Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An AMES study with the registered substance and two AMES study with a substance analogue (C8-C18 and C12) (performed according to OECD guideline 471) are available. Both studies are negative.


The results of a chromosome aberration study (OECD 473 guideline study) shows that the substance is not clastogenic in human lymphocytes under the experimental conditions of the study, however the results indicate that the substance may have the potential to disturb mitotic processes. A chromosome aberration study with the substance C8-C18 analogue shows the same results i.e. that the substance is not clastogenic in human lymphocytes under the experimental conditions of the study, however the results indicate that the substance may have the potential to disturb mitotic processes. A second chromomosome aberration study with the substance C12 analogue shows that that the substance does not disturb mitotic processe and cell cycle progression and is not clastogenic in human lymphocytes.


A mouse lymphoma assay conducted with the substance C8-C18 analogue in accordance with OECD 476 and according to GLP principles showed that the substance is not mutagenic in the TK mutation test system.


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


The endpoint conclusion is based on weight-of-evidence: relevant data show that two analogues are not mutagenic in vitro. This data is read-across to the registered substance.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 May 2021 - 07 June 2021
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
26 June 2020
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
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:
Type and composition of metabolic activation system:
- source of S9: male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 254 (500 mg/kg body weight)
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL; 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.
Test concentrations with justification for top dose:
FIRST EXPERIMENT: DIRECT PLATE ASSAY
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations were tested in triplicate. Based on the results of the dose-range finding test, the dose-range for the mutation assay with the tester strains, TA1535, TA1537 and TA98 was selected.
- TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (with and without metabolic activation).
- TA1535, TA1537 and TA98: 17, 52, 164, 512, 1600 and 5000 μg/plate. (with and without metabolic activation).
SECOND EXPERIMENT: PRE-INCUBATION ASSAY
Second Experiment: Pre-Incubation Assay
Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 μg/plate in all tester strains.
- TA1535, TA1537, TA100, TA98 and WP2uvrA: 17, 52, 164, 512, 1600 and 5000 μg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q water

The test item was corrected for the water content (50.6%). A correction factor of 2 was used. A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in Milli-Q water.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191;2-aminoanthracene
Remarks:
For details on positive control substances, see Table 1 and Table 2 in "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density: 10^9 cells/mL
- Test substance added in agar (plate incorporation) - Experiment 1; pre-incubation - Experiment 2

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Experiment 2 (pre-incubation assay): 30 ± 2 minutes
- Exposure duration/duration of treatment: 48 ± 4 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were evaluated.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The revertant colonies were counted automatically with the colony counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.
Rationale for test conditions:
according to guideline
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of
the tester strains, the positive response should be reproducible in at least one follow up
experiment.

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See explanation below
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
FIRST EXPERIMENT
Precipitation:
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain.

Cytotoxicity:
Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the absence and presence of S9-mix, where no toxicity was observed at any of the dose levels tested.

Mutagenicity:
In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item but with tester strain TA1537 in the absence of S9-mix, the lowest concentration of the test item induced a 3.7-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was within the historical control data range and not dose related and therefore considered not biologically relevant .

SECOND EXPERIMENT:
Precipitation:
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

Cytotoxicity:
Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the absence and presence of S9-mix, where no toxicity was observed at any of the dose levels tested.

Mutagenicity:
In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item but with tester strain TA1537 in the presence of S9-mix, the test item induced a 3.0-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was within the historical control data range and not dose related and therefore considered not biologically relevant.

ACCEPTABILITY CRITERIA
All bacterial strains showed negative responses over the entire dose-range, i.e. no biologically relevant, dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Details on results and historical control data are included in the attachments.
Conclusions:
Based on the results of an Ames test, performed according to OECD guideline 471 and in accordance with GLP principles, the test item is concluded to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

A bacterial reverse mutation test was performed according to OECD guideline 471 and in accordance with GLP principles. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation when tested up to and including the recommended top concentration. These results were confirmed in a follow-up experiment. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May 2021 - 19 August 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Physical appearance: yellow liquid
- Storage conditions: At room temperature
- Test item handling: No specific handling conditions required
Species / strain / cell type:
lymphocytes: Peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: healthy adult, non-smoking volunteers (approximately 18 to 35 years of age)
- Suitability of cells: Proposed test system in OECD test guideline.
- Average Generation Time and age of donor at time of AGT: Dose-range finding study: age 27, AGT = 14.3 h; First cytogenetic assay: age 24, AGT = 12.9 h; Cytogenetic assay 1A: age 25, AGT = 13.1 h; Second cytogenetic assay: age 27, AGT = 14.3 h

For lymphocytes:
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: No
- Mitogen used for lymphocytes: yes (phytohaemagglutinin)

MEDIA USED
- Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.

ENVIRONMENTAL CONDITIONS
- Humidity: 44 - 94% (actual range)
- CO2 concentration: 5.0 ± 0.5%
- Temperature: 35.0 - 37.6°C (actual range)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg)
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES.
The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin).
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).
Test concentrations with justification for top dose:
Selected test concentration were based on a dose-range finding test:

First Cytogenetic Assay :
- With and without S9-mix: 100, 500, 600, 700, 800, 900 and 1000 μg/mL culture medium (3 h exposure time, 24 h fixation time).

The experiment was repeated in cytogenetic assay 1A, with the following dose levels:
- Without S9-mix : 500, 700, 850, 900, 925, 950, 975 and 1000 μg/mL culture medium (3 h exposure time, 24 h fixation time).

Second cytogenetic Assay:
- Without S9-mix : 50, 150, 250, 300, 350, 400 and 500 μg/mL culture medium (24 h exposure time, 24 h fixation time).
Vehicle / solvent:
- Vehicle used: culture medium
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate cultures
- Number of independent experiments: 2

TREATMENT AND HARVEST SCHEDULE:
- First cytogenetic assay: Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of the test item for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay.
- Second cytogenetic assay: Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of the test item for 24 h in the absence of S9-mix. The cells were not rinsed after exposure but were fixed immediately after 24 h (24 h fixation time). Appropriate negative and positive controls were also included in the second cytogenetic assay.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: colchicine (0.5 μg/mL medium); last 2.5 - 3 h of the culture period
- Methods of slide preparation and staining technique used including the stain used: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa solution in Sörensen buffer pH 6.8.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations were calculated.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%).
Evaluation criteria:
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Key result
Species / strain:
lymphocytes: human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Inhibition of mitotic index of 50% or more was observed.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 8.165
- Data on osmolality: 329 mOsm/kg
- Precipitation and time of the determination: A concentration of 5000 μg/mL showed no precipitation in the culture medium after 3 hours exposure. After 24 hours exposure the test item precipitated in the culture medium at the concentration of 5000 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test blood cultures were treated with 156, 313, 625, 1250, 2500 and 5000 μg test item/mL culture medium with and without S9-mix.
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

STUDY RESULTS
First Cytogenetic Assay:
In the absence of S9-mix no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 900 μg/mL not enough cytotoxicity was observed (46%), whereas the next higher concentration of 1000 μg/mL was too toxic for scoring (93%).
The experiment was repeated in cytogenetic assay 1A.

The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix : 500, 975 and 1000 μg/mL culture medium (3 h exposure time, 24 h fixation time).
With S9-mix : 100, 600 and 900 μg/mL culture medium (3 h exposure time, 24 h fixation time).
Both in the absence and presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

The test item increased the number of polyploid cells both in the absence and presence of S9-mix in a dose dependent manner. The test item did not show a biologically relevant increase in cells with endoreduplicated chromosomes both in the absence and presence of S9-mix.

Second Cytogenetic Assay:
Further investigation (in cytogenetic assay 1A) showed that a concentration of 1000 μg/mL already precipitated in the culture medium after 24 hours exposure.
To obtain more information about the possible clastogenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to the test item in the absence of S9-mix for 24 hours.

Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 50, 250 and 300 μg/mL culture medium (24 h exposure time, 24 h fixation time).
At the 24 h continuous exposure time the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

EVALUATION OF THE RESULTS
The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database for MMC-C. Although in the presence of S9-mix the response of CP was above the upper control limits, these limits are 95% control limits and a higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
Conclusions:
An chromosome aberration study in human lymphocytes was performed according to OECD 473 and in accordance with GLP principles. Based on the results of this study it is concluded that the test substance is not clastogenic in human lymphocytes under the experimental conditions in this study. The test substance may have the potential to disturb mitotic processes.
Executive summary:

An chromosome aberration study in human lymphocytes was performed according to OECD 473 and in accordance with GLP principles. The possible clastogenicity of the test item was tested in two independent experiments.


In the first cytogenetic assay, the test item was tested up to 1000 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence of S9-mix and up to 900 μg/mL in the presence of 1.8% (v/v) S9-mix. Appropriate toxicity was reached at these dose levels.
In the second cytogenetic assay, the test item was tested up to 300 μg/mL for a 24 h continuous exposure time with a 24 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.
The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database for MMC-C. Although in the presence of S9-mix the response of CP was above the upper control limits, these limits are 95% control limits and a higher response is within the expected response ranges It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.
It was noted that the test item increased the number of polyploid cells both in the absence and presence of S9-mix in a dose dependent manner, after short term exposure. This may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations.
In conclusion, this test is valid and the test substance is not clastogenic in human lymphocytes under the experimental conditions described in this report. The test substance may have the potential to disturb mitotic processes.

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
Reason / purpose for cross-reference:
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 (amphoacetates C8-C18), in accordance with OECD 476 and according to GLP principles. It was concluded that the substance 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 (amphoacetates C8-C18), 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 substance is not mutagenic in the TK mutation test system. This result is read-across to this substance, member of the same chemical category. This result is read-across to the registered substance.

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

Genetic toxicity in vivo

Description of key information

An in vivo cytigenetic assay with a substance analogue (performed according to OECD guideline 475) is available. The presence of an increased number of polyploid cells in the first cytogenetic assay both in the absence and presence of S9-mix indicated that the test item has the potential to disturb mitotic processes. Therefore an in vivo mouse bone marrow cytogenetic assay was performed. The result of the in vivo study demonstrate that the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vivo.


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

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- In the dose range finding test, three males and three females were dosed with 2000 mg Miranol Ultra C32/kg body weight. The animals showed no clinical signs after dosing. Since there were no differences between sexes in toxicity only male animals were used in the main study, five male animals were used in each treatment group.

RESULTS OF DEFINITIVE STUDY
- The animals of the groups treated with 2000, 1000 and 500 mg Miranol Ultra C32/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs or mortality.
- No biologically relevant decrease in the mitotic index was observed in animals treated with Miranol Ultra C32. The inhibition of the mitotic index of the positive control was 10%.
- The scores for the number of aberrant cells (gaps included and excluded) and the number of the various types of chromosome aberrations at the various concentrations of Miranol Ultra C32 are attached.
- The number of cells with chromosome aberrations found in the animals dosed with the vehicle control was within the laboratory historical control data range. The positive control chemical produced a statistically significant increase in the frequency of aberrant cells. It was therefore concluded that the test conditions were appropriate for the detection of a clastogenic response. Miranol Ultra C32 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations, at both sampling times. No effects of Miranol Ultra C32 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed.

Table 1: Chromosome Aberrations in Bone Marrow Metaphases from Male and Female Mice Exposed to Vehicle


(12 – 18 hours sampling time)



























































































































































 


Dose (mg/kg)



 


Control (water for injection) 0



Total number of cells scored



Animal nr



7



8



9



10



11



 



No. of Cells scored



 


200



 


200



 


200



 


125



 


200



 


925



No. of Cells with aberrations (+ gaps) a)



 


0



 


0



 


0



 


0



 


0



 


0



No. of Cells with aberrations (- gaps)



 


0



 


0



 


0



 


0



 


0



 


0



g’



 



 



 



 



 



 



g”



 



 



 



 



 



 



b’



 



 



 



 



 



 



b”



 



 



 



 



 



 



m’



 



 



 



 



 



 



m”



 



 



 



 



 



 



exch.



 



 



 



 



 



 



dic



 



 



 



 



 



 



d’



 



 



 



 



 



 



misc.



poly



2poly



poly



 



4poly



 



total aberr (+ gaps)



0



0



0



0



0



 



total aberr (- gaps)



0



0



0



0



0



 



 


Table 2: Chromosome Aberrations in Bone Marrow Metaphases from Male and Female Mice Exposed to 2000 mg Miranol Ultra C32/kg body weight


(12 – 18 hours sampling time)



























































































































































 


Dose (mg/kg)



 


2000



Total number of cells scored



Animal nr



12



13



14



15



16



 



No. of Cells scored



 


142



 


174



 


200



 


200



 


200



 


916



No. of Cells with aberrations (+ gaps) a)



 


0



 


0



 


0



 


0



 


0



 


0



No. of Cells with aberrations (- gaps)



 


0



 


0



 


0



 


0



 


0



 


0



g’



 



 



 



 



 



 



g”



 



 



 



 



 



 



b’



 



 



 



 



 



 



b”



 



 



 



 



 



 



m’



 



 



 



 



 



 



m”



 



 



 



 



 



 



exch.



 



 



 



 



 



 



dic



 



 



 



 



 



 



d’



 



 



 



 



 



 



misc.



poly



poly



2poly



 



 



 



total aberr (+ gaps)



0



0



0



0



0



 



total aberr (- gaps)



0



0



0



0



0



 



 


Table 3: Chromosome Aberrations in Bone Marrow Metaphases from Male and Female Mice Exposed to 2000 mg Miranol Ultra C32/kg body weight


(36 – 44 hours sampling time)



























































































































































 


Dose (mg/kg)



 


2000



Total number of cells scored



Animal nr



17



18



19



20



21



 



No. of Cells


scored



 


200



 


160



 


200



 


200



 


200



 


960



No. of Cells with aberrations (+ gaps) a)



 


0



 


1



 


1



 


1



 


0



 


3



No. of Cells with aberrations (- gaps)



 


0



 


1



 


1



 


1



 


0



 


3



g’



 



 



 



 



 



 



g”



 



 



 



 



 



 



b’



 



1



1



1



 



 



b”



 



 



 



 



 



 



m’



 



 



 



 



 



 



m”



 



 



 



 



 



 



exch.



 



 



 



 



 



 



dic



 



 



 



 



 



 



d’



 



 



 



 



 



 



misc.



poly



 



 



poly



poly



 



total aberr (+ gaps)



0



1



1



1



0



 



total aberr (- gaps)



0



1



1



1



0



 



 


Table 4: Chromosome Aberrations in Bone Marrow Metaphases from Male and Female Mice Exposed to 1000 mg Miranol Ultra C32/kg body weight


(12 – 18 hours sampling time)



























































































































































 


Dose (mg/kg)



 


1000



Total number of cells scored



Animal nr



22



23



24



25



26



 



No. of Cells scored



 


200



 


200



 


200



 


200



 


200



 


1000



No. of Cells with aberrations (+ gaps) a)



 


0



 


0



 


0



 


0



 


0



 


0



No. of Cells with aberrations (- gaps)



 


0



 


0



 


0



 


0



 


0



 


0



g’



 



 



 



 



 



 



g”



 



 



 



 



 



 



b’



 



 



 



 



 



 



b”



 



 



 



 



 



 



m’



 



 



 



 



 



 



m”



 



 



 



 



 



 



exch.



 



 



 



 



 



 



dic



 



 



 



 



 



 



d’



 



 



 



 



 



 



misc.



 



 



2poly



2poly



 



 



total aberr (+ gaps)



0



0



0



0



0



 



total aberr (- gaps)



0



0



0



0



0



 



 


Table 5: Chromosome Aberrations in Bone Marrow Metaphases from Male and Female Mice Exposed to 500 mg Miranol Ultra C32/kg body weight


(12 – 18 hours sampling time)



























































































































































 


Dose (mg/kg)



 


500



Total number of cells scored



Animal nr



27



28



29



30



31



 



No. of Cells scored



 


200



 


200



 


200



 


200



 


200



 


1000



No. of Cells with aberrations (+ gaps) a)



 


0



 


0



 


0



 


0



 


0



 


0



No. of Cells with aberrations (- gaps)



 


0



 


0



 


0



 


0



 


0



 


0



g’



 



 



 



 



 



 



g”



 



 



 



 



 



 



b’



 



 



 



 



 



 



b”



 



 



 



 



 



 



m’



 



 



 



 



 



 



m”



 



 



 



 



 



 



exch.



 



 



 



 



 



 



dic



 



 



 



 



 



 



d’



 



 



 



 



 



 



misc.



poly



 



 



 



3poly



 



total aberr (+ gaps)



0



0



0



0



0



 



total aberr (- gaps)



0



0



0



0



0



 



 


Table 6: Chromosome Aberrations in Bone Marrow Metaphases from Male and Female Mice Exposed to Cyclophosphamide


(12 – 18 hours sampling time)



























































































































































 


Dose (mg/kg)



 


40 CP



Total number of cells scored



Animal nr



32



33



34



35



36



 



No. of Cells scored



 


200



 


200



 


200



 


200



 


200



 


1000



No. of Cells with aberrations (+ gaps) a)



 


44



 


31



 


23



 


85



 


30



***) 213



No. of Cells with aberrations (- gaps)



 


39



 


29



 


23



 


85



 


30



***) 206



g’



5



1



2



 



 



 



g”



2



1



2



 



 



 



b’



26



28



27



67



32



 



b”



6



2



1



15



4



 



m’



5



3



3



22



6



 



m”



3



1



 



5



1



 



exch.



5



2



6



13



5



 



dic



 



 



 



 



 



 



d’



 



 



 



 



1



 



misc.



5ma



5ma


poly



3ma


2poly



12ma


2poly



 



 



total aberr (+ gaps)



102



88



71



242



49



 



total aberr (- gaps)



95



86



67



242



49



 



 


Table 7: Summary Results





























































Dose mg/kg



Animal number



Fixation period



MI (percentage of control)



mean percentage of aberrant cells (%)



 



 



 



 



gaps incl



gaps excl.



 


0



 


7 - 11



 


12-18 h



 


100



 


0



 


0



2000



12 - 16



12-18 h



85



0



0



2000



17 - 21



36-44 h



96



0.3



0.3



1000



22 - 26



12-18 h



117



0



0



500



27 - 31



12-18 h



116



0



0



MI = mitotic index


*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

Conclusions:
Based on the results of a mouse bone marrow cytogenetic assay was performed according to OECD/EC guidelines and GLP principles, it is concluded that Miranol Ultra C32 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vivo.
Executive summary:

A mouse bone marrow cytogenetic assay was performed with Miranol Ultra C32 according to OECD/ EC guidelines and GLP principles. Male mice (5/group) were exposed to 500, 1000 or 2000 mg/kg bw/ day and bone marrow was sampled 12-18 (all doses, vehicle control group and positive control group (treated with cyclophosphamide) or 36-44 (highest dose only) hours after dosing. No mortality occurred, no clinical signs were noted in any of the mice. The number of cells with chromosome aberrations found in the vehicle control animals was within the laboratory historical control data range. The positive control animals treated with cyclophosphamide induced a statistically significant increase in the number of cells with chromosome aberrations, indicating that the test conditions were adequate. Miranol Ultra C32 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations, at both sampling times.


Based on these results it is concluded that Miranol Ultra C32 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vivo.

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

Additional information

Ames studies (OECD 471)


A bacterial reverse mutation test was performed according to OECD guideline 471 and in accordance with GLP principles. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation when tested up to and including the recommended top concentration. These results were confirmed in a follow-up experiment. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 


Chromosome aberration study (OECD 473)


An chromosome aberration study in human lymphocytes was performed according to OECD 473 and in accordance with GLP principles. The possible clastogenicity of the test item was tested in two independent experiments. In the first cytogenetic assay, the test item was tested up to 1000 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence of S9-mix and up to 900 μg/mL in the presence of 1.8% (v/v) S9-mix. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, the test item was tested up to 300 μg/mL for a 24 h continuous exposure time with a 24 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database for MMC-C. Although in the presence of S9-mix the response of CP was above the upper control limits, these limits are 95% control limits and a higher response is within the expected response ranges It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. It was noted that the test item increased the number of polyploid cells both in the absence and presence of S9-mix in a dose dependent manner, after short term exposure. This may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations. In conclusion, this test is valid and the test substance is not clastogenic in human lymphocytes under the experimental conditions described in this report. The test substance may have the potential to disturb mitotic processes.


Read-Across: Chromosome aberration study (OECD 473)


An in vitro chromosome aberration study was performed with with a substance analogue (amphoacetates C8-C18) according to OECD guideline 473 and GLP principles. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independent repeat experiments. Finally, it is concluded that this test is valid and that Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) is not clastogenic in human lymphocytes under the experimental conditions described in this report. Based on the presence of an increased number of polyploid cells in the first cytogenetic assay both in the absence and presence of S9-mix, the test item may have the potential to disturb mitotic processes. 


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


A mouse lymphoma assay was conducted with a substance analogue (amphoacetates C8 -C18) in accordance with OECD 476 and according to GLP principles. In the absence of S9-mix, the substance 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 substance 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, the substance is not mutagenic in the TK mutation test system. This result is read-across to this substance. The rationale to read across the data is attached in Section 13.


 


Read-Across: in vivo Chromosome aberration study (OECD 475)


A mouse bone marrow cytogenetic assay was conducted with a substance analogue (amphoacetates C8 -C18) in accordance with OECD 476 and according to GLP principles. Male mice (5/group) were exposed to 500, 1000 or 2000 mg/kg bw/ day and bone marrow was sampled 12-18 (all doses, vehicle control group and positive control group (treated with cyclophosphamide) or 36-44 (highest dose only) hours after dosing. No mortality occurred, no clinical signs were noted in any of the mice. The number of cells with chromosome aberrations found in the vehicle control animals was within the laboratory historical control data range. The positive control animals treated with cyclophosphamide induced a statistically significant increase in the number of cells with chromosome aberrations, indicating that the test  cnditions were adequate. The substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations, at both sampling times. Based on these results it is concluded that the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vivo. These results are read-across to the registered substance. The rationale to read across the data is attached in Section 13.


 


Conclusion


Based on the available study results on the registered substance and on a weight-of-evidence approach, taking into account all the available information of the analogue(s), it is considered justified to conclude that the substance has no mutagenic properties.

Justification for classification or non-classification

Based on the current data-set it is concluded that there are no indications that the registered substance has mutagenic properties and the substance does not need to be classified for mutagenicity in accordance with the CLP Regulation.