onto a glass slide. Two to four slides were prepared per animal. The slides were fixed in methanol, stained, and permanently mounted. Slides were coded and scored blindly. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:

The test article was considered to induce a positive response if a dose-responsive increase in mocronucleated polychromatic erythrocytes was observed and one or more doses were statistically-elevated relative to the vehicle control (p>0.05) at any sampling time.  If a single dose group was significantly elevated without a dose-response trend, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended.  The test article would be considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

Statistics:

Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical signs noted on the day of dosing included: lethargy in males and females at 60 mg/kg (highest dose).

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Pilot:Mortality occurred within 4 hours following dosing: 5/5 males and 5/5 females at 2000 mg/kg, and 2/2 males at 1000 mg/kg. There were no clinical observations noted.Toxicity:Mortality occurred within 3 days of dosing: 5/5 males and 5/5 females at 400, 600, and 800 mg/kg, and 3/5 males and 5/5 females at 200 mg/kg. Clinical signs included: lethargy in males and females at all dose levels, piloerection in males and females, and crusty eyes in males at 200 mg/kg. The LD50/3 was calculated by probit analysis to be approximately 73.7 mg/kg for male and female mice. Micronucleus Assay:There was no treatment-related mortality at any dose level. Clinical signs noted on the day of dosing included: lethargy in males and females at 60 mg/kg (highest dose). All other mice treated with test material and control articles appeared normal during the study. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test article-treated groups was not statistically increased relative to their respective vehicle control in either male of female mice, regardless of dose level or bone marrow collection time.

Conclusions:

Interpretation of results: negative
Under the conditions of the assay described in this report, 2-amino-2-methyl-1-propanol did not induce a significant increase in the incidence of micro-nucleated polychromatic erythrocytes in bone marrow, and was concluded to be negative in the micronucleus test using male and female ICR mice.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Remarks:

the tested substance is DDBSA one of the components of the compound

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

not specified

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

Details on exposure:

n this study, 40 male and 40 female mice were given a single oral dose by gavage of 1122 mg/kg LAS Acid (read across) and evaluated for chromosome aberrations. Only a single dose has been evaluated which was in the range of the acute oral LD50 value for LAS Acid in rats (LD50 = 1470 mg/kg). Furthermore, slight cytotoxicity has been observed after 48 hours.

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

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

Conclusions:

Interpretation of results: negative
No significant increases in the number of polychromatic erythrocytes with micronuclei were observed.

Executive summary:

 In this study, 40 male and 40 female mice were given a single oral dose by gavage of 1122 mg/kg LAS Acid (read across) and evaluated for chromosome aberrations. Only a single dose has been evaluated which was in the range of the acute oral LD50 value for LAS Acid in rats (LD50 = 1470 mg/kg). Furthermore, slight cytotoxicity has been observed after 48 hours. No statistically significant or biologically relevant increases in the number of polychromatic erythrocytes with micronuclei were observed; therefore the test material is considered negative for cytogenicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a weight of evidence approach based on in vitro and in vivo data on the components of the substance, it is concluded that the substance is not expected to show mutagenic effects.

Justification for classification or non-classification

Based on the data on the components the substance does not need to be classified according to CLP (Regulation EC No 1272/2008).