Alkylbenzenesulphonic acid compound with alkanolamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test, HPRT test and chromosome aberration test in vitro are available on the representative for the amine part (Mortelmans 1986, Hazleton 1994, Hazleton 1994). All three tests are negative.

For the acid the Ames test and the chromosome aberration test in vitro were negative (MOE 2008 and MOE 2008). In vivo micronucleus testing on the acid was negative as well.

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:

other: the tested substance is the acid part 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 the acid part 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 the acid .

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: the tested substance is the acid part 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

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

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: the tested substance is the amine of the compound

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study

Qualifier:

according to guideline

Guideline:

other: EEC: Directive 97/69/EEC, of July 31, 1992 adapting to technical progress for the seventeenth time Council Directive 67/548/EEC, Annex V, Part B- Methods for the determination of toxicity, B.10- Mutagenicity(in vitro mammalian cytogenetic test), EEC Publi

Deviations:

no

Remarks:

None specified

Qualifier:

according to guideline

Guideline:

other: OECD: Guidelines for Testing of Chemicals, ISBN 92-64-12221-4, Paris Cedex 16, France (1983)

Deviations:

no

Remarks:

none specified

Qualifier:

according to guideline

Guideline:

other: U.S. EPA: Health effects testing guidelines, 40 CFR Part 798 (1990),

Deviations:

no

Remarks:

none specified

Qualifier:

according to guideline

Guideline:

other: Shirasu, Y. The Japanese mutagenicity guidelines for pesticide registration, Mutation Res..205:393-395, 1988.

Deviations:

no

Remarks:

None specified

Qualifier:

according to guideline

Guideline:

other: Scott, D., Dean, B.J.. Danford, N.D., and Kirkland, D.J. Metaphase chromosome aberration assays, in "Basic Mutagenicity Tests: U.K.EMS Recommended Procedures", (Ed: Kirkland, D.J.), Cambridge University Press, New York, NY, 1990.

Deviations:

no

Remarks:

None specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

chromosomal aberration

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

from Sprague Dawley rats

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from AROCLOR 1254 treated male SD rats

Test concentrations with justification for top dose:

0,78.1, 156, 313, 625, 1250, 2500 and 5000 ug/ml

Vehicle / solvent:

RPMI 1640

Untreated negative controls:

yes

Remarks:

vehicle used as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

mitomycin C was the positive control agent in the nonactivation series

Details on test system and experimental conditions:

In the chromosomal aberration assays replicate cultures were used at each dose level, negative control, and at each of the three doses of thepositive control. In the first set of aberration assays with and without metabolic activation, the cultures were harvested approximately 24 hoursafter the termination of treatment. The assays with and without metabolic activation were repeated independently with a 24-hour harvest and 48-hour harvest after the termination of treatment. Chromosomal aberrations were analyzed for the cultures treated at three doses and from only one of the positive control doses.Cultures were incubated in 25 cm2 culture flasks with loose caps at about 37 ± 2°C in a humidified incubator, in an atmosphere of about 5% ± 1.5% C02. The medium was RPMI 1640 supplemented with approximately 10% heat inactivated fetal bovine serum, gentamycin (25 ug/ml), L-glutamine (2 mM), and phytohemagglutinin (PHA-P) at a final concentration of about 0.012 mg/ml. HEPES buffer (25 mM) was also added to the culture medium. The total culture volume was approximately 5 ml.

Evaluation criteria:

Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 42 (except for veryheavily damaged cells, where a count could not be made) were analyzed. Mitotic index was assessed by analyzing the number of metaphases in1000 cells and was expressed as a percentage of cells analyzed. One hundred cells from each replicate culture at three (from the 24-hour harvest) and one (from the 48-hour harvest) dose levels of the test article and from the negative control cultures were analyzedfor the different types of chromosomal aberrations. At least 25 cells were analyzed for chromosomal aberrations from one of the positive control cultures. For control of bias, all slides except for the positive controls were coded prior to analysis. The following factors were taken into account in the evaluation of the chromosomal aberrations data:1. The overall chromosomal aberration frequencies.2. The percentage of cells with any aberrations.3 . The percentage of cells with more than one aberration.4. Any evidence for increasing amounts of damage with increasing dose, i.e. a positive dose response.Chromatid and isochromatid gaps, if observed were noted in the raw data and were tabulated. They were not, however, considered in theevaluation of the ability of the test article to induce chromosomal aberrations since they may not represent true chromosomal breaks andmay possibly be induced by toxicity.A cell classified as "GT" is considered to contain at least 5 aberrations for statistical purposes but a ">" is also included in the tables for this classification to indicate that it is a minimum number.An acceptable test has a chromosomal aberration frequency in the positive control significantly higher than negative controls. Negative controls should be within reasonable limits of the laboratory historical values. A test chemical is considered positive if it induces a significant, dose-related, and reproducible increase.

Statistics:

The descriptive and comparative statistics on cytogenetic aberrations consider each of the analyzed cells as an observational unit. At each dose level, data from replicates were pooled. A two-way contingency table was constructed to analyze the frequencies of cytogenetic abnormalities. An overall Chi-square statistic, based on the table was partitioned into components of interest. Specifically, statistics were generated to test the global hypotheses: 1. no difference in the average number of cells with aberrations among the dose groups, and 2. no linear trend of increasing number of cells with aberrations with increasing dose. An ordinal metric (0, 1, 2, etc. ) will be used for the doses in the statistical evaluation. If either statistic is found to be significant at alpha = 0.01, versus an one-sided increasing alternative, pairwise tests control vs treatment were performed at each dose level and evaluated at alpha = 0.01 again versus an one-sided alternative.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No significant increase in cells with aberrations was observed at any of the concentrations tested either in the initial assay or confirmatory assay. The positive control chemicals induced significantly higher incidences of abnormal cells.  Concentration CheckTrial I Trial II Target Observed Target Observedmg/ml mg/ml mg/ml mg/ml0 0 0 07.81 6.4 --- ---15.6 16.0 --- ---31.3 32.2 31.2 35.962.5 62.8 62.5 75.0125 122 125 129250 244 250 238500 477 500 498

Remarks on result:

other: all strains/cell types tested

DIPA was considered to be negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes.

Conclusions:

Interpretation of results (migrated information): considered to be negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes.

Executive summary:

The clastogenic potential of the test substance was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated for 4 hours in the presence and absence of an external metabolic activation system (S9) with 0 (negative control), 78.1,156,313,625,1250, 2500, and 5000 pg DIPA per ml of culture medium. Cultures treated with mitomycin C or cyclophosphamide were used as positive controls for the non-activation and activation assays, respectively. In the first experiment, cultures were harvested 24 h after termination of treatment. Based upon the mitotic indices, cultures treated with 0,313,625, and 1250 pg/ml in the absence of S9 and cultures treated with 0,1250,2500, and 5000 pg/ml in the presence of S9 were selected for determining the incidence of chromosomal aberrations. In a confirmatory assay utilizing 2 harvest times (i.e., 24 and 48 h after termination of treatment), the incidence of chromosomal abnormalities was determined from cultures treated with 0, 313, 625, and 1250 pg/ml in the absence of S-9 at the first harvest and from cultures treated with 0 and 1250 pg/ml at the second harvest. The incidence of chromosomal abnormalities was determined from cultures treated with 0, 1250, 2500, and 5000 pg/ml in the presence of S-9 at the first harvest time, and from cultures treated with 0 and 5000 pg/ml at the second harvest. No significant increase in cells with aberrations was observed at any of the concentrations tested either in the initial assay or confirmatory assay. The positive control chemicals induced significantly higher incidences of abnormal cells. Hence, the test substance was considered to be negative in the vitro chromosomal aberration assay utilizing rat lymphocytes.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: the tested substance is the amine part of the compound

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

none specified

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Deviations:

no

Remarks:

none specified

Qualifier:

according to guideline

Guideline:

other: EEC: In vitro mammalian cell gene mutation test, Official Journal of teh European Communities, L133, 31, 61-63.1988

Deviations:

no

Remarks:

none specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The hypodiploid CHO-Kl cell line was originally derived from the ovary of a female Chinese hamster (Cricetulus sriseus) (Kao and Puck, 1968). Characteristics of the cell line were high clonability (approximately 85%) and rapid doubling time (11-14 hours). The particular clone used in this assay was CHO-Kl-BH. The BH subclone of CHO-Kl cells, isolated by Dr. A.W. Hsie (Oak Ridge National Laboratory, Oak Ridge, Tennessee), has been demonstrated to be sensitive to many chemical mutagens.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver from AROCLOR 1254 treated male SD rats

Test concentrations with justification for top dose:

313, 625, 1250, 2500 and 5000 ug/ml

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

The cleansed cells were plated at about 3 x 10E6 cells per T-75 (75 cm2) tissue culture flask on the day before dosing. The time between plating and treatment was about 18 hours. Cell cultures were treated in duplicate with test or control material for four hours at 37°C ± 1.5°C in a humidified atmosphere with about 5% CO2. Treatment was performed in the absence of serum. Cell cultures normally contain at least 4 x 10E6 cells by the time of treatment termination. After treatment, the cell monolayers were washed twice with phosphate buffered saline, trypsinized, and suspended in culture medium. Cell suspensions from each replicate for each dose level were counted using a Coulter Counter and each was replated at 1.5 x 10E6 cells into each of two 150-mm dishes and at 200 cells into each of three 60-mm dishes. The small dishes were incubated for seven to eight days to permit colony development and the determination of the cytotoxicity associated with each treatment. The large dishes were incubated for seven days to permit growth and expression of induced mutations. The mass cultures were subcultured every two or three days during the expression period to maintain logarithmic cell growth. At each subculture the cells from the two 150-mm dishes from each replicate of each dose level were trypsinized, combined, counted and reseeded at about 1.5 x 10E6 cells into each of two 150-mm dishes. Replicates for each dose level were maintained independently throughout the experiment and cell pooling was only performed within replicates.

The assay treatment conditions consisted of two vehicle controls , two positive controls and five different test material dose levels using two cultures per dose level. Several treated cultures may be eliminated during the expression period as long as five appropriate dose levels are left for analysis of mutant induction. If the dose range resulted in excessively toxic treatments, as few as three dose may be carried through the mutation assay.

At the end of the expression period (seven days), each culture was reseeded at 2 x 10E5 cells per 100-mm dish (12 dishes total) in mutant selection medium. Also, three 60-mm dishes were seeded at 200 cells each in culture medium to determine the cloning efficiency of each culture. After incubation for seven to ten days, at 37°C ± 1.5°C in a humidified atmosphere with about 5% CO, the colonies were fixed with alcohol, stained with Giemsa and counted to determine the number of TG-resistant colonies in mutant selection dishes and the number of colonies in the cloning efficiency dishes. The colonies were counted by eye, excluding those with approximately 50 cells or less.

A second, third and fourth nonactivation mutation assay was initiated with duplicate cultures after the completion and analysis of the results of the first mutation assay. The assay procedures were identical to those used in the first nonactivation assay except that the dose range was modified in Trial IV based on the results from previous trials.

Evaluation criteria:

Observation of a mutant frequency that meets the minimum criteria for a positive response in a single treated culture within a range of assayed concentrations is not sufficient evidence to evaluate a test article as a mutagen. The following test results must be obtained to reach this conclusion for either activation or nonactivation conditions: 1) A dose-related or toxicity-related increase in mutant frequency should be observed. It is desirable to obtain this relation for at least three doses, but this goal depends on the concentration steps chosen for the assay and the toxicity at which mutagenic activity appears. 2) Within group replicates were pooled for final analysis. If replicates were not similar, this is noted in the report. 3) A mutagenic dose-response in one mutation assay should be confirmed in the second mutation assay. While it is desirable to confirm significant mutagenesis in both trials , it may not always be possible due to normal assay variation and also due to the possible use of different dose levels in the two trials. 4) For some test articles, the correlation between toxicity and applied concentration is poor. The proportion of the applied article that effectively interacts with the cells to cause genetic alterations is not always repeatable or readily controlled. Conversely, measurable changes in the frequency of induced mutants may occur with concentration changes that cause only small changes in observable toxicity. Therefore, either parameter, applied
concentration or toxicity (percent survival), can be used to establish whether the mutagenic activity is related to an increase in effective treatment.
and 5) Treatments that reduce relative clonal survival to less than five percent may be included in the assay but will not be used as sufficient
evidence for mutagenicity as it relates to risk assessment.

Statistics:

The frequencies of mutants per 10E6 clonable cells were analyzed by Cochran-Armitage test for trend and (except for the positive control in the second activation assay) the Fisher-Irwin exact test for group comparisons for proportions. The positive control for the first activation assay was analyzed using an approximate continuity corrected 1-tailed test because large numbers were involved that exceeded the capacity of the computer. Within-group comparisons were made by Fisher-Irwin exact test. Within-group replicates were pooled for final analysis. Both tests must be significant to consider a dose level positive. In addition, the mutant frequency must meet or exceed 15 x 10E6 in order to compensate for the random fluctuations in the background mutant frequency.

Key result

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

Additional information on results:

Range finding Cytotoxicity Testing

After the selection of water as a suitable vehicle, a wide range of test article concentrations was tested for cytotoxicity both with and without S9 metabolic activation. Ten concentrations that spanned a 3-log concentration range were used. The applied doses ranged from 9.77 pg/ml to 5000 pg/ml. In addition, one negative (media) control and one vehicle control containing 10% water were used in the cytotoxicity assay.

Cells were seeded at 3 x 10E6 cells per T-75 flask, allowed to attach overnight (16 to 18 hours) and exposed to the test or control article for four hours at 37°C ± 1.5°C in a humidified atmosphere containing about 5% CO2. After treatment, cell monolayers were washed, trypsinized, counted and replated at 200 cells into each of three 60 mm dishes. The cells were incubated in F12 culture medium for seven additional days to allow colony development. Colonies were then fixed in alcohol, stained with Giemsa and counted manually, excluding those with approximately 50 cells or less. Cytotoxicity was expressed as a percentage of colony counts in treated cultures versus control cultures.

These results were used to select doses for the mutation assay.

Concentration check
Trial I Trial II Trial III Trial IV

Target Actual Target Actual Target Actual Target Actual
mg/ml mg/ml mg/ml mg/ml
0 ND 0 ND 0 ND 0 ND
3.13 2.52 3.13 3.04 3.13 3.27 --- ---
6.25 4.96 6.25 5.65 6.25 7.05 6.25 6.96
12.5 12.4 12.5 12.2 12.5 13.3 12.5 13.2
25.0 24.6 25.0 24.4 25.0 25.8 25.0 29.4
50.0 50.0 50.0 43.2 50.0 52.3 50.0 50.3

CHO/HGPRT Mutation Assay Without Activation

               Trial 1         Trial 3          Trial 4
Conc.         Mutants/        Mutants/          Mutants/
 (ug/ml)      10-6 Cells      10-6 Cells       10-6 Cells
VC A            10.7             4.5              3.4
VC B             6.2             4.1              5.7

PC A**          43.0            51.7             45.9
PC B            38.9              *             107.8

313 A           5.1              5.9               --
313 B           6.8              4.4               --

625 A           3.5              1.6               2.9
625 B           8.7              3.3               1.2

1250 A          8.0              5.5               6.9
1250 B           *               5.6               5.1

2500 A          5.9              4.4               6.1
2500 B          4.2               *                8.8

5000 A           *               1.9               6.3
5000 B           *               6.5               9.6
 
* Plate lost due to contamination.
**Significant increase: Fisher-Irwin Exact Test (replicates combined) and mutant frequency > or = to 15 x 10-6
VC=vehicle control; PC=positive control


CHO/HGPRT Mutation Assay With Activation

               Trial 1         Trial 3          
Conc.         Mutants/        Mutants/          
 (ug/ml)      10-6 Cells      10-6 Cells       
VC A            21.6             4.6              
VC B             5.8             8.9              

PC A**         198.2           131.6             
PC B           154.4           153.0             

313 A            3.0             5.2               
313 B            6.0             2.7               

625 A            5.8             3.0               
625 B            7.6             8.7               

1250 A           5.1             3.2               
1250 B           2.5             7.9               

2500 A           5.7             8.5               
2500 B          17.5             3.9                

5000 A           12.7            0.4               
5000 B           9.3             6.4               
 

VC=vehicle control; PC=positive control
**Significant increase: Fisher-Irwin Exact Test (replicates combined) and mutant frequency > or = to 15 x 10-6

Four independent mutation assays were performed with the test material using nonactivation conditions. The second trial was unacceptable due to contamination. Trial 1 is acceptable overall but is flawed since the high dose was contaminated.

Three independent mutation assays were performed with the test material using activation conditions. The second trial was unacceptable due to contamination.

Based upon the frequency of the TG-resistant mutants recovered in cultures treated with the test material, it was concluded that DIPA did not induce a mutagenic response in the assay system employed.

Conclusions:

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

The amine part did not induce a muatagenic response in the assay system employed

Executive summary:

The test substance was evaluated in the in vitro Chinese hamster ovary cell/ hypoxanthine-guanine-phosphoribosyl transferase (CHO/ HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two separate assays. The test material was assayed at concentrations ranging from 313 to 5000 pg/ml in the absence and presence of an externally supplied metabolic activation (S-9) system. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, 5-bromo-2'-deoxyuridine for assays without S-9 and 20-methylcholanthrene for assays with S-9. Negative control cultures were treated with the solvent (water) used to dissolve the test material. Based upon the frequency of TGr mutants recovered in cultures treated with the test material, it was concluded that the test substance did not induce a mutagenic response in the assay system employed.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: the tested substance is the amine part of the compound

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: standard protocol, non GLP, tables available on results of the test substance

Qualifier:

according to guideline

Guideline:

other: standardized protocol of NTP

Principles of method if other than guideline:

according to Zeiger E, Drake JW (1980) An environmental mutagenesis test development program. In Monsanto R, Bartsch H, Tomatis L (eds): "Molecular and cellular aspects of carcinogen screening tests". Lyon, IARC Scientific Publications No. 27: 303-313 and Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E (1983) Salmonella mutagenicity test results for 250 chemicals. Environ Mutagen 5: 3-142

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of culture media: Oxoid No.2 broth
- Properly maintained: yes
No additional data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat and hamster metabolic systems

Test concentrations with justification for top dose:

All strains with (10% HLI and 10% RLI) and without S9: 0, 100, 333, 1000, 3330 and 10000 μg/plate

Vehicle / solvent:

H2O

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: sodium azide for TA 1535 and TA 100; 4-nitro-o-phenylenediamine for TA98; 9-aminoacridine for TA1537; 2-aminoanthracene for all tests with metabolic activation

Details on test system and experimental conditions:

preincubation assay, preincubation time: 20 min., incubation time: 48 h; 5 dose levels; 3 plate/dose level; all assays were repeated no less than 1 week after completion of the initial test

Evaluation criteria:

Mutagenic response: a dose-related, reproducible increase in the number over the background, even if the increase was less than twofold

Statistics:

according to the protocol (no more information available)

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:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary dose-setting experiments were made to establish toxicity; toxic chemicals were tested up to a high dose which exhibited some degree of toxicity

 TA100

Dose (ug/plate)	Without metabolic activation* (SEM)	With metabolic activation, hamster S9* (SEM)	With metabolic activation, rat S9* (SEM)
0	126 (2.4)	125 (5.6)	109 (8.9)
100	120 (7.8)	121 (7.5)	123 (4.1)
333	125 (5.1)	119 (9.3)	124 (4.1)
1000	129 (5.5)	100 (0.9)	123 (2.3)
3333	118 (7.2))	105 (8.0)	123 (8.4)
10000	112 (3.9)	127 (1.9)	128 (3.8)
Positive control	1250 (73.4)	1249 (68.2)	567 (58.5)

*: mean values; SEM: standard error of mean

 

TA1535

Dose (ug/plate)	Without metabolic activation* (SEM)	With metabolic activation, hamster S9* (SEM)	With metabolic activation, rat S9* (SEM)
0	27 (2.3)	11 (0.7)	14 (1.6)
100	27 (3.5)	11 (1.5)	10 (1.5)
333	25 (2.6)	7 (1.9)	8 (0.9)
1000	27 (0.9)	11 (0.9)	12 (2.2)
3333	28 (2.4)	9 9 (0.9)	10 (1.9)
10000	7 (2.9)	15 (0.6)	14 (1.5)
Positive control	7.34 (47.3)	161 (5.9)	151 (7.1)

*: mean values; SEM: standard error of mean

TA1537

Dose (ug/plate)	Without metabolic activation* (SEM)	With metabolic activation, hamster S9* (SEM)	With metabolic activation, rat S9* (SEM)
0	7 (0.3)	8 (1.2)	5 (1.3)
100	4 (0.7)	5 (0.9)	5 (2.0)
333	5 (1.0)	9 (0.3)	4 (1.3)
1000	4 (1.2)	7 (1.3)	8 (1.2)
3333	6 (3.6)	9 (1.7)	7 (2.0)
10000		5 (0.6)	7 (1.0)
Positive control	536 (34.7)	130 (3.8)	98 (6.8)

*: mean values; SEM: standard error of mean

TA98

Dose (ug/plate)	Without metabolic activation* (SEM)	With metabolic activation, hamster S9* (SEM)	With metabolic activation, rat S9* (SEM)
0	18 (2.8)	27 (3.0)	29 (0.9)
100	17 (1.8)	23 (3.3)	31 (1.7)
333	16 (2.1)	27 (0.3)	29 (1.5)
1000	18 (3.2)	25 (2.4)	27 (1.7)
3333	14 (0.9)	32 (2.0)	29 (2.1)
10000	14 (1.3)	28 (2.1)	25 (3.5)
Positive control	926 (22.7)	932 (31.7)	866 (14.9)

*: mean values; SEM: standard error of mean

 

Conclusions:

Not mutagenic in Salmonella typhimurium strains

Executive summary:

The amine part was tested for its mutagenic activity in the Salmonella/microsome assay in Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation; the substance showed no dose-related, reproducible increase in the number of revertants over background in any of the strains both with and without metabolic activation. The substance is considered not mutagenic in bacteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

For the acid and its sodium salt, in vivo micronucleus testing was negeative (1991 and 1976). A dominant lethal test on the sodium salt of the acid was negative as well.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Remarks:

the tested substance is a structural analogue of the salt

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method: 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:

not specified

Type of assay:

mammalian germ cell cytogenetic assay

Species:

rat

Strain:

other: Wistar and SD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan, Inc.
- Age at study initiation: 4 weeks
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

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

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): feed powder CE-2

Duration of treatment / exposure:

9 months

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0.9%, 450 mg/kg bw d
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes

Tissues and cell types examined:

femur bone marrow cells

Details of tissue and slide preparation:

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

Evaluation criteria:

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

Statistics:

Rohrborn's method.

Key result

Sex:

male

Genotoxicity:

negative

Negative controls validity:

valid

Additional information on results:

No increase in chromosome aberrations was noted.

Chromosome Aberrations

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

 

Conclusions:

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

Executive summary:

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

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Remarks:

the tested substance is a structural analogue of the salt

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method: Seven male mice received LAS in the diet for 9 months. Each of the male mice was then mated with 2 female mice that had not been given LAS. The pregnant mice were laparotomized on day 13 of gestation to determine the numbers of luteal bodies, implantations, surviving fetuses, and dead fetuses.

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

other: JCL-ICR

Sex:

male

Details on test animals or test system and environmental conditions:

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


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

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): feed powder CE-2

Duration of treatment / exposure:

9 months

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0.6%, 300 mg/kg bw d
Basis:
nominal in diet

No. of animals per sex per dose:

7

Control animals:

yes

Statistics:

Rohrborn's method.

Key result

Sex:

male

Genotoxicity:

negative

Negative controls validity:

valid

Additional information on results:

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

Dominant Lethal Assay Results

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

 

Conclusions:

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

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: the tested substance is the acid part 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 (migrated information): 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)

Justification for classification or non-classification

Based on the available information, there is no need to classify the substance according to CLP (Regulation EC No 1272/2008)