Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C7 odd-numbered alkyl) derivs. and sodium hydroxide and chloroacetic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

One reliable study (Klimisch 1, OECD 471, GLP) with the registered substance is available, which showed no mutagenic activity.

An MLA and a chromosome aberration study are available, performed with two analogues. Both studies had a negative outcome. This data is read across to Amphoacetates C8.

In order to substantiate the read across, the representative mono- and di-acetate structures with C8 chain length were investigated with DEREK NEXUS (report attached in Section 13). No substructures were found in the amphoacetate representative structures that relate to mutagenicity or clastogenicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September-December 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP- and OECD 471-compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

- OECD guideline No. 471, adopted on 21st July 1997,
- Commission Directive No. 2000/32/EC, B13/14, 8 June 2000.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Trade name: Miranol J2M Freeze Dried
Other name: DV 7581
Purity: 100%
Chemical active ingredient: Disodium caprylo amphoacetate
CAS No.: 68608-64-0
Batch No.: R-0469-203-19
Appearance: granular whitish powder
Storage conditions: at room temperature
Expiry date: August 2005

Target gene:

Histidine operon

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Liver post-mitochondrial fraction of Aroclor 1254 intraperitoneally-treated rats (S9 mix)

Test concentrations with justification for top dose:

Experiments without S9 mix:
- 156.3, 312.5, 625, 1250 and 2500 μg/plate, for the TA 98 strain in the first experiment and for the TA 1537 and TA 102 strains in both experiments,
- 312.5, 625, 1250, 2500 and 5000 μg/plate, for the TA 1535 and TA 100 strains in both experiments and for the TA 98 strain in the second experiment.
Experiments with S9 mix:
- 312.5, 625, 1250, 2500 and 5000 μg/plate, for all the strains.
in both mutagenicity experiments.
Based on the levels of toxicity observed in the preliminary assay.

Vehicle / solvent:

Water for injections, batch No. FVC01A (Fresenius Kabi, Sèvres, France)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 mix: sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mitomycin C / With S9 mix: 2-Anthramine

Details on test system and experimental conditions:

The test item was tested in a preliminary test and two mutagenicity experiments.
In the preliminary assay, to assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.
The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
The preincubation method was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate. After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK).

Evaluation criteria:

Treatment of results:
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), are presented in tabular form.

Acceptance criteria:
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the historical data of the testing facility,
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Without S9 mix:

A moderate to marked toxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 1535, TA 1537 and TA 102 strains and at 5000 μg/plate in the TA 98 and TA 100 strains.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

With S9 mix:

A moderate toxicity was noted in the TA 1535 strain in the first experiment (using the direct plate incorporation method).

A moderate to marked toxicity was noted at 5000 μg/plate in all the strains in the second experiment (using the preincubation method).

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

Conclusions:

Miranol J2M Freeze Dried did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

The mutagenic potential of disodium caprylo amphodiacetate, as Miranol J2M Freeze Dried, was assessed in the Salmonella typhimurium microsomal assay according to OECD 471 test guideline and in compliance with Good Laboratory Practice.

The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

A preliminary toxicity assay was performed to define the 5 dose levels to be used. The test substance, a slight pale yellow solid, was then tested in 2 independent experiments performed according to the direct plate incorporation method, except for the second test with S9 mix, which was performed according to the preincubation method (60 min, 37°C).

The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test article was dissolved in water. Because the test substance was toxic in the preliminary assay, the choice of the highest dose level for the main assay was based on the level of toxicity:

-         156.3, 312.5, 625, 1250 and 2500 µg/plate, for TA 98 strain in the first experiment and for the TA 1537 and TA 102 strains in both experiments without S9 mix

-         312.5, 625, 1250, 2500 and 5000 µg/plate, for the TA 1535 and TA 100 strains in both experiments and for the TA 98 strain in the second experiment without S9 mix

-         312.5, 625, 1250, 2500 and 5000 µg/plate, for all the strains in both experiments with S9 mix

 

All determinations were made in triplicate. Simultaneous negative (solvent) and positive controls were used in all experiments.

 

A moderate to marked toxicity was noted without S9 mix at dose-levels ≥ 2500 µg/plate in the TA 1535, TA 1537 and TA 102 strains and at 5000 µg/plate in the TA 98 and TA 100 strains. With S9 mix, a moderate toxicity was noted in the TA 1535 strain in the first experiment (using the direct plate incorporation method) and a moderate to marked toxicity was noted at 5000 µg/plate in all the strains in the second experiment (using the preincubation method).

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. Positive controls gave the expected increases in the number of revertants, with and without S-9 mix.

 

Under such experimental conditions, Miranol J2M Freeze Dried did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration 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:

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

Conclusions:

A chromosome aberration study with Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-2-(C11 alkyl) derivs. and sodium hydroxide and chloroacetic acid (aqueous solution; amphoacetates C12) 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. This result is read across to the registered substance.

Executive summary:

In accordance with OECD 473 (1998), EU Method B.10 (2008) and according to GLP principles, a chromosome aberration study with Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-2-(C11 alkyl) derivs. and sodium hydroxide and chloroacetic acid (aqueous solution; amphoacetates C12) 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. This result is read across to the registered substance.

Reference 3

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 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 Amphoacetates C8, member of the same chemical category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A mouse bone marrow cytogenetic assay was performed with substance analogue Amphoactetates C8-C18 (Miranol Ultra C32)

according to OECD/EC guidelines and GLP principles. The results demonstrate that the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vivo. This result is read across to Amphoacetates C8.

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:

key study

Justification for type of information:

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

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.

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 substance analogue Amphoactetates C8-C18 (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 substance analogue Amphoactetates C8-C18 (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. This result is read across to Amphoacetates C8.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The mutagenic potential of disodium caprylo amphodiacetate, as Miranol J2M Freeze Dried, was assessed in the Salmonella typhimurium microsomal assay according to OECD 471 test guideline and in compliance with Good Laboratory Practice. The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The choice of the highest dose level for the main assay was based on the level of toxicity. All determinations were made in triplicate. Simultaneous negative (solvent) and positive controls were used in all experiments. A moderate to marked toxicity was noted without S9 mix at dose-levels ≥ 2500 µg/plate in the TA 1535, TA 1537 and TA 102 strains and at 5000 µg/plate in the TA 98 and TA 100 strains. With S9 mix, a moderate toxicity was noted in the TA 1535 strain in the first experiment (using the direct plate incorporation method) and a moderate to marked toxicity was noted at 5000 µg/plate in all the strains in the second experiment (using the preincubation method). The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. Positive controls gave the expected increases in the number of revertants, with and without S-9 mix. Under such experimental conditions, Miranol J2M Freeze Dried did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

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 Amphoacetates C8.

In accordance with OECD 473 (1998), EU Method B.10 (2008) and according to GLP principles, a chromosome aberration study with substance analogue Amphoacetates C12 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. This result is read across to Amphoacetates C8.

In order to substantiate the read across, the representative mono- and di-acetate structures with C8 chain length were investigated with DEREK NEXUS (report attached in Section 13). No substructures were found in the amphoacetate representative structures that relate to mutagenicity or clastogenicity.

Justification for classification or non-classification

Based on the available data, Amphoacetates C8 is not classified for genetic toxicity.