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Genetic toxicity in vitro

Description of key information

Two in vitro studies (Klimisch score 1) are available (Ames test and Chromosome aberration test). A third in vitro test, a mouse lymphoma assay (Klimisch score 1), was performed with a substance analogue and the result is read across to the registered substance.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 21, 2000 - July 19, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine (S. typhimurium) and tryptophan (E. coli)
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:
Rat liver S9-mix induced by phenobarbitone and betanaphthoflavone
Test concentrations with justification for top dose:
Concentrations were expressed in terms of material as received (aqueous solution)
Toxicity test: 50, 158, 500, 1580 and 5000 µg/plate
Experiment 1, plate incorporation method: 313, 625, 1250, 2500 and 5000 µg/plate (TA1535, TA1537, TA98 and WP2 uvrA) and 156, 313, 625, 1250 and 2500 µg/plate (TA100)
Experiment 2, preincubation method: 313, 625, 1250, 2500 and 5000 µg/plate (TA1535, TA1537, TA98 and WP2 uvrA) and 156, 313, 625, 1250 and 2500 µg/plate (TA100)
Experiment 3, preincubation method: 39.1, 78.1, 156, 313, 625 and 1250 µg/plate (TA1535 and TA1537) and 39.1, 78.1, 156, 313 and 625 µg/plate (TA100 without S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: Water is regarded as compatible with the Ames test. The minimum required solubility for the test article of 5% (corresponding with 5000 μg/plate) was reached.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: See section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: First experiment: in agar (plate incorporation); second and third experiment: preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: Toxicity test single plate, main experiments three replicate plates

DETERMINATION OF CYTOTOXICITY
- Method: the number of revertants and the background growth

Evaluation criteria:
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. The effect must be reproduced in an independent experiment.
Statistics:
The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions.
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:
See section Additional information on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See section Additional information on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING STUDY:
Moderate toxicity, as indicated by reduction in revertant numbers and thinning of the background lawn, was observed in TA1535 and TA1537 at the highest dose-level tested, both in the absence and presence of S9 metabolic activation. A more pronounced effect was observed in TA100. Reduction in revertant colonies was observed at the two highest concentrations in WP2uvrA, in the absence of S9 metabolism.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: slight toxicity, as indicated by thinning of the background lawn, was observed at the highest dose level in strain TA1535, TA1537 and TA100.
Experiment 2: moderate thinning of the background lawn was observed at the highest dose-level with TA98 and WP2uvrA tester strains, while marked toxicity was observed with TA1535, TA1537 and TA100 tester strains at most of the concentrations tested.
Experiment 3: toxicity, as indicated by reduction in revertant numbers and thinning of the background lawn, was observed at the highest dose-level only in strain TA1535, TA1537 and TA100, both in the absence and presence of S9 metabolism.
Conclusions:
Based on the outcome of a Salmonella typhimurium reverse mutation assay and an Escherichia coli reverse mutation assay, it is concluded that the test item is not mutagenic with or without metabolic activation.
Executive summary:

The mutagenic potential of the test material was assessed in Salmonella typhimurium bacterial strains and Escherichia coli , according to EC method B.13-B.14 and modified OECD guideline 471, and in compliance with Good Laboratory Practice.

The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98, TA 100 and tryptophan-requiring E.coli mutant WP2uvrA were used in the presence and the absence of metabolic activation (+/- S9 -mix). All determinations were made in triplicate. The first experiment was conducted using a plate-incorporation method at 5 doses levels up to 5000 µg/plate. The second and third experiment were performed using a pre-incubation method. Simultaneously negative (solvent) and positive controls were used in all experiments.

In a preliminary range-finding assay, cytotoxicity was seen.

In the plate-incorporation assay, 4 strains were treated at dose levels 313 to 5000 µg/plate, while TA100 was treated at 156 to 2500 µg/plate. Slight toxicity was observed at the highest dose level with TA1535, TA1537 and TA100, but there was no increase in the revertant numbers in any of the strains tested.

The second assay was performed with a pre-incubation step and using the same dose-range. Moderate thinning of the background lawn was observed at the highest dose level with TA98 tester strain, while marked toxicity was observed with TA1535, TA1537 and TA100 at the 3 highest dose levels. A third experiment was then performed with these 3 strains using a lower dose-range (39.1- 625 µg/plate without S9-mix, and 78.1-1250 µg/plate only for TA1535 and TA1537 in the presence of S9 -mix).

The test material did not cause any two-fold or greater increase in the mean number of revertant colonies in the test plates, both in the absence and the presence of metabolic activation. Positive controls gave the expected increases in the number of revertants, with and without S9 mix.

Under the conditions of this study, the test material did not demonstrate any in vitro mutagenic activity in these bacterial test systems.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
A mouse lymphoma assay was conducted with Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) in accordance with OECD 476 and according to GLP principles. It is concluded that the chemical is not mutagenic in the TK mutation test system. This result is read across to the registered substance.
Executive summary:

A mouse lymphoma assay was conducted with Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) in accordance with OECD 476 and according to GLP principles. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) did not induce a significant increase in the mutation frequency in the first experiment.

This result was confirmed in an independent experiment with modifications of the S9-mix concentration added for metabolic activation. In conclusion, the chemical is not mutagenic in the TK mutation test system. This result is read across to the registered substance.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October, 31 2011 - March 9, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
other: International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Federal Register 61:18198-18202, April 24, 1996 (S2A document recommended for adoption at step 4 of the ICH process)
Deviations:
no
Qualifier:
according to
Guideline:
other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Federal Register 62:16026-16030, November 21, 1997 (S2B document recommended for adoption at step 4 of the ICH process)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes (HPBL)
Details on mammalian cell type (if applicable):
Peripheral blood lymphocytes were obtained from a healthy non-smoking 24-year-old adult female on 01 November 2011 for the preliminary toxicity assay and from the same donor on 15 November 2011 for the definitive assay and 03 January 2012 for the confirmatory assay. The donor had no recent history of radiotherapy, viral infection or the administration of drugs.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
The dosing preparations were adjusted for the surfactant content of the substance, using a correction factor of 3.3. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light.

Expressed as surfactant content:

Dose range finding test:
Without S9-mix, 4 hr exposure; 20 hr fixation: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/mL
Without S9-mix, 20 hr exposure; 20 hr fixation: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/mL
With S9-mix, 4 hr exposure; 20 hr fixation: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/mL

First cytogenetic test (definitive assay):
Without S9-mix, 4 hr exposure; 20 hr fixation: 30, 65, 130, 146, 162, 190, 200 and 250 µg/mL
Without S9-mix, 20 hr exposure; 20 hr fixation: 30, 65, 130, 146, 162 and 190 µg/mL
With S9-mix, 4 hr exposure; 20 hr fixation: 30, 65, 130, 146, 162, 190, 200 and 250 µg/mL

Second cytogenetic test (confirmatory assay):
With S9-mix, 4 hr exposure; 20 hr fixation: 30, 65, 125, 140, 155, 170, 185 and 200 µg/mL

Note: expressed as solid content the tested concentrations need to be multiplied by a factor of 1.27
Vehicle / solvent:
- Vehicle used: water (obtained from Gibco, Lot no. 1029809)
- Justification for choice of vehicle: Test compound was stable in water and soluble in culture medium (up to 50 mg/mL)
Negative solvent / vehicle controls:
yes
Remarks:
(water)
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 44-48 hr
- Exposure duration: 4 hr (with and without S9-mix), 20 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hr
SPINDLE INHIBITOR (cytogenetic assays): Colcemid®
STAIN: Giemsa
NUMBER OF REPLICATIONS: duplicates in two independent experiments
NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 500 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test substance would be considered to induce a positive response when the percentages of cells with aberrations were increased in a dose-responsive manner, with one or more dose levels being statistically significant and clearly outside the historical solvent control data (p ≤ 0.05). However, values that are statistically significant and fall within or just outside the range of historical solvent control values may be judged as not biologically significant. Test substances not demonstrating a statistically significant increase in aberrations would be concluded to be negative. In the event of a positive response only at the high dose level with at least 50% reduction in cell growth relative to the respective solvent control and no evidence of dose response in one or more treatment conditions, the test substance will be considered to induce positive response at a cytotoxic dose level.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Fisher's Exact test. Fisher's Exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's Exact test at any test substance dose level, the Cochran-Armitage test was used to measure dose-responsiveness.
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes (HPBL)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/mL
- Effects of pH: No. The pH of the highest dose level of test substance in treatment medium was approximately 7.5.
- Effects of osmolality: Yes, the osmolality in treatment medium of the highest dose level tested, 5000 μg/mL, was 292 mmol/kg.
The osmolality of the solvent (water) in the treatment medium was 236 mmol/kg. The osmolality of the test substance dose level in the treatment medium exceeded the osmolality of the solvent by more than 20%. The osmolality in treatment medium of the second highest dose level, 1500 μg/mL was measured (270 mmol/kg) and was found to be acceptable.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 500 µg/ml and above in the absence and presence of S9, 4 hr treatment/20 hr fixation and at dose levels of 150 µg/ml and above in the absence of S9 for the continuous treatment of 20 hr

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures were within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

Definitive Chromosome Aberration Assay

- Cytogenetic analysis of the non-activated 4-hour exposure group: At the highest test dose level evaluated microscopically for chromosome aberrations, 250 μg/mL, mitotic inhibition was 55%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 65, 146 and 250 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) group was statistically significant (16.0%) (p ≤ 0.01, Fisher's Exact test).

- Cytogenetic analysis of the S9 -activated 4 -hour exposure group: At the highest test dose level evaluated microscopically for chromosome aberrations, 162 μg/mL, mitotic inhibition was 51%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 30, 65 and 162 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) group was statistically significant (12.0%) (p ≤ 0.01, Fisher's Exact test).

- Cytogenetic analysis of the non-activated 20-hour exposure group: At the highest test dose level evaluated microscopically for chromosome aberrations, 146 μg/mL, mitotic inhibition was 58%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 30, 65 and 146 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) group was statistically significant (12.0%) (p ≤ 0.01, Fisher's Exact test).

Confirmatory Chromosome Aberration Assay

- Cytogenetic analysis of the S9-activated 4-hour exposure group: At the highest test dose level evaluated microscopically for chromosome aberrations, 155 μg/mL, mitotic inhibition was 48%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 65, 125 and 155 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) group was statistically significant (20.0%) (p ≤ 0.01, Fisher's Exact test).

Conclusions:
A chromosome aberration study with the substance was performed according to OECD 473 guideline and GLP principles, in peripheral human lymphocytes in two independent experiments with and without metabolic activation. It is concluded that the substance is not clastogenic in human lymphocytes.
Executive summary:

In accordance with OECD 473 (1998), EU Method B.10 (2008) and according to GLP principles, a chromosome aberration study with the substance (dissolved in water) was performed in human peripheral blood lymphocytes in two independent experiments with and without metabolic activation. The dosing preparations were adjusted for the surfactant content of the substance and were set based on the observed toxicity in the dose range finding study (no precipitation was observed up to and including the top dose of 5000 µg/mL).

In the first cytogenetic assay, the cells were treated for 4 hr (with and without S9-mix) and for 20 hr (without S9-mix) with the substance at doses ranging from 30 to 250 µg/mL. In the second cytogenetic assay, the cells were treated for 4 hr (with S9 -mix) with the substance at doses ranging from 30 to 200 µg/mL.

Toxicity was reached at the dose levels selected for scoring. In all exposure groups, the percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Based on the results of this study, it is concluded that the substance does not disturb mitotic processes and cell cycle progression and is not clastogenic in human lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-May-2010 to 13-Jul-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Species strain
- Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 100, 333, 1000, 3330 and 5000 µg/mL
Without S9-mix, 24 hours treatment: 100, 333, 1000, 3330 and 5000 µg/mL and 0.3, 1, 3, 10 and 33 µg/mL
Experiment 1 (doses selected for measurement of mutation frequency):
Without S9-mix, 3 hours treatment: 0.1, 1, 5, 10, 33, 50, 75 and 100 µg/mL
With (8% (v/v) S9-mix, 3 hours treatment: 33, 100, 125, 150, 175, 200 and 225 µg/mL
Experiment 2 (doses selected for measurement of mutation frequency):
Without S9-mix, 24 hours treatment: 0.08, 0.8, 4, 8, 17, 25, 34 and 42 µg/mL
With (12% (v/v) S9-mix, 3 hours treatment: 4, 42, 84, 125, 146, 168, 210 and 230 μg/mL
(additional concentrations were not evaluated as there was a sufficient number of analysable concentrations and toxicity at higher doses)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI 1640 culture medium
- Justification for choice of solvent/vehicle: Test compound was soluble in RPMI 1640 culture medium and this has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Positive control substance:
cyclophosphamide
Remarks:
with S9: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 100 µg/mL and higher in the absence of S9, 3 hours treatment; at dose levels of 333 µg/mL and higher in the presence of S9, 3 hours treatment; at dose levels of 100 µg/mL and higher in the absence of S9, 24 hours treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 84 and 90% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 89 and 92% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment, respectively.

In the first experiment in the presence of S9-mix (3h treatment), there were only 7 analysable concentrations obtained, due to toxicity. However these conditions were repeated in the second experiment where 8 analysable concentrations were evaluated. This slight deviation was not considered to alter the study integrity.

In the second experiment in the presence of S9-mix, the recommended toxic range of approximately 10-20% RTG was not covered in the conditions with S9-mix. But the RTG was 8%, and a mutation frequency similar to that of the next inferior dose within a very narrow dose range, and not more than 2-fold compared to the negative control. The test conditions were similar to those of the first experiment where an appropriate RTG range was obtained, except for the metabolic activation concentration which was higher in experiment 2.
Conclusions:
A mouse lymphoma assay was conducted with Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) in accordance with OECD 476 and according to GLP principles. It is concluded that the chemical is not mutagenic in the TK mutation test system.
Executive summary:

A mouse lymphoma assay was conducted with Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) in accordance with OECD 476 and according to GLP principles. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) did not induce a significant increase in the mutation frequency in the first experiment.

This result was confirmed in an independent experiment with modifications of the S9-mix concentration added for metabolic activation. In conclusion, thee chemical is not mutagenic in the TK mutation test system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames studies (OECD 471)

Two studies are available with the substance (C12 -alkyl derivatives). In the key study, the mutagenic potential of the substance (aqueous solution) was assessed in Salmonella typhimurium bacterial strains and Escherichia coli, in accordance with OECD 471 and according to GLP principles. The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98, TA 100 and tryptophan-requiring E.coli mutant WP2uvrA were used in the presence and the absence of metabolic activation. All determinations were made in triplicate. The first experiment was conducted using a plate-incorporation method at 5 dose levels up to 5000 µg/plate (aqueous solution) . The second and third experiments were performed using a pre-incubation method. Simultaneously negative (solvent) and positive controls were used in all experiments. In a preliminary range-finding assay, toxicity was seen. In the plate-incorporation assay, 4 strains were treated at dose levels 313 to 5000 µg/plate, while TA100 was treated at 156 to 2500 µg/plate. Slight toxicity was observed at the highest dose level with TA1535, TA1537 and TA100, but there was no increase in the revertant numbers in any of the strains tested. The second assay was performed with a pre-incubation step and using the same dose-range. Moderate thinning of the background lawn was observed at the highest dose level with TA98 tester strain, while marked toxicity was observed with TA1535, TA1537 and TA100 at the 3 highest dose levels. A third experiment was then performed with these 3 strains using a lower dose-range (39.1- 625 µg/plate without S9-mix, and 78.1-1250 µg/plate only for TA1535 and TA1537 in the presence of S9 -mix). The substance (aqueous solution) did not cause any two-fold or greater increase in the mean number of revertant colonies in the test plates, both in the absence and the presence of metabolic activation. Positive controls gave the expected increases in the number of revertants, with and without S9 mix. The supporting study shows comparable results but one strain missing. Under the conditions of the studies, the substance did not demonstrate any in vitro mutagenic activity in these bacterial test systems.

 

Chromosome aberration study (OECD 473)

In accordance with OECD 473 (1998), EU Method B.10 (2008) and according to GLP principles, a chromosome aberration study with the substance (dissolved in water) was performed in human peripheral blood lymphocytes in two independent experiments with and without metabolic activation. The dosing preparations were adjusted for the surfactant content of the substance and were set based on the observed toxicity in the dose range finding study (no precipitation was observed up to and including the top dose of 5000 µg/mL). In the first cytogenetic assay, the cells were treated for 4 hr (with and without S9-mix) and for 20 hr (without S9-mix) with the substance at doses ranging from 30 to 250 µg/mL. In the second cytogenetic assay, the cells were treated for 4 hr (with S9 -mix) with the substance at doses ranging from 30 to 200 µg/mL. Toxicity was reached at the dose levels selected for scoring. In all exposure groups, the percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Based on the results of this study, it is concluded that the substance does not disturb mitotic processes and cell cycle progression and is not clastogenic in human lymphocytes.

 

Read-Across: in vitro gene mutation assay (OECD 476)

A mouse lymphoma assay was conducted with a substance analogue (C8 -C18 alkyl derivatives) in accordance with OECD 476 and according to GLP principles. In the absence of S9-mix, the source chemical did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, the source chemical did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications of the S9-mix concentration added for metabolic activation. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In conclusion, as the source chemical is not mutagenic in the TK mutation test system, also the registered substance is not mutagenic in the TK mutation test system.

The rationale to read across the data is attached in Section 13.


Short description of key information:
The following 3 studies were performed with the substance: two Ames studies (one key and one supporting) in accordance with OECD 471 and according to GLP principles and a chromosome aberration study in human peripheral blood lymphocytes in accordance with OECD 473 and according to GLP principles. A mouse lymphoma assay conducted with a substance analogue in accordance with OECD 476 and according to GLP principles was considered appropriate to support the absence of mutagenic potential of the substance.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, it is concluded that there are no indications that the substance has genotoxic properties and therefore the substance is not classified for mutagenicity in accordance with the CLP Regulation.