2-methylpentane-1,5-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro data for the submission substance indicate that this substance has no potential to induce gene mutations in bacteria or mammalian cells. Furthermore, no cytogenic effects (i.e. chromosomal aberrations) were observed.

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:

weight of evidence

Study period:

From 22 FEB 1990 to 09 APR 1990

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Does not meet important criteria of todays standard methods.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only strains TA1537 (Salmonella typhimurium) and E.coli WP2 uvrA tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

0, 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled-deionized water
- Justification for choice of solvent/vehicle: to maintain consistency with earlier testing

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA 1537 and WP2 uvrA: 2-aminoanthracene

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA 1537: ICR-191 Acridine

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: WP2 uvrA: methyl methanesulfonate

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: at least two replicates per dose group

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of the bacterial background lawn

Evaluation criteria:

Evaluation of test results
For a test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article as specified below:

strain TA 1537:
Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value.

Strain WP2 uvrA: Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Criteria for determination of a valid test:
1) tester strain integrity must be guaranteed (Salmonella strain: rfa wall mutation, and characteristic number of spontanous revertants, E.coli: Characteristic number of spontanous revertants)
2) tester strain titers must be greater than 0.6 x 10 exp 9
3) positive control values must be 3-fold the number of revertants over the mean value of the respective vehicle control

Statistics:

Average number of revertants from three plates was calculated.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slightly reduced background geowth in the highest concentration without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

ADDITIONAL INFORMATION ON positive control validity: In experiment I and II the positive control substance in tester strain TA1537 in the absence of metabolic activation system gave not the expected increase in revertant mutant colonies (at least 3fold to be valid). Therefor TA1537 was retested in the absence of metabolic activation system in two further and independent experiments. Experiment III and IV were performed in the same way as experiment I and II (ten concentrations of test material, vehicle control, positive control, each concentration tested in duplicate). In both experiments the positive control gave the expected results, thus being valid.

Conclusions:

Under the conditions of the test, the test substance was not mutagenic to Salmonella typhimurium TA 1537 and E.coli WP2 uvrA.

Executive summary:

In an Ames test similar to OECD 471 the test substance, MPMD, was tested on its mutagenic activity. Therefore different concentrations (0, 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/plate) were incubated with Salmonella typhimurium TA 1537 and E.coli WP2 uvrA. No mutagenic activity was detected with and without metabolic activation. The respective positive control substances gave the expected results.

Reference 2

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:

From 29 JUL 2003 to 10 OCT 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Pursuant to the Netherlands GLP Compliance Monitoring Programme and according to Directive 88/320/EEC

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK)-locus on chromosome 11 of cultured mouse lymphoma L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum, sodium pyruvate and penicillin/streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment I (with and without metabolic activation): 10, 6.5, 4.2, 2.7, 1.8, 1.2, 0.58, 0.29, 0.15, 0.073, 0.036, 0.018, and 0.009 and 0.0 mmol/l
Experiment II (without metabolic activation): 6.3, 5.4, 4.6, 3.9, 2.7, 1.9, 1.3, and 0.0 mmol/l
in parallel: 6.3, 5.4, 4.6 mmol/l with pH adjusted solutions
Experiment II (with metabolic activation): 10, 8.5, 7.2, 6.1, 4.3, 3.0, and 2.1 mmol/l
in parallel: 10, 8.5, 7.2 mmol/l with pH adjusted solutions

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 h (experiment I), 4 h (experiment II)
- Expression time (cells in growth medium): 44-48 h
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: In experiment I single cultures (except for the vehicle control which had two cultures) were used. In experiment II duplicate cultures were used.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)

OTHER: pH adjustment was done using 5N HCl (starting with stock solution of 25 mmol/l). pH was adjusted to 7.9 at a concentration of 10 mmol/l in the final culture medium.

Evaluation criteria:

A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 100 mutants per 1,000,000 clonable cells (Aaron et al, 1994; Clive et al., 1995). A response was considered to be equivocal if the induced mutant frequency was more than 50 mutants per 1,000,000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity was considered to be an artefact and not indicative of genotoxicity.

The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concen¬trations was observed.

The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test points.

Statistics:

No statistical analysis was performed.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

details see below

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the presence of S9-mix the relative initial growth and the RTG were decreased above 4.3 mmol/l. The relative total growth at the highest concentration evaluated for mutagenicity was 11 ± 3%.

Vehicle controls validity:

not valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

details see below

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The relative total growth at the highest concentration was not decreased, but the initial cell yield was below 10%. After pH adjustment at the highest concentration (5.4 mmol/l) less toxicity was observed and no increase of the mutant frequency.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
Historical data on negative and positive controls are available.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Dytek A (MPMD) was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix, the initial cell yield was decreased above a concentration of 2.7 mmol/l; the relative total growth (RTG) was not decreased in any culture that survived the treatment. The initial cell yield at the highest concentration evaluated for mutagenicity (5.4 mmol/l) was 7.9 ±3.6%. After pH-adjustment Dytek A (MPMD) was less cytotoxic; the initial cell yield at 5.4 mmol/l was 56 ±2%. In the presence of S9-mix, the initial cell yield and RTG were decreased above 4.3 mmol/l Dytek A (MPMD). The RTG at the highest concentration evaluated for mutagenicity (7.2 mmol/l) was 11 ± 3%. After pH-adjustment Dytek A (MPMD) was less cytotoxic. The RTG at 7.2 mmol/l was 64 ±13% and at 10 mmol/l it was 67 ±0.3%.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Mutagenicity

In the absence of S9-mix in the first assay the mutant frequency was increased by more than 50 mutants per 1,000,000 clonable cells compared to the negative control at two single concentrations of Dytek A (MPMD). At 4.2 and 1.8 mmol/l the mutant frequencies were increased by 51 and 76 mutants per 1,000,000 clonable cells, respectively, above the negative control. In the second assay, in which duplicate cultures were used, no increase of the mutant frequency was observed at any dose level. The RTG at the highest concentration was not decreased, but the initial cell yield was below 10%. After pH adjustment at the highest concentration (5.4 mmol/l) less toxicity was observed and no increase of the mutant frequency.

In the presence of S9-mix in the first assay in a single culture at 6.5 mmol/l the mutant frequency was increased by 72 mutants per 1,000,000 clonable cells above the negative control. In the second assay at 3.0 mmol/l the mutant frequency of one of the duplicate cultures was increased by 646 mutants per 1,000,000 clonable cells above the negative control. In the accompanying culture at 3.0 mmol/l and in all other cultures tested, no increase of the mutant frequency was observed. The RTG at the highest concentration was 11 ± 3%. No clear explanation could be found for the extreme high value of the mutant frequency in the single culture at 3.0 mmol/l. The results in the same concentration range considered, in both the first and second assay, the inexplicable value is regarded to be an artefact. After pH adjustment, the mutagenicity could be evaluated at higher concentrations. At 10 mmol/l no increase of the mutant frequency was observed.

The observed increases of the mutant frequencies in single cultures in the first assay were not confirmed in the second assay. Therefore these increases were

considered to be fortuitous and not indicative of mutagenicity.

Colony sizing

In both the absence and the presence of S9-mix the number of large and small colonies formed at concentrations causing an increase in mutant frequency was more or less equal.

Positive and negative controls

Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of the S9-mix, respectively. Treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls. The negative controls were within acceptable ranges.

Conclusions:

Under the conditions of the test the test substance did not induce mammalian cell gene mutations in a mouse lymphoma cell line.

Executive summary:

In an OECD 476 guideline study induction of mammalian cell gene mutations in vitro has been investigated in mouse lymphoma L5178Y cells in the presence (rat liver S9) and absence of metabolic activation. Concentration tested were in Experiment I (with and without metabolic activation): 10, 6.5, 4.2, 2.7, 1.8, 1.2, 0.58, 0.29, 0.15, 0.073, 0.036, 0.018, 0.009 and 0.0 mmol/l in Experiment II (without metabolic activation): 6.3, 5.4, 4.6, 3.9, 2.7, 1.9, 1.3, and 0.0 mmol/l (in parallel: 6.3, 5.4, 4.6 mmol/l with pH adjusted solutions) and in Experiment II (with metabolic activation): 10, 8.5, 7.2, 6.1, 4.3, 3.0, and 2.1 mmol/l (in parallel: 10, 8.5, 7.2 mmol/l with pH adjusted solutions). The test item did not induce gene mutations in concentrations up to 10 mmol/l under the tested conditions. Treatment with the respective positive control substances yielded the expected significant increase in mutant frequency compared to negative controls. Negative controls were within the accepted range.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 07 DEC 1987 to 14 DEC 1987

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Does not meet important criteria of todays standard methods.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only Salmonella strains TA 1535, TA 97, TA 98 and TA 100 tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

0, 10, 50, 100, 500, 1000, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled, deionized water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide: TA 100 and TA 1535, ICR-191 Acridine: TA 97, 2-nitrofluorene. TA 98

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: all concentrations were analysed in duplicate, 2 independent tests

DETERMINATION OF CYTOTOXICITY
- Method: evaluationof the bacterial background lawn

Evaluation criteria:

A test sample is classified as a POSITIVE when:
A. The number of induced revertants at one or more of the test sample concentrations studied is at least two times greater than the number of revertants in the solvent control. These dose levels must have a probability of less than 0.01 that the number of induced revertants are the same as the spontaneous revertant number.
AND
B. There is a dose—response relationship.

A test sample is classified as a NEGATIVE when:
A. The probability is greater than 0.05 that the number of revertants at each test sample concentrations studied are not greater than the number of revertants in the solvent control.
OR
B. There is no dose—response relationship.

A test sample is classified as EQUIVOCAL when:
A. Neither of the criteria for a positive or negative is satisfied.

Statistics:

Doses (concentrations: with and without activation, were ranked and results from a strain ware analyzed individually by multiple linear regression. Comparisons were made between each dose/concentration and the solvent control (0 rank), using the mean square error estimate. All comparisons were at the 95% level of confidence (alpha = 0.05).

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A spot test in a closed system was performed prior to toxicity screening to determine the most appropriate testing method. The results of the spot test were negative indicating the sample could be tested using standard toxicity and plate incorporation assays.
Dytek A Amine exhibited toxicity to strain TA 98 without and with activation at 1000 and 5000 ug/plate, respectively. Based an these results, 1000 µg/plate without activation and 2500 µg/plate with activation were chosen as the highest doses for the mutagenicity assays.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the conditions of the test, the test substance was not mutagenic to Salmonella typhimurium TA 1535, TA 97, TA 98 and TA 100.

Executive summary:

In an Ames test similar to OECD 471 the test substance, MPMD, was tested on its mutagenic activity. Therefore different concentrations (0, 10, 50, 100, 333, 500, 1000 µg/plate) were incubated with Salmonella typhimurium TA 1535 TA 97, TA 98 and TA100. No mutagenic activity was detected with and without metabolic activation.The respective positive control substances gave the expected results.

Reference 4

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:

From 11 JAN 1989 to 03 MAR 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

none

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: PRMI 1640 medium

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

0, 0.3, 0.6, 1.2, and 2.3 mg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period of cells: 47 -54 h
- Exposure duration: 3 h
- Expression time (cells in growth medium): 22 h (2 h with colcemid)
- Fixation time (start of exposure up to fixation or harvest of cells): 25 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): giemsa

NUMBER OF REPLICATIONS: All test concentrations and solvent controls were evaluated in duplicate cultures both with and without metabolic activation

NUMBER OF CELLS EVALUATED: 50 cells for determination of cytotoxicity, 100 cells for chromosome aberration studies (50 from each duplicate culture) for each trial with and without metabolic activation. 50 cells for positive control substance

DETERMINATION OF CYTOTOXICITY
- Method: average generation time (AGT)
- concentrations tested: 0, 1.0, 1.2, 1.5, 2.0 and 2.3 mg/ml

Evaluation criteria:

Acceptability criteria:
The complete chromosome aberration assay was based on the results of two independent, valid trials with activation and two without activation. An individual trial was valid if both of the folloving criteria were met:
The aberration frequencies of the negative control feil in the range of historical control data.
The percent abnormal cells in the positive indicator was statistically significantly greater than that of the solvent control cuitures.

Statistics:

For each trial, the proportion of abnormal cells and the proportion of cells with more than one aberration were evaluated using a Fisher Exact Test (Conover, 1971) to compare each treatment level with the solvent control, where statistical significance was judged at the 5% level. A Cochran-Armitage test for linear trend (dose-response) (Snedecor and Cochran, 1980) was performed where appropriate; significance was judged at the 1% level. Chromatid and isochromatid gaps were excluded from the calculations; they are listed separately in the appropriate appendices.

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
A sample of Dytek A was diluted (in an ice bath) with 5N HCl to yield a 230 mg/mL stock solution of pH 9.8. Using a treatment volume of 1%, this yielded a maximal Dytek A concentration of 2.3 mg/mL in the treatment medium. The resulting pH of the medium was 7.6 (as compared to 7.4 without activation and 7.5 with activation in untreated medium). At 2.3 mg Dytek A/mL, the osmolality of the treatment medium was 362 and 385 mOsm/kg H20 under nonactivated and activated conditions, respectively. These values were approximately 45 mOsm/kg H20 greater than control cultures. The use of higher test concentrations was judged inappropriate. Osmolality and pH were measured using a Micro Diagnostics freezing point depression osmometer (Model A0-10), and a Fisher Accumete pH meter (Model 815MP), respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the condotions of the test, the test item did not induce chromosome aberrations in human lymphocytes.

Executive summary:

In a study similar to OECD guideline 473 induction of chromosome aberrations by the test item has been investigated in human lymphocyte cells in vitro in the presence (Aroclor 1254 induced rat liver S9) and absence of metabolic activation. Concentrations tested were 0, 0.3, 0.6, 1.2, and 2.3 mg/ml. Cells were exposed to the test material for 3 hours and fixation time was 25 hours. The test item did not induce chromosome aberrations in tests concentrations up to 2.3 mg/ml under these test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No specific information available.

Additional information

In vitro data

In an Ames test similar to OECD test guideline 471 MPMD was tested on its mutagenic activity. Therefore different concentrations (0, 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/plate) were incubated with Salmonella typhimurium TA 1537 and E.coli WP2 uvrA. No mutagenic activity was detected with and without metabolic activation. 

In an additional Ames test Salmonella typhimurium TA 1535, TA 97, TA 98 and TA100 were used. No mutagenic activity was detected with and without metabolic activation as well. In combination these two studies can be used in a weight of evidence strategy to fulfil this endpoints requirement.

Gene mutation in mammalian cells has been investigated in one reliable study (OECD test guideline 476) with MPMD (Mouse Lymphoma Assay). Negative results were obtained in the presence and absence of metabolic activation. Under the test conditions MPMD did not induce gene mutations in concentrations up to 10 mmol/L. In the test the positive control yielded the expected increase of mutant frequencies and the negative control values were within acceptable ranges.

There is one reliable in vitro study (according to OECD test guideline 473) for MPMD on the induction of chromosome aberration in mammalian cells. Within these tests negative results were obtained with and without metabolic activation.

Justification for classification or non-classification

In a reliable set of bacterial mutation assays, gene mutation studies in mammalian cells and in vitro chromosomal aberration tests, the test item is considered to be non-mutagenic and is not a clastogenic agent.

Based on the available data no classification according to Regulation (EC) No. 1272/2008 on mutagenicity is warranted.