Registration Dossier
Registration Dossier
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EC number: 202-928-3 | CAS number: 101-25-7
Toxicological information
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
REACH_positive | S.t. TA98, 100, 1535, 1537, 1538, E. coli WP2 | OECD 471 | with and without | JETOC #key study#
REACH_negative | S.t. TA98, 100, 1535, 1537, 1538 | OECD 471 | with and without | Elsevier (323ff) #key study#
REACH_positive | CHO | SCE OECD 479 | with and without | Elsevier (538ff) #key study#
REACH_positive | L5178Y | OECD 476 | with and without | Elsevier (580ff) #key study#
REACH_negative | Saccharomyces cerevisiae | OECD 480 | with and without | Elsevier (456ff) #key study#
Link to relevant study records
- 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:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Method of Ames et al. (1975), Maron and Ames (1983) and Matsushima et al. (1980)
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- extra pure reagent (Tokyo Kasei Kogyo Co. Ltd), Lot AI01
- Species / strain / cell type:
- S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 mix (sodium phenobarbital and 5,6-benzoflavone induced)
- Test concentrations with justification for top dose:
- 0, 50, 100, 200, 500, 1000, 2000, 5000 µg/plate
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- other: Solvent control served as negative control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with S9
- Untreated negative controls:
- other: Solvent control served as negative control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- sodium azide
- other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
- Remarks:
- without S9
- Details on test system and experimental conditions:
- Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were obtained from Dr. B.N. Ames (University of California, Berkeley, CA, USA), and Escherichia coli WP2uvrA were from Dr. M. Ishizawa (Kyushu University, Fukuoka, Japan). To a stationary culture of the tester strains, which had been checked for the genetic markers according to Ames et al. (1975) and Maron and Ames (1983), dimethyl sulfoxide (DMSO) was added to give a final concentration of 8.2% (v/v).
The cultures were stored in aliquots of 0.2mL at -80°C. The frozen permanents were thawed and inoculated to nutrient broth (OXOID, # 2) at 1 / 500-1 / 1000 inoculation size of thawed
solution. The cultures were incubated for 10-14 hours (tester strains grew up to early stationary phase) at 37°C in a shaking water bath before starting the tests. - Evaluation criteria:
- Two-hold rule criteria was used for data evaluation (Ames et al., 1975). The chemicals are considered to be mutagenic when a dose-related increase in revertant colony count is observed and the number of revertant colonies per plate with the test substance is more than twice that of the negative control (solvent control) and when a reproducibility of test result is observed.
Mutagenic potency was calculated by following equation and maximum value of mutagenic potency was expressed as a specific acitivity on the data sheet:
mutagenic potency (induced revertants / mg test substance) = (number of induced revertants on the dose X - number of revertant on the solvent control) + mg of test chemical on the dose X. - Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- Under the conditions of this test, the test item was positive causing gene mutations by base pair changes in the genome of the tester strain S. typhimurium TA100 with and without metabolic activation.
- 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:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Ames et al. (1977), plate incorporation method
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- pure reagent (ICI), trace 0.08% trinitrosotrimethylenetriamine
- Species / strain / cell type:
- S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 mix (Aroclor 1254 induced)
- Test concentrations with justification for top dose:
- 0.1, 1, 10, 100, 500, 2000 µg/plate
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- other: Solvent control served as negative control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- Nutrient broth cultures of Salmonella typhimurium strains TA1535, TA1537, TA1538, and TA100, containing 7.5% DMSO, were stored in screw-cap vials in a liquid nitrogen refrigerator.
Strain T A98 was not stored in liquid nitrogen since we found that, under the described storage conditions, the spontaneous mutation rate increased to unacceptable levels (60-100 revertants per plate). The strain was stored as a stab culture in Oxoid nutrient agar and subcultured every 2 months. The presence of the plasmid and the rfa and his mutations were checked during subculture.
Cultures of TA1535, TA1537, and TA1538 were grown for 16 hr in Oxoid Nutrient Broth No. 2 (25 mL) at 37°C in a shaking water bath. Strains TA98 and TA100 were incubated for 16 hr without shaking, to allow transfer of the plasmid to any of the cells that had lost it during storage; then the cultures were shaken for 3 hr at 37°C.
The cultures were tested for the presence of the rfa mutation and the plasmid (TA98 and TA100) as described by Ames et al. (1977), and their responses to standard mutagens (N-methyl-N-nitro-N-nitrosoguanidine, 2-nitrofluorene, 9-aminoacridine) were confirmed. The cultures were stored at 4-6°C for up to 5 days.
A 10 mg/mL solution of the test compound in DMSO was prepared immediately before use. Further dilutions were made in DMSO as required. - Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- Under the conditions of this test, the test item was negative.
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
- GLP compliance:
- not specified
- Type of assay:
- sister chromatid exchange assay in mammalian cells
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 mix (Aroclor 1254 induced)
- Test concentrations with justification for top dose:
- -S9: 5, 10, 20, 40, 80 µg/mL
+S9: 25, 50, 100, 200, 400 µg/mL - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO (1%)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-dimethylnitrosamine
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Each assay was conducted in duplicate in the presence and in the absence of an Aroclor-induced rat liver metabolic activation system.
- Evaluation criteria:
- 1) testing was performed to the maximum concentration expected to yield sufficient M2 metaphases for analysis, to the limits of solubility, or, because of limited chemical availability, to 1000 μg/mL;
2) at least one cytogeneticist observed at least twice the negative control SCE frequencies in 8 positive control sample;
3) SCE frequency analyses by at least two cytogeneticists were in general agreement.
A chemical was considered to have elicited a positive response if the variance between treatment groups was significantly greater (p<0.05; two-tailed) than the variance within treatment groups for either SCEs per chromosome or per cell.
A chemical was considered negative if the variance between treatment groups was not significantly different from the variance within treatment groups. - Statistics:
- The mean SCE frequency per cell for each concentration of the test chemical and for the controls was determined by dividing the total number of SCEs observed by the number of cells analyzed. Similarly, the mean SCE frequency per chromosome was determined by dividing the number of SCEs observed by the number of chromosomes counted. Confidence limits were set by determining the standard error of the mean (assuming a Poisson distribution).
These means then were evaluated by a one-way analysis of variance. The mean SCE frequencies per chromosome and per cell that each scorer observed
for the same treatment group (variance within sample) were compared with the mean SCE frequencies observed for the other treatment groups and for the negative control (variance between samples). An F test then was performed to determine whether the between-sample variance (the effect of the chemical) was significantly greater than the within-sample variance (the variance between scorers). - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- Under the conditions of this test, the test item was positive.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- 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
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 mix (Aroclor 1254 induced)
- Test concentrations with justification for top dose:
- 29.4, 42, 60, 85.8, 122.5, 175, 250 µg/mL
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- CELL LINE
L5178Y mouse lymphoma cells, heterozygous for thymidine kinase, TK +/- , were used as target cells. The cells were cleansed of homozygous (TK -/-) cells with methotrexate, and stocks were stored in a liquid nitrogen freezer. The stock cultures were checked routinely for mycoplasma contamination. - Evaluation criteria:
- The mutation frequency was calculated by dividing the number of mutant cells per milliliter of original suspension culture by the number of viable cells per milliliter of original suspension culture. Therefore, the mutation frequency was the ratio of mutant cells to surviving cells at each test concentration. The mutation frequency of the solvent control samples was subtracted from the mutation frequency of each treated sample, so the results were expressed as induced mutation frequencies.
An acceptable assay was one in which:
1) the compound was tested to the level of approximately 10% total relative growth or to the limits of solubility or to 3,000 μg/mL,
2) the relative plating efficiency of the solvent control was 60 to 130%,
3) the mutation frequency of the solvent control was no higher than 140 x 10E-6,
4) the mutation frequency of the positive control was at least twice that of the solvent control.
The test results were considered positive if the induced mutation frequency of at least one concentration was approximately twice that of the solvent control, or higher. A dose-response relationship was also a criterion. However, because compound availability was limited, this criterion was not always applied.
The results were considered negative if the test was valid but none of the treated samples showed an induced mutation frequency at least twice that of the solvent control. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- Under the conditions of this test, the test item was positive.
- Endpoint:
- genetic toxicity in vitro, other
- Remarks:
- Gene Mutation Assay in Yeast
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 480 (Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay)
- GLP compliance:
- not specified
- Type of assay:
- gene mutation assay in fungi
- Species / strain / cell type:
- Saccharomyces cerevisiae
- Additional strain / cell type characteristics:
- other: Strain D4
- Remarks:
- Heteroallelic (ade2, trp5) Diploid
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 mix (Aroclor 1254 induced)
- Test concentrations with justification for top dose:
- -S9: 0.33, 1, 3.33, 10, 33.33, 100, 333.33 µg/plate
+S9: 0.33, 1, 3.33, 10, 33.33, 100, 333.33 µg/plate - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- N-dimethylnitrosamine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- ethylmethanesulphonate
- other: 2-Anthramine (+ S9)
- Details on test system and experimental conditions:
- Three plates per dose
- Evaluation criteria:
- 1. Compounds that exhibited two- or-more-fold increase in the number of convertants compared to the controls were recorded as positive (+).
2. If the number of convertants were equal to or less than the controls, the compound was classified as negative (-).
3. If a compound exhibited less than twofold increase in the number of convertants but the increase was consistent in 1 hr and 3 hr experiments, the response was recorded as (±).
4. All doubtful ones marked as (?) either exhibited inconsistent responses or needed further testing but could not be retested because of lack of time.
5. Strong positives were those that exhibited a dose-relation increase in convertant numbers. - Key result
- Species / strain:
- Saccharomyces cerevisiae
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- Under the conditions of this test, the test item was negative.
Referenceopen allclose all
Toxicity (*) was observed in all tester strains at 5000 µg/plate and in TA98 (+/-), TA100 (+), TA1535 (+/-), TA1537 (+/-) and TA1538 (+/-) at 2000 µg/plate.
Number of revertants per plate, without S9
| µg/plate | TA100 | TA1535 | E.coli WP2uvrA | TA98 | TA1537 | TA1538 |
| 0 (DMSO) | 179 | 36 | 24 | 26 | 16 | 13 |
| 50 | 202 | 23 | 22 | 25 | 15 | 13 |
| 100 | 179 | 16 | 18 | 14 | 16 | 8 |
| 200 | 165 | 19 | 25 | 17 | 24 | 8 |
| 500 | 263 | 16 | 30 | 22 | 313 | 11 |
| 1000 | 419 | 13 | 39 | 30 | 12 | 8 |
| 2000 | 0 * | 0 * | 32 | 0 * | 0 * | 0 * |
| 5000 | 0 * | 0 * | 0 * | 0 * | 0 * | 0 * |
| pos. control | 1068 | 269 | 375 | 277 | 234 | 297 |
Number of revertants per plate, with S9
| µg/plate | TA100 | TA1535 | E.coli WP2uvrA | TA98 | TA1537 | TA1538 |
| 0 (DMSO) | 199 | 12 | 26 | 39 | 24 | 18 |
| 50 | 183 | 11 | 21 | 25 | 22 | 24 |
| 100 | 222 | 9 | 22 | 31 | 22 | 14 |
| 200 | 243 | 11 | 24 | 31 | 19 | 18 |
| 500 | 452 | 19 | 40 | 34 | 20 | 22 |
| 1000 | 825 | 15 | 51 | 61 | 12 | 13 |
| 2000 | 588 | 7 * | 57 | 62 * | 3 * | 6 * |
| 5000 | 0 * | 0 * | 18 * | 0 * | 0 * | 0 * |
| pos. control | 501 | 203 | 646 | 288 | 305 | 252 |
Mean SCEs without metabolic activation
| dose group | Mean SCEs/cell (scorer A) |
Mean SCEs/cell (scorer B) |
Mean SCEs/chromosome (scorer A) |
Mean SCEs/chromosome (scorer B) |
| negative control (1% DMSO) | 11.96 | 11.84 | 0.622 | 0.599 |
| DNPT 5 µg/mL | 12.20 | 14.96 | 0.630 | 0.739 |
| DNPT 10 µg/mL | 15.72 | 13.48 | 0.819 | 0.675 |
| DNPT 20 µg/mL | 14.64 | 18.76 | 0.766 | 0.940 |
| DNPT 40 µg/mL | 18.08 | 21.48 | 0.948 | 1.078 |
| DNPT 80 µg/mL | 33.52 | 39.56 | 1.768 | 1.970 |
| positive control (EMS) | 30.00 | 39.76 | 1.559 | 2.004 |
Mean SCEs with metabolic activation
| dose group | Mean SCEs/cell (scorer A) |
Mean SCEs/cell (scorer B) |
Mean SCEs/chromosome (scorer A) |
Mean SCEs/chromosome (scorer B) |
| negative control (1% DMSO) | 12.68 | 13.00 | 0.630 | 0.681 |
| DNPT 25 µg/mL | 13.52 | 15.12 | 0.686 | 0.778 |
| DNPT 50 µg/mL | 14.52 | 16.44 | 0.722 | 0.855 |
| DNPT 100 µg/mL | 16.28 | 17.88 | 0.821 | 0.914 |
| DNPT 200 µg/mL | 25.32 | 26.96 | 1.300 | 1.425 |
| DNPT 400 µg/mL | * | * | * | * |
| positive control (DMN) | 30.08 | 35.36 | 1.498 | 1.830 |
* insufficient metaphases for analysis
Summary Results Mutation Frequency (MF) DNPT
| S9 | NC MF (*10E-6) |
PC MF (*10E-6) |
dose range [g/mL] |
rel. total growth [%] |
max repsonse: conc. [µg/mL] |
max response: rel. tot. growth [%] |
max repsonse: MF (*10E-6) |
mutagenic response |
| - | 24 | 415 | 29.4 - 250 | 72.4 - 0.2 | 122.5 | 43.3 | 163 | + |
| + | 79 | 431 | 29.4 - 250 | 104.7 - 3.5 | 175.0 | 37.3 | 223 | + |
Mean Number of Convertants without metabolic activation (1h preincubation assay)
| Dose [µg/plate] | mean number | SD (three plates) |
| NC / Solvent control | 59.00 | +/- 5.00 |
| 0.33 | 61.67 | +/- 17.79 |
| 1 | 60.33 | +/- 16.44 |
| 3.33 | 61.33 | +/- 13.61 |
| 10 | 57.67 | +/- 2.89 |
| 33.33 | 49.00 | +/- 7.21 |
| 100 | 47.00 | +/- 6.08 |
| 333.33 | 69.00 | +/- 16.97 |
Mean Number of Convertants with metabolic activation (1h preincubation assay)
| Dose [µg/plate] | mean number | SD (three plates) |
| NC / Solvent control | 34.67 | +/- 5.51 |
| 0.33 | 33.00 | +/- 7.81 |
| 1 | 24.00 | +/- 5.57 |
| 3.33 | 28.33 | +/- 5.51 |
| 10 | 26.67 | +/- 5.51 |
| 33.33 | 32.33 | +/- 5.67 |
| 100 | 31.33 | +/- 8.62 |
| 333.33 | 32.67 | +/- 4.73 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
REACH_negative | mouse (male) | micronucleus test | Richardson et al.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- GLP compliance:
- not specified
- Type of assay:
- mammalian erythrocyte micronucleus test
- Specific details on test material used for the study:
- DNPT supplied by Organics Division, ICI PLC, Blackley, Manchester, Great Britain
- Species:
- mouse
- Strain:
- C57BL
- Remarks:
- /6J
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- ANIMALS
Male C57Bl/6J mice (6-8 weeks old) were used for both the main study and the LD50 determinations.
HUSBANDRY
On arrival the mice were housed 5 per cage on mobile mouse racks and given food (Porton Combined Diet [PCD] supplied by BP Nutrition Ltd., Stepfield, Witham, Essex, Great Britain) and water ad libitum.
The animal cell was maintained at a mean temperature of 21°C (range 20-22°C), a relative humidity mean of 44% (range 29-58%) with 22 air changes/h.
Lighting was cycled with 12 h light and 12 h dark each 24 h. - Route of administration:
- intraperitoneal
- Vehicle:
- not specified
- Details on exposure:
- Animals were dosed with 2 consecutive daily doses administered 24 h apart using doses equivalent to 80% and 50% of the LD50/7 by intraperitoneal injection.
The lower dose level was used to ensure a test result in the event of deaths in the top-dose group and to provide additional data in the event of a positive result. - Duration of treatment / exposure:
- 24 h
- Frequency of treatment:
- 2 consecutive daily doses administered 24 h apart
- Post exposure period:
- 24-48 h
- Dose / conc.:
- 160 other: mg/kg
- No. of animals per sex per dose:
- - Negative control a: 10 (24h) / 11 (48h)
- DNPT: 7 (24h) / 6 (48h)
- Positive control: 9 (48h) - Control animals:
- yes
- Positive control(s):
- Cyclophosphamide (75 mg/kg)
- Tissues and cell types examined:
- polychromtic erythrocytes (PCEs) from bone marrow (femur)
- Details of tissue and slide preparation:
- The animals were killed by cervical dislocation either 24 or 48 h after the last dose of compound. Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush wetted with a solution of albumen (6% w/v in water) was dipped into the marrow canal. 3-4 streaks of marrow suspension were then applied to appropriately labelled clean dry microscope slides.
The slides were allowed to air-dry and then stained in Leishman's stain using an Ames Hema-Tek staining machine (Miles Laboratories Inc., Ames Division, Stoke Court, Stoke Poges, Slough, Bucks., Great Britain). Slides were coded and scored blind: 500 polychromatic erythrocytes (PCEs) were examined for each animal and the number containing micronuclei scored. The ratio of polychromatic to mature erythrocytes was also determined as a measure of cytotoxicity.
Slides prepared from animals dosed at 50% of the LD50/7 were removed before coding. In the event of a positive result being observed with any of the test chemicals, the relevant slides from the low-dose groups were randomised with positive and negative control slides and scored for the presence of micronuclei. - Statistics:
- The results were analysed for significant differences from the control groups using a one-sided Student's ' t ' test.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- 80% of the LD50 based on preliminary toxicity test
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- DNPT gave negative results at both the sampling times used.
The system positive control (cyclophosphamide) gave a greatly elevated and statistically significant increase in micronuclei. - Conclusions:
- Under the conditions of this test, the test item did not show clastogenic activity.
- Executive summary:
A number of biocidal chemicals were tested for clastogenic activity in the micronucleus test using C57Bl/6J mice.
N, N-dinitrosopentamethylenetetramine (DNPT) was tested as the negative control.
DNPT did not increase the incidence of micronuclei.
Reference
Incidence of micronuclei/1000 cells (2 sampling times)
| Substance | Dose | 24h sampling | 48h sampling |
| NC (Control a) | 1.6 +/- 2.1 | 1.4 +/- 1.6 | |
| DNPT | 160 mg/kg | 1.4 +/- 1.5 | 1.3 +/- 1 |
| PC (Cyclophosphamide) | 75 mg/kg | - | 64.1 +/- 18 |
There was no undue influence of cytotoxicity on the results generated, since the ratios of PCEs to mature erythrocytes did not deviate from the normally expected range.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
DNPT was submitted to an international collaborative program for evaluation of selected short-term tests for discrimination between carcinogens and non-carcinogens. For this reason, several in vitro studies were executed with DNPT. Only tests which are are sufficiently documented and which correspond to former or current OECD Test Guidelines were included.
Negative results were obtained in several bacterial reverse mutation tests using Salmonella tester strains with and without metabolic activation. A positive outcome has been observed in a single Ames study in Salmonella strain TA 100 with and without metabolic activation and in Escherichia coli strain WP2uvrA with metabolic activation. DNPT yielded negative results in a gene mutation assay with Saccharomyces cerevisiae.
Mutagenic effects were detected with and without metabolic activation in a mouse lymphoma gene mutation test using the thymidine kinase locus.
DNPT induced sister chromatid exchanges in CHO cells in absence and presence of metabolic activation and chromosome aberrations in CHL cells with and without metabolic activation. No effects were detected in a test on unscheduled DNA Synthesis in HeLa cells in vitro.
The positive effects seen in the sister chromatid exchanges and chromosome aberrations were not confirmed in an in vivo micronucleus test.
Overall, test results are equivocal but do not support classification as mutagenic based on CLP criteria.
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