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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item showed no mutagenic effect neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation when tested up to a cytotoxic concentration of 1000 μg/plate in the Salmonella typhimurium strains TA 98, TA 100, TA 1535 TA 1537 and TA 1538 and the Escherichia coli strain WP2 uvr A.

The test item was tested in a study equivalent to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test). In the direct method (24- and 48-hour treatments), no difference was observed between any of the test article groups and the negative control (non-treatment or solvent) groups with respect to the frequency of cells with structural aberrations or the frequency of cells with numerical aberrations. The percentage of cells with structural aberrations in the positive control (MMC) group was 45.5% for the 24-hour treatment and 64.0% for the 48-hour treatment. Due to cytotoxicity, there were less than 50 metaphase cells per dish (TOX) in the 160μg/mL group in the direct method with 48-hour treatment. In the metabolic activation method (with and without S9 mix), no difference was observed between any of the test article groups and the negative control (non-treatment or solvent) groups with respect to the frequency of cells with numerical aberrations. In the metabolic activation method without S9 mix, no difference was observed between any of the test article groups and the negative control (non-treatment or solvent) groups with respect to the frequency of cells with structural aberrations. However, the frequency of cells with structural aberrations in the 625μg/mL group with S9 mix was 23.0%, which was assessed as positive; even though no concentration dependency was observed. The percentage of cells with structural aberrations in the positive control (DMN) group was 73.5% in the presence of S9 mix.

Based on these results it was concluded that the test item induced chromosome aberrations (structural aberrations) in Chinese hamster lung fibroblasts (CHL/IU) in the presence of a metabolic activation system.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-2013
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 (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Ajinomoto Co., Inc.
- Lot/batch No.of test material: 110825
- Expiration date of the lot/batch: 25.08.2014
- Purity: 100%
Species / strain / cell type:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
3.16 - 1000 µg/plate
Vehicle / solvent:
water for injection
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-amino-anthracene
Details on test system and experimental conditions:
The test item was completely dissolved in water for injection and the vehicle served as the negative control.
Prior to the main test two preliminary cytotoxicity tests (plate incorporation test, without and with metabolic activation) were carried out in test strain TA 100.
In the main study 6 different concentrations of the test item were tested, with half-log intervals between plates (i.e. 3.16, 10.0, 31.6, 100, 316 and 1000 µg per plate).
Evaluation criteria:
In this study, the test item was considered to show a positive response if:
the number of revertants is significantly increased compared to the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and Escherichia coli and 3-fold of the solvent control for TA 1535, TA 1537 and TA 1538 in both independent experiments
a significant concentration (log value)-related effect is observed
positive results have to be reproducible and the histidine or tryptophan independence of the revertants has to be confirmed by streaking random samples on histidine- or tryptophan-free agar plates.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.
Statistics:
yes
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 µg/plate
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test item showed no mutagenic effect neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation when tested up to a cytotoxic concentration of 1000 µg/plate in the Salmonella typhimurium strains TA 98, TA 100, TA 1535 TA 1537 and TA 1538 and the Escherichia coli strain WP2 uvr A.
Executive summary:

In a study performed according to OECD Guideline 471 with GLP comliance, the test item was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and in the Escherichia coli strain WP2 uvr A in two independent experiments, each carried out without and with metabolic activation. The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced cytotoxicity was noted starting at a concentration of 1000 µg/plate. Hence, 1000 µg/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test, respectively. Six concentrations ranging from 3.16 to 1000 µg/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation cytotoxicity was noted at the top concentration of 1000 µg/plate, in all Salmonella typhimurium strains and in the Escherichia coli strain WP2 uvr A.

No mutagenic effect was observed tested up to a cytotoxic concentration of 1000 µg/plate, in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: In Vitro Mammalian Chromosome Aberration Test
Specific details on test material used for the study:
Name (IUPAC name): Sodium cocoyl glycinate (sodium cocoyl aminoacetate)
BML registration number: BML-4051
Lot No.: 970925
Purity: 28 wt%
Name of impurity and its concentration: Sodium cocoate (3.0 wt%)
Appearance at room temperature: Clear liquid
Solubility: Water (30% or more); Physiological saline (55 mg/mL or more)
Stability in solvent: Three months in water, in a dark place at room temperature
Storage: Refrigerated, shielded conditions
Species / strain / cell type:
other: Chinese hamster lung fibroblasts (CHL/IU)
Metabolic activation:
with and without
Test concentrations with justification for top dose:
up to 625 ug/mL (based on a preliminary study)
Vehicle / solvent:
physiol. saline
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
other: N-nitrosodimethylamine
Details on test system and experimental conditions:
Preliminary cytotoxicity test conducted to determine the concentrations for the main test
Direct method
1.2 * 10E+4 cells were seeded in a plastic dish containing 2.0 mL of the culture medium. The dish was incubated for three days. The highest concentration of the test article was set at 5.0 mg/mL, and 10 serial dilutions (2-fold dilutions) were prepared (0.010, 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL). 0.2 mL of the test article solution was added to a dish (two dishes per concentration), and the dish was incubated for 48 hours. Physiological saline was added to a dish instead of the test article solution for the negative control. After incubation, the culture medium was removed from the dish. The surface of the cells was washed with physiological saline, and the cells were fixed with a 10% formalin solution and stained with 0.1% crystal violet. Cell density in each concentration was measured by using monolayer cultured cells densimeter based on the staining and cell density in the negative control (regarded as 100%).
Metabolic activation method
Cells were seeded in the same manner as in the direct method and incubated for three days. The culture medium in the dish was removed and 2.0 mL of S9 mix dilution (6-fold dilution, final concentration of S9 mix: 5%) was added to the dish, and then 0.2 mL of the test article solution was added. The concentrations of the test article solution and the number of dishes per concentration were the same as in the direct method. The combined solution was mixed well and incubated for 6 hours at 37°C. Next, the culture medium was removed from the dish and the cells were washed with physiological saline. 2.0 mL of a fresh culture medium was added, and the dish was incubated for a further 18 hours. The cells were fixed and stained, and cell density was measured in the same manner as in the direct method.

Main study
Rationale for the concentrations of the test article: The concentrations of the test article in the main test were determined in accordance with the results of the cytotoxicity test. In addition to the direct method, the metabolic activation method (with rat liver S9) was conducted in order to examine the effects of metabolites. Based on the results, IC50 was calculated: 0.105 mg/mL for the direct method and 0.420 mg/mL for the metabolic activation method. Thus, the highest concentration of the test article for the direct method was determined as 160 ug/mL, and a total of four concentrations (increment: 40 ug/mL) were set. The highest concentration of the test article for the metabolic activation method was set at 625 ug/mL, and a total of four concentrations (serial dilutions with a geometric ratio of 2) were set.
Direct method
2 * 10E+4 cells were seeded in a plastic dish (60 mm in diameter) containing 5.0 mL of the culture medium. The dish was incubated for three days. The highest concentration of the test article was set at 160 ug/mL based on the results of the cytotoxicity test, and a total of four concentrations (increment: 40 ug/mL; namely, 40, 80, 120 and 160 ug/mL) were set. 0.5 mL of the test article solution was added to a dish (two dishes per concentration). In preparing the negative controls, physiological saline was added instead of the test article solution in the solvent group, and no solution was added in the non-treatment group. In preparing the positive controls, MMC (0.05 ug/mL) was added instead of the test article solution. Chromosome specimens were prepared following treatment (24- or 48-hour treatment).
Metabolic activation method
Cells were incubated in the same manner as in the direct method. The highest concentration of the test article was set at 625 ug/mL based on the results of the cytotoxicity test, and a total of four concentrations (serial dilutions with a geometric ratio of 2; namely, 78, 156, 313 and 625 ug/mL) were set. For the test system with S9 mix, 2.5 mL of the culture medium was removed from the dish, and then 0.5 mL of S9 mix (2.5 mL of the culture medium + 0.5 mL of S9 mix, the final concentration of S9 mix: 5%) and 0.3 mL of the test article solution was added to the dish. For the test system without S9 mix, 2.0 mL of the culture medium was removed from the dish, and 0.3 mL of the test article solution was added to the dish. In preparing the negative controls, physiological saline was added instead of the test article solution in the solvent group, and no solution was added in the non-treatment group. In preparing the positive controls, DMN (0.4 mg/mL) was added instead of the test article solution. Two dishes were used per concentration. The combined solutions were mixed well and incubated for 6 hours at 37°C. The entire volume of the culture medium was removed from the dish, and the cells were washed with physiological saline. 5.0 mL of a fresh culture medium was added to the dish and the dish was incubated for a further 18 hours. Chromosome specimens were prepared following incubation.
Evaluation criteria:
The following criteria were set to evaluate the frequency of aberrant cells in each concentration group. The frequencies of cells with structural aberrations were calculated, both for the case in which cells with gaps were included, and for the case in which cells with gaps were excluded. Assessment was based on the frequency of cells with structural aberrations including gaps.
Negative: < 5%
Equivocal: > 5 < 10%
Positive: > 10%
When there were less than 50 metaphase cells per dish due to cytotoxicity, the specimen was excluded from assessment.
Statistics:
No statistical analysis was conducted for assessment.
Key result
Species / strain:
other: Chinese hamster lung fibroblasts (CHL/IU)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: Chinese hamster lung fibroblasts (CHL/IU)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test item induced chromosome aberrations (structural aberrations) in Chinese hamster lung fibroblasts (CHL/IU) in the presence of a metabolic activation system.
Executive summary:

The test item was tested in a study equivalent to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test). In the direct method (24- and 48-hour treatments), no difference was observed between any of the test article groups and the negative control (non-treatment or solvent) groups with respect to the frequency of cells with structural aberrations or the frequency of cells with numerical aberrations. The percentage of cells with structural aberrations in the positive control (MMC) group was 45.5% for the 24-hour treatment and 64.0% for the 48-hour treatment. Due to cytotoxicity, there were less than 50 metaphase cells per dish (TOX) in the 160μg/mL group in the direct method with 48-hour treatment. In the metabolic activation method (with and without S9 mix), no difference was observed between any of the test article groups and the negative control (non-treatment or solvent) groups with respect to the frequency of cells with numerical aberrations. In the metabolic activation method without S9 mix, no difference was observed between any of the test article groups and the negative control (non-treatment or solvent) groups with respect to the frequency of cells with structural aberrations. However, the frequency of cells with structural aberrations in the 625μg/mL group with S9 mix was 23.0%, which was assessed as positive; even though no concentration dependency was observed. The percentage of cells with structural aberrations in the positive control (DMN) group was 73.5% in the presence of S9 mix.

Based on these results it was concluded that the test item induced chromosome aberrations (structural aberrations) in Chinese hamster lung fibroblasts (CHL/IU) in the presence of a metabolic activation system.

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

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus study using the bone marrow of male CD-1 mice was conducted, where groups of 6 mice received doses of 400, 200, 100 or 50 mg/kg bw via intraperitoneal injections (twice with 24 -hour interval). The bone-marrow smears were prepared 24 hours following the final administration. Four deaths were observed in the 400 mg/kg bw group and one death in the 200 mg/kg bw group. A decrease in locomotor activity and bradypnea were observed at >= 50 mg/kg bw, piloerection was observed at >= 100 mg/kg bw and hypothermia, lacrimation and prone position at >= 200 mg/kg bw. A decrease in body weight was observed at >= 100 mg/kg bw one day following the first administration and onwards. The percentage of MNPCE in the MMC (positive control) group 24 hours following single intraperitoneal administration was 4.77%, which is clearly higher than that in the negative control group, 0.07%. The number of MNPCE was 286, which was assessed as positive and exceeded the number of MNPCE estimated on the basis of the historical control data. The number of MNPCE in the negative control group was within the range estimated on the basis of the historical control data. Thus, it was judged that this study was valid.

The percentage of MNPCE in the 400, 200, 100 and 50 mg/kg bw groups 24 hours following the final administration of the test item was 0.10, 0.08, 0.07 and 0.18%, respectively. These values were within the range estimated on the basis of the historical control data for the negative control. The result of the Kastenbaum and Bowman assessment was negative. The proportion of PCE to total erythrocytes was significantly lower in the 100 and 200 mg/kg bw groups than that in the negative control group.

Based on these results it can be concluded that the test item inhibits the growth of erythroblasts at >= 100 mg/kg bw, but does not have the potency of eliciting micronuclei in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: Mammalian Bone Marrow Chromosome Aberration Test
Specific details on test material used for the study:
Sodium cocoyl glycinate (clear liquid, Lot No. 970925, purity: 28.0% w/w) provided by Ajinomoto Co., Inc.
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
For this test 7-week-old male ICR (Crj: CD-1) SPF mice were purchased from Japan Biomaterial Center. The mice were produced by Charles River Japan, Inc.. The animals were kept in the laboratory for eight days for quarantine and acclimatization. Following physical examination, the mice were used in the tests with an age of 8 weeks. The range of body weights at the first administration was 34.5 to 39.1 g in the dose-finding test and 33.2 to 40.6 g in the main test. The animals were stratified with the body weight on the final day of the acclimatization period, and then randomly assigned to one of the test groups using random numbers produced by a computer. Six mice were assigned to each group in both the dose-finding test and the main test. A total of 36 male mice were used in each test. Six mice (the whole group) were kept in one cage.
The animals were kept in aluminum box-type cages. The temperature was set at 20 to 26°C, and relative humidity was set at 40 to 70%. The lighting period was set to 12 hours and the ventilation cycle was set at 10 to 15 cycles per hour.
Autoclaved floor chips were placed on the floor of the cage.
The animals had free access to commercially available radiation-sterilized solid food and tap water from a water bottle.
Route of administration:
intraperitoneal
Vehicle:
physiol. saline
Details on exposure:
The required amount of the test item was weighed out and diluted with physiological saline to prepare 400, 200, 100 and 50 mg/kg solutions (4, 2, 1 and 0.5 % [w/v]) for use in the main test.
Duration of treatment / exposure:
The animals received intraperitoneal administration twice (24-hour interval)
Frequency of treatment:
see above
Post exposure period:
see below
Remarks:
50-400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6
Control animals:
yes
Positive control(s):
The animals in the positive control group received a single intraperitoneal administration of mitomycin C at 2 mg/kg bw and were put down 24 hours following administration. The dose and time at which they were put down were based on the historical control data.
Tissues and cell types examined:
micronuclei / bone-marrow smears
Details of tissue and slide preparation:
Preparation of bone-marrow smears
The mice that were alive at the completion of the treatment period were put down humanely by dislocating the neck bone. The right femur was removed and bone marrow cells were washed out using a small volume (0.5 to 1.0 mL/femur) of fetal bovine serum. The bone marrow cells were centrifuged at 1000 rpm for five minutes. The supernatant was removed and the bone marrow cells in the test tube were mixed well using a Pasteur pipette to prepare a suspension of even consistency. One drop of the suspension was placed on a slide, and a cover glass was used to prepare a bone-marrow smear. The smear was dried at room temperature, fixed using methanol for five minutes, and stained using Giemsa’s staining solution for 30 minutes. Next, the smear was washed with Sörensen phosphate buffer and placed in a 0.004% citric acid solution for several seconds. The smear was washed with distilled water and dried naturally. Two smears were prepared per mouse, and the code number was written on each smear.

Observation of micronuclei
The bone-marrow smear were observed using a 100× oil immersion objective lens and a 10× ocular lens (total: 1000×), and in the order of the code. Information on the test solution for each bone-marrow smear was concealed (blind observation). Erythrocytes were classified as either polychromatic erythrocytes (PCE) or normochromatic erythrocytes (NCE). One thousand PCE were observed per bone-marrow smear and the percentage of micronucleated PCE (MNPCE) was calculated. One thousand erythrocytes (PCE and NCE [total erythrocytes]) were observed per animal (smear) and the proportion of the PCE to total erythrocytes was calculated to examine the cytostatic effect of the test solution on erythroblasts.
Evaluation criteria:
The percentages of MNPCE in the negative control group and the positive control group were compared with historical control data for the negative control. If the following criteria were met, this study was regarded as valid:
The number of MNPCE in the negative control group in this study does not exceed the number of MNPCE estimated on the basis of the historical control data.
The number of MNPCE in the positive control group in this study exceeds the number of MNPCE estimated on the basis of the historical control data.
Statistics:
The test results were assessed using the Kastenbaum and Bowman assessment table. In addition, in order to examine the cytostatic effect of the test item on bone marrow cells, the proportions of PCE to total erythrocytes in the test item and positive control groups were each compared to that in the negative control group using Statistic Library 1, Statistical Analysis Version 5. When three or more groups were compared, the comparisons were carried out in the following manner. Bartlett’s test for homogeneity was conducted for distribution among the groups. If the distribution in the groups was equivalent, one-way analysis of variance (ANOVA) was carried out. If ANOVA revealed a significant difference among the groups, a Dunnett’s test was performed to compare the mean values of the negative control and the test article (or positive control) groups. If the distribution was not equivalent, a Kruskal-Wallis rank-test was performed. If the rank-test revealed a significant difference, a Dunnett’s test (a comparison of the mean values of the negative control and the test article or positive control groups) was performed.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test item was shown to inhibit the growth of erythroblasts at dose levels of >= 100 mg/kg bw, but does not have the potency of eliciting micronuclei in vivo.
Executive summary:

An in vivo micronucleus study using the bone marrow of male CD-1 mice was conducted, where groups of 6 mice received doses of 400, 200, 100 or 50 mg/kg bw via intraperitoneal injections (twice with 24 -hour interval). The bone-marrow smears were prepared 24 hours following the final administration. Four deaths were observed in the 400 mg/kg bw group and one death in the 200 mg/kg bw group. A decrease in locomotor activity and bradypnea were observed at >= 50 mg/kg bw, piloerection was observed at >= 100 mg/kg bw and hypothermia, lacrimation and prone position at >= 200 mg/kg bw. A decrease in body weight was observed at >= 100 mg/kg bw one day following the first administration and onwards. The percentage of MNPCE in the MMC (positive control) group 24 hours following single intraperitoneal administration was 4.77%, which is clearly higher than that in the negative control group, 0.07%. The number of MNPCE was 286, which was assessed as positive and exceeded the number of MNPCE estimated on the basis of the historical control data. The number of MNPCE in the negative control group was within the range estimated on the basis of the historical control data. Thus, it was judged that this study was valid.

The percentage of MNPCE in the 400, 200, 100 and 50 mg/kg bw groups 24 hours following the final administration of the test item was 0.10, 0.08, 0.07 and 0.18%, respectively. These values were within the range estimated on the basis of the historical control data for the negative control. The result of the Kastenbaum and Bowman assessment was negative. The proportion of PCE to total erythrocytes was significantly lower in the 100 and 200 mg/kg bw groups than that in the negative control group.

Based on these results it can be concluded that the test item inhibits the growth of erythroblasts at >= 100 mg/kg bw, but does not have the potency of eliciting micronuclei in vivo.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The genetic toxicity of the test item was investigated in three studies, i.e. bacterial gene mutation assay, in vitro mammalian chromosome aberration test and in vivo micronucleus study with mice. In total there was no indication for a mutagenic/genotoxic potential of the test item. Therefore, there is no need for classification and labelling of the test item according to CLP Regulation 1272/2008/EG.