Reaction product of ammonium molybdate and C12-C24-diethoxylated alkylamine (1:5-1:3)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in-vitro: OECD 471 bacterial reverse mutation assay, non-mutagenic

Genetic toxicity in-vitro: OECD 473 chromosome abberation test, non-mutagenic

Genetic toxicity in-vitro: OECD 476 mouse lymphona assay, non-mutagenic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 June 1993 - 18th June 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

bacteria, other: Salmonella Typhimurium TA 1535, TA 1537, TA 98, TA 100 Escherichia coli WP2, WP2 uvrA

Metabolic activation system:

Aroclor induced S9 rat liver fraction.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 33 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 33 ... 5000 µg/plate

Vehicle / solvent:

Solvent: DMSO/dichloromethane 9/1

Species / strain:

other: as specified above

Metabolic activation:

with

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 2500 µg/plate)

Species / strain:

other: as specified above

Metabolic activation:

without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 2500 µg/plate)

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

Executive summary:

In a GLP compliant, guideline bacterial reverse mutation assay the test substance did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test susbtance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 July 1993 - October 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: V79 cells of the Chinese hamster

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum
(FCS; Boehringer Mannheim, D-68261 Mannheim).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced S9 rat liver fraction

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 3 ... 30 µg/ml
Concentration range in the main test (without metabolic activation): 1 ... 15 µg/ml

Vehicle / solvent:

The test article was suspended in a dilution of dichloromethane and DMSO (1 + 9) resulting in a homogenous suspension

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Ethylmethanesulfonate was the positive control used without metabolic activation and Cyclophosphamide was the positive control used with metabolic activation

Details on test system and experimental conditions:

The test substance was assessed for its potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments.

The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. The treatment interval was 4 h with and without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following concentrations were evaluated (18 h: 3 concentrations; 28 h: highest evaluable concentration):
Experiment I
without S9 mix:
18 h: 1.0, 3.0, 10.0 µg/ml
28 h: 10.0 µg/ml

with S9 mix:
18 h: 3.0; 10.0; 30.0 µg/ml
28 h: 30.0 µg/ml

Experiment II:
without S9 mix:
18 h: 1.0, 3.0, 10.0 µg/ml
28 h: 15.0 µg/ml

with S9 mix:
18 h: 3.0, 10.0, 20.0 µg/ml
28 h: 6.0 µg/ml

The test article was suspended in a dilution of dichloromethane and DMSO (1 + 9) resulting in a homogenous suspension. The concentrations
mentioned in this report are nominal concentrations because of a precipitation of the test article starting at concentrations of 10 µg/ml.

The concentration range of the test article had been determined in a pre-test using the colony forming ability and a qualitative evaluation of cell densitiy and morphology as indicator for toxicity response. After treatment with concentrations starting at 50.0 µg/ml (with S9 mix) and 25.0 µg/ml (without S9 mix) toxic effects could be observed.

In the cytogenetic experiments the test article was tested up to cytotoxic concentrations evidenced by reduction of the mitotic index.

Evaluation criteria:

A test article is classified as mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.

A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.

This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Species / strain:

other: as specified above

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Approx. 50 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: as specified above

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Approx. 25 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

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

In conclusion, under the experimental conditions reported, the test article did not induce structural chromosomal aberrations as determined by
the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, the test substance is considered to be non-mutagenic in this chromosome aberration test.

Executive summary:

In a GLP compliant, guideline Chromosome Aberration assay in Chinese Hamster V79 cells in vitro the test substance did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test substance is considered to be non-mutagenic in the chromosome aberration test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2008 and 16 February 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quaity of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 19 August 2008; Date of signature: 4 March 2009

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: R10 Media (RPMI 1640 with Glutamax 1 and HEPES buffer (20 mM) supplemented with penicillin, streptomycin, sodium pyruvate, amphotericin B, and 10% donor horse serum). R0 Media (RPMI 1640 without donor horse serum) and R20 Media (RPMI 1640 with 20% donor horse serum).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

The test material was treated as a complex mixture and found to be fully soluble at 500mg/mL in Tetrahydrofuran (THF). Tetrahydrofuran was, therefore, selected as the vehicle. However, THF is considered to be toxic to L5178Y cells above 50µl per 20mL culture and was therefore dosed at this level to give a maximum final concentration of 1250 µg/mL of test material in the solubility test.

NOTE 1: Based on the substance identity, the test material is 75% active ingredient (the reaction to make Additiv 104 is done in naphthenic base oil (EC# 265-155-0) which cannot be removed from the resulting mixture, and which therefore forms part (ca. 25% w/w) of the resulting mixture. However, in this study, a correction factor of 75% was applied when formulating the test concentrations, and as such all reported dose levels relate to the 'active' ingredient concentration of the test material only.

PRELIMINARY TOXICITY TEST: The investigated dose range was approximately 3.42 to 875 µg/mL (based on 75% active component), for all three of the exposure groups.

MUTAGENICITY TEST - VEHICLE AND POSITIVE CONTROLS: Used in parallel with the test material. Solvent (THF) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 126K0758 at 400 µg/mL for Experiment 1 and 150 µg/mL for Experiment 2, was used as positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch AO164185 at 2 µg/mL was used as positive control in the presence of metabolic activation (+ 2% S9 mix).


MUTAGENICITY TEST - EXPERIMENT 1: Four hour treatments performed in duplicate (A and B), both with and without metabolic activation, at eight dose levels, ranging from 1.72 to 110 µg/mL (- S9 mix) and 6.88 tp 220 µg/mL (+ S9 mix; 2%).

MUTAGENICITY TEST - EXPERIMENT 2: Twenty four hour treatment (- S9 mix) performed at eight dose levels ranging from 0.44 to 21 µg/mL. Four hour treatment (+ S9 mix; 1%) at eight dose levels, ranging from 10 to 110 µg/mL.

Vehicle / solvent:

Vehicle: Tetrahydrofuran (THF).
The test material was found to be fully soluble at 500 mg/mL in THF. No marked change in pH when the test material in THF (50 µL per 20 mL culture media) was dosed into media and the osmolality did not increase more than 50 mOsm.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (THF) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (THF) treatment groups were used as the vehicle controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With meabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in R0, R10 and R20 medium (0%, 10% and 20% donor horse serum-supplemented RPMI 1640 medium)

Evaluation criteria:

Please see method section.

Statistics:

The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS. Please see method section for more details.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes
- Effects of osmolality: No increase by more than 50mOsm
- Evaporation from medium:
- Water solubility:
- Precipitation: A precipitate of the test material was observed at and above 27.34 µg/mL in the 4-h exposure groups and at and above 109.38 µg/mL in the 24-h exposure group
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: The dose range of the test material used in the preliminary toxicity test was approximately 3.42 to 875 µg/ml. In all three of the exposure groups there was a marked reduction in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. A precipitate of the test material was observed at and above 27.34 µg/ml in the 4-hour exposure groups, and at and above 109.38 µg/ml in the 24-hour exposure group. In the subsequent mutagenicity experiments the maximum dose was limited by toxicity. Results of the preliminary toxicity tests are shown in the next section 'Any other information on results incl. tables'.

COMPARISON WITH HISTORICAL CONTROL DATA: The normal range for mutant frequency per survivor is 50-200 x 10-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controlsresults should ideally be within this strange, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10-6 mutant frequency per survivor are not normally acceptable.
Positive controls should induce at least three to five increases in mutant frequency greater than the corresponding vehicle control.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1
The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.
There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any significant decrease in (%V) viabilities in either of the exposure groups, therefore indicating that residual toxicity had not occurred. Optimum levels of toxicity were achieved in the presence of metabolic activation and near optimum levels of toxicity were achieved in the absence of metabolic activation. The excessive toxicity observed at 110 µg/ml in the absence of metabolic activation, and 220 µg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Table 3 and Table 6).

Neither of the vehicle control mutant frequency values were outside the range of 50 to 200 x 10-6 viable cells that is acceptable for L5178Y cells at Harlan Laboratories Ltd, UK. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). A precipitate of test material was observed at and above 27.5 µg/ml in the absence of metabolic activation, and at and above 55 µg/ml in the presence of metabolic activation.

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2
The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13.

As was seen previously there was evidence of dose-related reductions in % RSG and RTG values in cultures dosed with the test material in both the absence and presence of metabolic activation. There was no evidence of any significant reductions in (%V) viabilities, therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. Optimum levels of test material-induced toxicity were achieved in the presence of metabolic activation. In the absence of metabolic activation optimum levels were not achieved. This was considered to be due to the test material having a very sharp onset of toxicity and the maximum dose level being tested being close to this point. However, with no evidence of a response in any of the exposure groups, the test material was considered to have been adequately tested. Both positive controls induced acceptable levels of toxicity (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test material. It was also noted that the lowering of the S9 concentration to 1% S9 in this second experiment resulted in slightly greater levels of toxicity than those observed in the presence of 2% S9 in the first experiment.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test material did not induce any reproducible statistically significant or dose-related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). A precipitate of test material was observed at and above 20 µg/ml in the presence of metabolic activation. The precipitate observations in the absence of metabolic activation varied from those of the preliminary toxicity test with a precipitate of the test material observed at and above 7 µg/ml. However, the purpose and integrity of the study was considered unaffected.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

All tables are attached or in the section below.

Remarks on result:

other: strain/cell type: heterozygous at the thymidine kinase locus

Remarks:

Migrated from field 'Test system'.

Preliminary Toxicity Test The dose range of the test material used in the preliminary toxicity test was 3.42 to 875 µg/ml. The results for the Relative Suspension Growth (%) were as follows: Dose (µg/ml) Relative Suspension Growth (%) (-S9 mix) 4h exposure (+S9 mix) 4h exposure (-S9 mix) 24h exposure 0 100 100 100 3.42 90 108 87 6.84 124 77 38 13.67 78 118 23 27.34 54 94 0 54.69 49 71 0 109.38 0 43 0 218.75 0 8 0 437.5 0 0 0 875 0 2 0

Conclusions:

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

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction. The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.  Methods. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation. The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 1.72 to 110 µg/ml in the absence of metabolic activation and 6.88 to 220 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 0.44 to 21 µg/ml in the absence of metabolic activation, and 10 to 120 µg/ml in the presence of metabolic activation. Results. The maximum dose level used was limited by test material-induced toxicity. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.  The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment using a dose range that induced optimum or near optimum levels of toxicity.   Conclusion. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro genetic toxicity studies according to OECD protocols and to accetable quality standards show no evidence of gene mutation or chromosomal aberrations. On the basis of the negative in vitro results there is currently no justification for further in vivo genetic toxicology testing.

Justification for selection of genetic toxicology endpoint:

GLP compliant guideline studies each with a klimisch score of 1