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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key, equivalent to OECD TG 471 (bacterial Reverse Mutation Assay), S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and TA 1538, with and without metabolic activation: Negative (TSCATS, 1977)

Key, CA / SCE Assay, well-documented study, which meets basic scientific principles (non-GLP, non OECD TG study) CHO with and without metabolic activation: Negative (Natarajan et al., 1981)

Key, mouse lymphoma L5178Y/tk+/- cells well-documented studies, which meets basic scientific principles, 5 µL/mL: negative (NTP, Technical report, 1992)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Principles of method if other than guideline:
- Principle of test: Standard plate incorporation assay (Ames, 1975)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
DNA polymerase A deficient
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Ames test with and without metabolic activation with S-9 mix prepared from liver homogenate of Aroclor pre-treated male Sprague-Dawley rats.
Test concentrations with justification for top dose:
9400, 24000, 47000, 94000, 190000, 470000 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
In the assay with metabolic activation. Tested in concentrations of 5, 10 and 100 µg/plate
Positive controls:
yes
Positive control substance:
other: N-Methyl-N'-nitro-N-nitrosoguanidine
Remarks:
In the assay without metabolic activation. Tested in 2 µg/plate (TA1535, TA100)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
In the assay without metabolic activation. Tested at 50 µg/plate (TA1537).
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
In the assay without metabolic activation. Tested at 25 µg/plate (TA1538 and TA98).
Negative solvent / vehicle controls:
yes
Remarks:
A negative control run in parallel in the assay with and without metabolic activation.
Details on test system and experimental conditions:
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Plates are incubated at 37 °C for 48 hours.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- toxicity indicated by sparse background lawn
Rationale for test conditions:
Cytotoxicity of the test sample as measured in strain TA1535 is the basis for selecting concentrations to be used in the present test. Concentrations of test sample that give less than 50 % of control survival are normally not selected for the assay.
Evaluation criteria:
Evaluation criteria: a chemical was classified as non-mutagenic if the reversion frequency was less than 2 times the spontaneous frequency.
Statistics:
No statistical analysis was performed. However, in the present assay 470000 µg/plate was chosen because this experiment was done to aid in evaluating a recent Utah Biological Testing Service report, that concluded that DMF is mutagenic in 4 of the Ames strains of Salmonella typhimurium.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
DMF is not mutagenic even at concentrations that are cytotoxic.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 190000 and 470000 µg/plate without metabolic activation and at 470000 µg/plate with metabolic activation.

DMF is not mutagenic even at concentrations that are cytotoxic. Cytotoxicity occurred at 190000 and 470000 µg/plate without metabolic activation and at 470000 µg/plate with metabolic activation.

Positive and negative controls did work well in the present investigation.

Conclusions:
DMF is not mutagenic even at concentrations that are cytotoxic.
Executive summary:

Study design

This non-GLP study is an in vitro gene mutation test in bacteria, conducted similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay). The Ames test was performed with and without metabolic activation with S-9 mix prepared from liver homogenate of Aroclor pre-treated male Sprague-Dawley rats. The Plates are incubated at 37 °C for 48 hours.

Cytotoxicity of the test sample as measured in strain TA1535 is the basis for selecting concentrations to be used in the present test. Concentrations of test sample that give less than 50 % of control survival are normally not selected for the assay.

Results

Using S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 DMF is not mutagenic even at concentrations that are cytotoxic (Cytotoxicity occurred at 190000 and 470000 µg/plate without metabolic activation and at 470000 µg/plate with metabolic activation). Positive controls (with metabolic activation: 2 -Aminoanthracene and without metabolic acitvation: N-Methyl-N'-nitro-N-nitrosoguanidine; 9-Aminoacridine; 2-Nitrofluorene) and the negative control did work well in the present investigation.

Conclusion

DMF is not mutagenic even at concentrations that are cytotoxic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Chromosome Aberration/ Sister Chromatid Exchange Assay
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented publication which meets basic scientific principles.
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test: Determination of the frequencies of i.e. gaps, breaks and exchanges and sister chromatid exchanges.
GLP compliance:
not specified
Type of assay:
other: Chromosome Aberration/ Sister Chromatid Exchange Assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- S-9 mix was prepared from Aroclor-induced rat liver homogenates
Test concentrations with justification for top dose:
1.67, 3.33, 6.67 µg/mL (about 1.6, 3.2, 6.3 mg/mL)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Remarks:
with S-9 and without S-9 mix
Details on test system and experimental conditions:
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: CHO cells grown in the dark for one cell cycle and supplemented with BrdU
- Exposure duration/duration of treatment: cell were treated for 1 h with various concentrations of DMF in the presence or absence of S9 fraction.
- Harvest time after the end of treatment (sampling/recovery times): After treatment the cells were washed and allowed to recover for ≥ 12 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: After the colcemid block the cells were prepared for staining.
- Some preparations from each treatment were stained with Giemsa stain for the determination of the frequencies of chromosome aberrations.
- For determination of the frequencies of sister chromatid exchanges preparations were first stained with Hoechst 33258 and stained with Giemsa solution in order to obtain sister chromatid differentiation.
- 25 to 50 cells were scored for the frequencies of sister chromatid exchanges. 50 to 100 cells were scored for aberrations for each point, i.e. gaps, breaks and exchanges.
- Two controls, one with S-9 and one without S-9 mix treated with the solvent DMSO were included in the test.

Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified

DMF was found to be negative in both tests, in the presence or absence of S-9 mix during treatment.

Conclusions:
Interpretation of results: negative
DMF was found to be negative in both tests, in the presence or absence of S-9 mix during treatment.
Executive summary:

Study design

In this well documented study the frequencies of chromosome aberrations and sister chromatid exchanges induced in CHO cells were determined. It is a non-GLP, non-OECD Guideline study.

In the study CHO cells were grown in the dark for one cell cycle and supplemented with BrdU were treated for 1 h with various concentrations of DMF in the presence or absence of S9 fraction (S-9 mix was prepared from Aroclor-induced rat liver homogenates). After treatment the cells were washed and allowed to recover for 12 h. After the colcemid block the cells were prepared for staining. Some preparations from each treatment were stained with Giemsa stain for the determination of the frequencies of chromosome aberrations. For determination of the frequencies of sister chromatid exchanges preparations were first stained with Hoechst 33258 and stained with Giemsa solution in order to obtain sister chromatid differentiation. 25 to 50 cells were scored for the frequencies of sister chromatid exchanges. 50 to 100 cells were scored for aberrations for each point, i.e. gaps, breaks and exchanges. Two controls, one with S-9 and one without S-9 mix treated with the solvent DMSO were included in the test.

Results

DMF was found to be negative in both tests, in the presence or absence of S-9 mix during treatment.

Conclusion

Interpretation of results: negative

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Mouse Lymphoma Assay
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well documented report, which meets scientific standards
Principles of method if other than guideline:
- Principle of test: NTP Technical report. The chemical was tested in 3 laboratories for induction of gene mutations in mouse lymphoma L5178Y/tk+/- cells. The experimental protocols and statistical methods are presented in detail by McGregor et al. (1988), Mitchell et al. (1988), and Myhr and Caspary (1988).
- Short description of test conditions: Mouse lymphoma L5178Y/TK ± cells were incubated with DMF for 4 h. All treatment levels within an experiment, including concurrent positive and solvent controls. After incubation period, cells were resuspended in fresh medium and further incubated for an additional 2 days to express the mutant phenotype. After the 2-day expression period, cells were plated in medium and soft agar supplemented with trifluorothymidine for selection of TFT-resistant cells (TK-/-); cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 °C in 5 % CO2 for 10 - 12 days.
- Parameters analysed / observed: cytotoxicity and mutagenic response
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
Mouse lymphoma L5178Y/TK± cells
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system
- Source of S9: from the livers of Aroclor 1254 induced Fischer 344 rats
Test concentrations with justification for top dose:
Two studies: up to a maximum dose of 5 μg/mL (Mitchell et al.,1988, and Myhr and Caspary, 1988). In a third study the highest dose was 5 mg/mL (McGregor et al.,1988)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water or DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
methylmethanesulfonate
other: Methylcholanthrene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of independent experiments : 3 McGregor et al. (1988), Mitchell et al. (1988), and Myhr and Caspary (1988).

METHOD OF TREATMENT/ EXPOSURE:
Mouse lymphoma L5178Y/TK ± cells were maintained at 37 °C as suspension cultures in Fischer's medium. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Incubation with DMF was for 4 hours; the medium plus chemical then was removed, and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): After treatment (4h), cells were incubated for an additional 2 days to express the mutant phenotype.
- Selection time (if incubation with a selective agent): After the 2-day expression period, cells were plated in medium and soft agar supplemented with trifluorothymidine for selection of TFT-resistant cells (TK-/-); cells were plated in non-selective medium and soft agar to determine cloning efficiency
- Fixation time (start of exposure up to fixation or harvest of cells): 4h exposure, 2d expression period, 10 - 12 d selection period
- Method used: agar or microwell plates for the mouse lymphoma assay: non-selective medium and soft agar
Evaluation criteria:
Data were evaluated statistically for both trend and peak response (Myrh and Caspary, 1991). Both responses had to be significant (P < 0.05) for a chemical to be considered capable of inducing mutation; a single significant response led to a "questionable" conclusion, and the absence of both a trend and a peak response resulted in a "negative" call.
Statistics:
Data were evaluated statistically for both trend and peak response (Myrh and Caspary, 1991). Both responses had to be significant (P < 0.05) for a chemical to be considered capable of inducing mutation; a single significant response led to a "questionable" conclusion, and the absence of both a trend and a peak response resulted in a "negative" call.
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
Mitchell et al., 1988; Myhr and Caspary, 1988
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
other: marginal mutagenic response
Cytotoxicity / choice of top concentrations:
other: reduced relative total growth
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified

In 2 of the laboratories, DMF, tested up to a maximum dose of 5 μg/mL, was non-toxic and non-mutagenic with or without induced S9 (Mitchell et al., 1988; Myhr an Caspary, 1988). Results from the third laboratory showed a marginal mutagenic response at the highest dose tested (5 mg/mL) in all three trials conducted without S9 (McGregor et al., 1988). Evidence of toxicity, in the form of reduced relative total growth, occurred at 5 mg/mL, the dose which consistently showed a small increase in the number of mutant colonies.

Conclusions:
Results from 2 studies were negative. In these studies DMF was tested up to a maximum dose of 5 μL/mL and was non-toxic and non-mutagenic with or without induced S9 (Mitchell et al., 1988; Myhr and Caspary, 1988). In the third study, a marginal increase in mutant colonies was observed after treatment with DMF in the absence of S9 (only at the highest dose of 5 mL/mL) (McGregor et al., 1988).
The result of the third test can only be included in the evaluation to a limited extent, since the maximum dose of 5 mL/mL exceeds by far the maximum dose specified in the OECD TG 490 (2 µL/mL).
Executive summary:

Study design

The present NTP Technical report (1992) provides acceptable well documented studies, which meet scientific standards.

In the studies N,N-Dimethylformamide (DMF) was tested in 3 laboratories for induction of gene mutations in mouse lymphoma L5178Y/TK+/- cells. The experimental protocols and statistical methods are presented in detail by McGregor et al. (1988), Mitchell et al. (1988), and Myhr and Caspary (1988). DMF was supplied to the laboratory as a coded aliquot. Mouse lymphoma L5178Y/TK ± cells were maintained at 37 °C as suspension cultures in Fischer's medium. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Incubation with DMF was for 4 hours; the medium plus chemical then was removed, and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 2-day expression period, cells were plated in medium and soft agar supplemented with trifluorothymidine for selection of TFT-resistant cells (TK-/-); cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37°C in 5 % CO2 for 10 - 12 days.

Results and conclusion

Results from 2 studies were negative. In these studies DMF was tested up to a maximum dose of 5 μL/mL and was non-toxic and non-mutagenic with or without induced S9 (Mitchell et al., 1988; Myhr and Caspary, 1988). In the third study, a marginal increase in mutant colonies was observed after treatment with DMF in the absence of S9 (only at the highest dose of 5 mL/mL) (McGregor et al., 1988).

The result of the third test can only be included in the evaluation to a limited extent, since the maximum dose of 5 mL/mL exceeds by far the maximum dose specified in the OECD TG 490 (2 µL/mL).

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

Genetic toxicity in vivo

Description of key information

WoE, WHO, Technical Report Series, no. 482, intraperitoneal, 380 mg/kg bw, conception and number of live implantation sites: Negative (BASF, 1976a)

WoE, (non-OECD TG study), rat, inhalation, 30 and 300 ppm: Negative (TSCATS, 1978)

WoE, (non-GLP, non-OECT TG study), mouse, intraperitoneal, 0.2, 20, 2000 mg/kg bw/day: Negative (Antoine et al., 1983)

WoE, (non-OECD TG study), mouse, approx. 404, 808, 1615 kg/kg: Negative (Kirkhart et al., 1981)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented report which meets basic scientific principles.
Qualifier:
according to guideline
Guideline:
other: According to WHO: "evaluation and testing of drugs for mutagenicity: Principles and Problems", WHO, Technical Report Series, no. 482.
Principles of method if other than guideline:
- Short description of test conditions: DMF administration once by i.p. injection as an aqueous solution at 0.1 mL/animal at a dose of 380 mg/kg to 20 male NMRI mice. After ~20h male mice were mated with 3 untreated virgin female mice (1:3) for the duration of 7 days. Female animals were sacrificed after a gestation period of 10-17 days.
- Parameters analysed / observed: The implantations were evaluated. Male mice were supposed to macroscopic evaluation.
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: WIGA; Sulzfeld, Germany.
- Age at study initiation: 12-14 weeks.
- Weight at study initiation: male: 30 g (mean); female: 27 g (mean).
- Assigned to test groups randomly under following basis: similar weight.
- Housing: male: single; female: 3 per cage.
- Diet: Altromin-R, ad libitum
- Water: water ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2 °C
- Humidity (%): 55±5 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle/solvent used: water
Details on exposure:
In two studies DMF was administered once by i.p. injection as an aqueous solution at 0.1 mL/animal at a dose of 380 mg/kg to 20 male NMRI mice. Positive control was 2,3,5-Tris-ethyleneimino-benzoquinone-1,4 at a dose of 0.125 mg/kg in the first and at a dose of 0.056 mg/kg in the second study. Negative (untreated) control groups run in parallel. Twenty male mice each were used for the positive and the negative control group.
Frequency of treatment:
single dose
Post exposure period:
8 weeks: About 20 h after substance application, the male mice were mated with 3 untreated virgin female mice (1:3) for the duration of 7 days. Then the females were removed and replaced with three new untreated virgin females. This mating procedure continued for 7 consecutive weeks. Female animals were sacrificed 18 days after the first day of mating with the male rat, resulting in a gestation period of 10-17 days.
Dose / conc.:
380 mg/kg bw/day
Remarks:
intraperitoneal; conversation in mg/kg is based on the density: d= 0.94 g/cm3
No. of animals per sex per dose:
male: 20
female: 480
Control animals:
yes, concurrent no treatment
Positive control(s):
2,3,5-Tris-aethylenimino-benzochinon-1,4
- Route of administration: intraperitoneal
- Doses / concentrations: 0.125 and 0.056 mg/kg bw
Tissues and cell types examined:
males: macroscopic evaluation
females: evaluation of implantations
(For more details see: 'Any other information')
Details of tissue and slide preparation:
no data
Evaluation criteria:
no information given
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Please see: 'Any other information'

In the first study no substance-related effect on conception and number of live implantations was observed. In the second week of mating a significantly increased number of dead implantations (mutagenicity index) was observed in the females mated with DMF treated males when compared to untreated controls. However, according to the authors the mutagenicity index was still in the range of normal values that occurred also in the concurrent untreated control group. Whereas in the positive control a significant decrease in the mean number of implantation sites and a significant increase in the percentage of dead implantation sites (mutagenicity index) were observed during the first three mating intervals.

In the second study again no substance-related effect on conception and number of live implantation sites was seen but there was a statistically (mathematically) significant increase in the number of dead implantations in the 1st and 2nd week of mating in comparison to the untreated control. However, according to the authors, this was not due to the test substance since this effect was also observed at other time points and occurred in untreated controls, too. In the positive control a significant decrease in the mean number of implantation sites was observed in the first week of mating and a significant increase in the percentage of dead implantation sites (mutagenicity index) was observed in the first three mating intervals.

Conclusions:
In the first and in the second study, no substance-related effect on conception and number of live implantation sites was seen.
Executive summary:

Study design

A well-documented test was performed according to WHO: "evaluation and testing of drugs for mutagenicity: Principles and Problems", WHO, Technical Report Series, no. 482.

In two studies DMF was administered once by i.p. injection as an aqueous solution at 0.1 mL/animal at a dose of 380 mg/kg to 20 male NMRI mice. Positive control was 2,3,5-Tris-ethyleneimino-benzoquinone-1,4 at a dose of 0.125 mg/kg in the first and at a dose of 0.056 mg/kg in the second study. Negative (untreated) control groups run in parallel. Twenty male mice each were used for the positive and the negative control group. About 20 h after substance application, the male mice were mated with 3 untreated virgin female mice (1:3) for the duration of 7 days. Then the females were removed and replaced with three new untreated virgin females. This mating procedure continued for 7 consecutive weeks. Female animals were sacrificed 18 days after the first day of mating with the male rat, resulting in a gestation period of 10-17 days. All animals were observed daily for clinical symptoms. Body weight of the male mice was determined before treatment, after treatment and weekly thereafter. All male mice were killed at the end of the last mating period (week eight) and macroscopically examined. For the female animals the conception rate was calculated for each mating interval (derived from pregnant animals in relation to mated females). Moreover implantation sites/female animal were recorded and mean number of implantation sites/dam were calculated. Implantation sites were further distinguished as live and dead fetuses and deciduoma (early resorption sites still macroscopically discernible) and early resorptions discernible only by the staining method according to Salewski.

Results

In the first study no substance-related effect on conception and number of live implantations was observed. In the second week of mating a significantly increased number of dead implantations (mutagenicity index) were observed in the females mated with DMF treated males when compared to untreated controls. However, according to the authors the mutagenicity index was still in the range of normal values that occurred also in the concurrent untreated control group. Whereas in the positive control a significant decrease in the mean number of implantation sites and a significant increase in the percentage of dead implantation sites (mutagenicity index) was observed during the first
three mating intervals.
In the second study again no substance-related effect on conception and number of live implantation sites was seen but there was a statistically (mathematically) significant increase in the number of dead implantations in the 1st and 2nd week of mating in comparison to the untreated control. However, according to the authors, this was not due to the test substance since this effect was also observed at other time points and occurred in untreated controls, too. In the positive control a significant decrease in the mean number of implantation sites was observed in the first week of mating and a significant increase in the percentage of dead implantation sites (mutagenicity index) was observed in the first three mating intervals.

Conclusion

In the first and in the second study, no substance-related effect on conception and number of live implantation sites was seen.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study which meets basic scientific principles.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Evaluation of mutagenicity in test system after premating DMF-exposure.
- Short description of test conditions: Male rats were exposed to DMF-vapour. At least 2 h after the last exposure, two untreated virgin females were placed into each male's cage for 7 days. This mating procedure continued for six consecutive weeks. Females were sacrificed after a gestation period of 11-18 days. At completion of the study the males were sacrificed and necropsy was performed.
- Parameters analysed / observed: During the mating intervals: clinical signs, mortality, body weight. At completion of the study: Pregnancy rates and implantation,iImplantation efficiency, fetal death, uterine implants, histopathology of reproductive organs
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
other: Sprague-Dawley (CRCD)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Males used were about 8 weeks and females about 6 weeks of age
Route of administration:
inhalation
Details on exposure:
no details given
Duration of treatment / exposure:
5 d
Frequency of treatment:
6 h/d
Post exposure period:
six week post-treatment period
Dose / conc.:
30 ppm
Remarks:
ca. 0.09 mg/L
Dose / conc.:
300 ppm
Remarks:
ca. 0.91 mg/L
No. of animals per sex per dose:
Each group (two DMF-exposed) and the control groups contained 10 proven fertile male rats. Number of female animals not specified.
Control animals:
yes, concurrent no treatment
Positive control(s):
triethylenemelamine saline solution
- Justification for choice of positive control(s):
- Route of administration: i.p.
- Doses / concentrations: 0.5 mg/kg and a volume of 1 mL/kg administered once two hours prior to mating
Tissues and cell types examined:
At completion of the study the males were sacrificed and necropsy was performed. The following tissues of 5 males/group were preserved: seminal vesicles, epididymides, testes, prostate and from all males any abnormal lesions or tissue masses. Histopathological examination was carried out on seminal vesicles, epididymides, testes and prostate.
Regarding in-life observations, please see 'Any other information on materials and methods'
Details of tissue and slide preparation:
10 fertile male rats/dose group or control group
Evaluation criteria:
no details given
Statistics:
no details given
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Additional information on results:
For more details see: 'Any other information'

All male animals survived until termination of the study. During treatment and during the six week post-treatment mating period all animals were unremarkable. Mean body weights for males exposed to 30 and 300 ppm DMF were slightly lower than the negative control already during pre-treatment. This trend persisted during treatment and post-treatment. Body weight gain during the post-treatment mating period was slightly lower than negative control for the males exposed to 300 ppm DMF.

Pregnancy rates and implantation efficiency values of females mated to males exposed to DMF were considered comparable to the negative control throughout the post-treatment period. In contrast, pregnancy rates for the females mated to males of the positive control were lower than the negative control at mating weeks 2, 3 and 4, only at week 4 this difference was statistically significant.

Implantation efficiency values were significantly lower at weeks 2-6. Early fetal death data were significantly increased during the entire post-treatment mating period. Fetal death data for the DMF-treated groups were slightly higher than negative control at week 2 for both treated groups and week 5 for the 30 ppm and week 6 for the 300 ppm group.

According to the authors, at each interval the increase in fetal deaths was, in part, attributable to a single female that had uterine implants comprised entirely of early fetal deaths. These increases were not considered indicative of a dominant lethal mutagenic response since the second female of the pair mated with the DMF-treated male had high numbers of uterine implants which in most cases were all viable fetal swellings. Histopathology of male reproductive organs revealed no alterations to treatment.
Thus, premating DMF-exposure of the rats for 5 consecutive days did not result in mutagenic effects in the test system.

Conclusions:
Premating DMF-exposure of the rats for 5 consecutive days did not result in mutagenic effects in the test system.
Executive summary:

Study design

The present study was carried out on the basis of basic scientific principles and was well documented.

In the study male rats were exposed to DMF-vapour (30 und 300 ppm). At least 2 h after the last exposure, two untreated virgin females were placed into each male's cage for 7 days. This mating procedure continued for six consecutive weeks. Females were sacrificed after a gestation period of 11-18 days. At completion of the study the males were sacrificed and necropsy was performed.

Results

All male animals survived until termination of the study. During treatment and during the six week post-treatment mating period all animals were unremarkable. Mean body weights for males exposed to 30 and 300 ppm DMF were slightly lower than the negative control already during pre-treatment. This trend persisted during treatment and post-treatment. Body weight gain during the post-treatment mating period was slightly lower than negative control for the males exposed to 300 ppm DMF. Pregnancy rates and implantation efficiency values of females mated to males exposed to DMF were considered comparable to the negative control throughout the post-treatment period. In contrast, pregnancy rates for the females mated to males of the positive control were lower than the negative control at mating weeks 2, 3 and 4, only at week 4 this difference was statistically significant. Implantation efficiency values were significantly lower at weeks 2-6. Early fetal death data were significantly increased during the entire post-treatment mating period.
Fetal death data for the DMF-treated groups were slightly higher than negative control at week 2 for both treated groups and week 5 for the 30 ppm and week 6 for the 300 ppm group.

According to the authors, at each interval the increase in fetal deaths was, in part, attributable to a single female that had uterine implants comprised entirely of early fetal deaths. These increases were not considered indicative of a dominant lethal mutagenic response since the second female of the pair mated with the DMF-treated male had high numbers of uterine implants which in most cases were all viable fetal swellings. Histopathology of male reproductive organs revealed no alterations to treatment.

Conclusion

Premating DMF-exposure of the rats for 5 consecutive days did not result in mutagenic effects in the test system.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study which meets basic scientific principles.
Qualifier:
according to guideline
Guideline:
other: According to the methodology of Schmid W.: Chem. Mutagens, Vol. 4, 31-53, (1976).
Principles of method if other than guideline:
- Short description of test conditions: Young (8-12 weeks old) male Balb/c mice (5 per treatment group) were injected i.p. with 0.2, 20 and 2000 mg DMF/kg. Positive controls received 100 mg/kg cyclophosphamide. A negative control (not further specified) run in parallel.
- Parameters analysed / observed: Preparations of bone marrow cells were made 30 h after treatment. Cells were examined for the presence of micronuclei.
GLP compliance:
not specified
Type of assay:
other: micronucleus assay
Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-12 weeks
Route of administration:
intraperitoneal
Details on exposure:
no information given
Duration of treatment / exposure:
no information given
Frequency of treatment:
no information given
Dose / conc.:
0.2 mg/kg bw/day
Dose / conc.:
20 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5 / treatment group
Control animals:
yes
Positive control(s):
cyclophosphamide
- Doses / concentrations: 100 mg/kg
Tissues and cell types examined:
Preparations of bone marrow cells
Details of tissue and slide preparation:
no details given
Evaluation criteria:
no details given
Statistics:
no details given
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified

There was no increase in cells with micronuclei in the DMF-treated animals, whereas the positive control showed a clear increase.

Conclusions:
There was no increase in cells with micronuclei in the DMF-treated animals.
Executive summary:

Study design

The present study was carried out on the basis of basic scientific principles.

In the study, young (8-12 weeks old) male Balb/c mice (5 per treatment group) were injected i.p. with 0.2, 20 and 2000 mg DMF/kg.

Positive controls received 100 mg/kg cyclophosphamide. A negative control (not further specified) run in parallel Preparations of bone marrow cells were made 30 h after treatment.

Results and conclusion

There was no increase in cells with micronuclei in the DMF-treated animals.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study which meets basic scientific principles.
Qualifier:
according to guideline
Guideline:
other: Modification of the method of Schmid W.: Chem. Mutagens, Vol. 4, 31-53, (1976).
Principles of method if other than guideline:
- Short description of test conditions: DMF dissolved in DMSO was administered i.p. once at 5 mL/kg to male ICR mice (weight of 18-30 g). Groups of 8 animals each were administered 50, 25 or 12.5 % of the LD50, i.e. 1.70, 0.85 or 0.425 mL/kg. The negative control received 5 mL/kg of DMSO and the positive control animals were given 1,500 mg/kg of trimethylphosphate in DMSO at 5mL/kg. Femoral bone marrow smears were made from 4 animals each per group at 30 h and at 48 h according to the method of Schmid W. (1976).
- Parameters analysed / observed: For each animal scored, 1000 polychromatophilic erythrocytes were examined for the presence of micronuclei. The ratio of polychromatophilic erythrocytes to normochromatic erythrocytes was determined for the first four animals in each group. Since there was no significant difference in the value obtained at 30 h from that at 48 h, the results have been pooled.
GLP compliance:
not specified
Type of assay:
other: micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 18-30 g
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: DMF was dissolved in DMSO
Duration of treatment / exposure:
30 h and at 48 h
Dose / conc.:
0.425 other: mL/kg
Remarks:
~ 404 mg/kg
Dose / conc.:
0.85 other: mL/kg
Remarks:
~ 808 mg/kg
Dose / conc.:
1.7 other: mL/kg
Remarks:
~ 1615 mg/kg
Tissues and cell types examined:
- Polychromatophilic erythrocytes were examined for the presence of micronuclei.
- The ratio of polychromatophilic erythrocytes to normochromatic erythrocytes was determined.
Details of tissue and slide preparation:
According to the method of Schmid W. (1976)
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified

Under the conditions of the present test, DMF was not mutagenic.

Conclusions:
Under the conditions of the present test, DMF was not mutagenic.
Executive summary:

Study design

The present, well-documented study was carried out on the basis of basic scientific principles.

In the study, DMF dissolved in DMSO was administered i.p. once at 5 mL/kg to male ICR mice (weight of 18-30 g). Groups of 8 animals each were administered 50, 25 or 12.5 % of the LD50, i.e. 1.70, 0.85 or 0.425 mL/kg.

The negative control received 5 mL/kg of DMSO and the positive control animals were given 1,500 mg/kg of trimethylphosphate in DMSO at 5mL/kg. Femoral bone marrow smears were made from 4 animals each per group at 30 h and at 48 h according to the method of Schmid W. (1976). For each animal scored, 1000 polychromatophilic erythrocytes were examined for the presence of micronuclei. The ratio of polychromatophilic erythrocytes to normochromatic erythrocytes was determined for the first four animals in each group. Since there was no significant difference in the value obtained at 30 h from that at 48 h, the results have been pooled.

Results and conclusion

Under the conditions of the present test, DMF was not mutagenic.

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

Additional information

In vitro studies

A non-GLP study was conducted similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay).

In the study DMF was negative in the standard plate-incorporation Ames-Test with Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538, both with and without metabolic activation (rat liver S-9 mix) and even when tested at cytotoxic concentrations (concentrations tested: 9400 – 470 000 μg/plate (TSCATS: OTS 0520905, 1977).

Negative results were also obtained with and without metabolic activation in the SCE assay with CHO-cells (conc. tested: 0.0625 -1 mg/mL), the UDS Test in human diploid fibroblasts (conc. tested: up to 9220 mg/L) and a chromosome aberration/SCE assay (conc. tested 1.6, 3.2, 6.3 mg/mL) again with CHO-cells (Evans and Mitchell, 1981; TSCATS: OTS 0516796, 1981; Natarajan and Kesteren-van Leeuwen, 1981). Although the vast majority of mutagenicity and genetic toxicity studies led to negative results, single in vitro investigations with a positive result were reported in the literature. In a study of Chen et al., 1984, DMF was tested positive in a V79 Metabolic Cooperation Assay.

A NTP Technical report (1992) provides three studies of assessing mutagenic potency of DMF in vitro in mammalian cells, in mouse lymphoma L5178Y/TK+/- cells.

Results from 2 studies were negative. In these studies DMF was tested up to a maximum dose of 5 μL/mL and was non-toxic and non-mutagenic with or without induced S9 (Mitchell et al., 1988; Myhr and Caspary, 1988). In the third study, a marginal increase in mutant colonies was observed after treatment with DMF in the absence of S9 (only at the highest dose of 5 mL/mL) (McGregor et al., 1988).

The result of the third test can only be included in the evaluation to a limited extent, since the maximum dose of 5 mL/mL exceeds by far the maximum dose specified in the OECD TG 490 (2 µL/mL).

In vivo studies

In two dominant lethal assays (one in NMRI mice by single i.p. administration of the substance at a dose of about 380 mg/kg bw, and another one in Sprague-Dawley rats by inhalation exposure over a period of 5 days, 6 h/d at concentrations of 30 and 300 ppm (about 90 and 910 mg/m³)) DMF exposure did not result in mutagenic effects (BASF, 1976 a; TSCATS: OTS 0516779, 1978).

In two Micronucleus assays in which Balb/c or ICR mice were treated by a single i.p. administration of DMF (Balb/c mice were treated with doses of 0.2, 20 and 2000 mg/kg bw and the ICR mice were treated with 12.5, 25 or 50 % of the LD50, i.e. doses of about 404, 808 and 1615 mg/kg bw) no increases in micronuclei in bone marrow were seen (Antoine et al., 1983; Kirkhart, 1981).

Justification for classification or non-classification

DMF does not induce chromosome aberrations or gene mutations in various test systems in vivo and in vitro. In assessable in vitro studies in mouse lymphoma L5178Y/TK ± cells DMF was tested non-toxic and non-mutagenic.

The classification is not warranted according to the criteria of Regulation (EC) No 1272/2008 (CLP Regulation).