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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Diamine methylated is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. Diamine methylated is not clastogenic or aneugenic in human lymphocytes and is not mutagenic in the mouse lymphoma L5178Y test system.

All studies were performed under GLP according to current guidelines.

Link to relevant study records

Referenceopen allclose all

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:
28-Mar-2016 to 28-Apr-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without S9-mix: 0.18, 0.52, 1.7, 5.4, 17 and 52 μg/plate.
With S9-mix: 0.52, 1.7, 5.4, 17, 52 and 164 μg/plate.

Experiment 2:
Without S9-mix: 2.6, 4.7, 8.4, 15, 27 and 48 μg/plate.
With S9-mix: 8.4, 15, 27, 48, 86 and 154 μg/plate.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:

Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive control substance:
2-nitrofluorene
Remarks:
without S Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
sodium azide
Remarks:
without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance 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 (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100, WP2uvrA) or more or a three-fold (TA1535, TA1537, TA98) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
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 independently repeated experiment.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

CYTOTOXICITY RANGE-FINDING/SCREENING STUDIES:
TA1535: without S9: 52 µg/plate and with S9: 164 µg/plate
TA1537: without S9: 52 µg/plate and with S9: 164 µg/plate
TA98: without S9: 52 µg/plate and with S9: 164 µg/plate
TA100: without S9: 52 µg/plate and above and with S9: 164 µg/plate and above
WP2uvrA: without S9: 52 µg/plate and above and with S9: 164 µg/plate and above

COMPARISON WITH HISTORICAL CONTROL DATA:
Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 27 µg/plate and above and with S9: 154 µg/plate
TA1537: without S9: 27 µg/plate and above and with S9: 154 µg/plate
TA98: without S9: 27 µg/plate and above and with S9: 154 µg/plate
TA100: without S9: 27 µg/plate and above and with S9: 154 µg/plate
WP2uvrA: without S9: 45 µg/plate and with S9: 154 µg/plate

Conclusions:
It is concluded that Diamine methylated is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
Executive summary:

Diamine methylated 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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

The negative control values were within the laboratory historical control data ranges.

 

The 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, except the response for TA98 in the first experiment (absence of S9-mix).The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the validity of the study.

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:
24-Aug-2016 to 25-Oct-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 1.25, 2.5, 5, 10, 20 and 80 µg/mL
Without S9-mix, 24 hours treatment: 1.25, 2.5, 5, 10, 20 and 80 µg/ml

Experiment 1, 3 hours treatment
Without S9-mix: 0.1, 0.5, 1, 1.5 and 2 μg/ml exposure medium.
With S9-mix: 0.5, 2.5, 5, 9, 10, 11, 12 and 14 μg/ml exposure medium.

Experiment 2, 24 hours treatment
Without S9-mix: 0.25, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 μg/ml exposure medium.

The test item was tested up to cytotoxic dose levels.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: cyclophosphamide 10 µg/mL
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

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

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

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

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

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a)The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b)The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c)The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d)The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10-6). At least 40% of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10-6).


DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

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

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

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
- Effects of osmolality: No
-The test item precipitated in the exposure medium at the concentration of 80 μg/ml.

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, the relative suspension growth was 23% at the test item concentration of 5 μg/ml compared to the relative suspension growth of the solvent control. No or hardly any survival was observed at test item concentrations of 10 μg/ml and above. The test item precipitated in the exposure medium at the concentration of 80 μg/ml.
In the presence of S9-mix, the relative suspension growth was 19% at the test item concentration of 10 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at the item concentrations of 20 and 80 μg/ml. The test item precipitated in the exposure medium at the concentration of 80 μg/ml.
In the prolonged treatment, the relative suspension growth was 2% at the test item concentration of 1.25 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 2.5 μg/ml and above. The test item precipitated in the exposure medium at the concentration of 80 μg/ml.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the effect of Diamine methylated was evaluated up to the dose levels of 2 µg/ml giving a RTG value of 12%. Above this dose level the RTG was below the acceptable limit of 10%.
In the presence of S9-mix, the relative total growth of the highest test item concentration was 33% compared to the total growth of the solvent controls.
In the prolonged treatment, the effect of Diamine methylated was evaluated up to the dose levels of 1 µg/ml giving a RTG value of 30%. Above this dose level the RTG was below the acceptable limit of 10%.

Remarks on result:
other: strain/cell type: Test system L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.
Conclusions:
In conclusion, the test item is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Although the mutation frequencies of the solvent control cultures in the first and second experiment observed in the absence of S9-mix, were just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The growth rate over the two-day expression period for cultures treated with ethanol was between 12 and 18 (3 hour treatment) and 93 (24 hour treatment).

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.

In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26-Feb-2016 to 10-Jul-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
-Name of test material (as cited in study report):Diamine methylated
-Substance type:Amber liquid
-Physical state:Liquid
-Purity:100%
-Batch/Lot number:211401A Morris
-Expiration date of the lot/batch:24 March 2018

Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

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

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test/First cytogenetic assay:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 0.5, 1 and 5 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation:0.52, 1.7, 5.4, 17 and 52 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 0.5, 1 and 5 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 Migrated to IUCLID6: MMC-C 0.25 µg/mL for a 3 hours exposure period and 0.15 µg/mL for a 24 hours exposure period
Positive control substance:
other: colchicine: 0.1 µg/mL
Remarks:
without S9
Positive control substance:
cyclophosphamide
Remarks:
with S9 Migrated to IUCLID6: 15 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)
Evaluation criteria:
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with an Cochran Armitage 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 or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
Key result
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 5 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding, toxicity was observed at the highest tested dose of 52 µg/ml
In the cytogenetic assay, no toxicity was observed up to and including the highest precipitating dose of 5 µg/ml
COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the 95% control limits of the distribution of the historical negative control database .

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.
Remarks on result:
other: no remarks
Conclusions:
It is concluded that this test is valid and that Diamine methylated is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

The effect of Diamine methylated was studied on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity and aneugenicity of Diamine methylated was tested in accordance to OECD 487 in two independent experiments. Diamine methylated was dissolved in ethanol.

In the first cytogenetic assay, Diamine methylated was tested up to 5 µg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. Diamine methylated precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Diamine methylated was again tested up to 5 µg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix.

The number of mono- and binucleated cells with micronuclei 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, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Diamine methylated did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Finally, it is concluded that this test is valid and that Diamine methylated is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, genototoxicity by Diamine methylated is not expected. In physiological circumstances, the Diamine methylated has a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures.Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and transfer to the nucleus to interact with DNA.

Additional information

For each endpoint bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity a GLP compliant study is available.

 

Diamine methylated 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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

 

The effect of Diamine methylated was studied on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity and aneugenicity of Diamine methylated was tested in accordance to OECD 487 in two independent experiments. Diamine methylated was dissolved in ethanol.

In the first cytogenetic assay, Diamine methylated was tested up to 5 µg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. Diamine methylated precipitated in the culture medium at this dose level. In the second cytogenetic assay, Diamine methylated was again tested up to 5 µg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix.

The number of mono- and binucleated cells with micronuclei 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, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Diamine methylated did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Finally, it is concluded that this test is valid and that Diamine methylated is not clastogenic or aneugenic in human lymphocytes under the experimental conditions of this study.

 

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Although the mutation frequencies of the solvent control cultures in the first and second experiment observed in the absence of S9-mix, were just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The growth rate over the two-day expression period for cultures treated with ethanol was between 12 and 18 (3 hour treatment) and 93 (24 hour treatment).

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.

In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

Justification for classification or non-classification

Diamine methylated was found to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, not clastogenic or aneugenic in human lymphocytes and not mutagenic in the mouse lymphoma L5178Y test system.

All data therefore indicate that Diamine methylated is not genotoxic.