Registration Dossier
Registration Dossier
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EC number: 288-312-5 | CAS number: 85711-52-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial reverse mutation assay in vitro, OECD 471: not mutagenic with and without metabolic activation
in vitro chromosome aberration, OECD 473: not mutagenic with and without metabolic activation
gene mutation assay in cultured mammalian cells, OECD 490: not mutagenic with and without metabolic activation
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- other: read across from analogue substance
- Adequacy of study:
- key study
- Study period:
- 28 September 2015 - 27 October 2015
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Test item without emulsifier was investigated.
Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Physical state: pale yellowish solid at 20 °C
Batch No.: K8 4309 L481
Expiry date of batch: 09 March 2017
Purity: 100 % (UVCB)
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light - Target gene:
- Salmonella typhimurium
Strains / Genotype
TA1537 / his C 3076; rfa-; uvrB-;
TA98 / his D 3052; rfa-; uvrB-;R-factor
TA1535 / his G 46; rfa-; uvrB-
TA100 / his G 46; rfa-; uvrB-;R-factor
Escherichia coli
Strain / Genotype
WP2uvrA / trp-; uvrA-
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 1.5 to 5000 µg/plate.
Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.
- Vehicle / solvent:
- The test item was considered to have formed the best doseable suspension in sterile distilled water (12.5 mg/mL), therefore, this solvent was selected as the vehicle.
- Untreated negative controls:
- yes
- Remarks:
- sterile distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- other: 2-Aminoanthracene (with metabolic activation), Benzo(a)pyrene (with metabolic activation)
- Details on test system and experimental conditions:
- The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A number of manual counts were performed, predominantly due to colonies spreading and artefacts on the plates, thus distorting the actual plate count.
- Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Experiment 1 and 2 - plate incorporation test and in the preincubation test.
The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/beta-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed by
centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test. - Remarks on result:
- other:
- Remarks:
- Experiment 1 & 2
- Conclusions:
- The test item was considered to be non-mutagenic under the conditions of this test.
- Executive summary:
In a key Ames test performed with registered substance, the test item was examined in the four Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and one Escherichia coli strain in two independent experiments, each carried out without and with metabolic activation (P Thompson, 2016). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was suspended in sterile distilled water. Sterile distilled water was used as vehicle control. In a preliminary test, no cytotoxicity was noted at concentrations up to 5000 µg test item/plate, hence 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test. In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, no increase in revertant colony numbers as compared with control counts was observed up to a concentration of 5000 µg test item/plate, in any of the 5 test strains. A test item precipitate (powdery in appearance) was observed at 5000 µg/plate in the first mutation test (plate incorporation method) and initially from 500 µg/plate in the second mutation test (pre-incubation method), this observation did not prevent the scoring of revertant colonies. In conclusion, negative results were obtained for both bacterial mutagenicity studies tested both with and wihtout metbabolic activation.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- other: read across from analogue substance
- Adequacy of study:
- key study
- Study period:
- 14 October 2015 - 25 November 2015
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- Test item without emulsifier was investigated.
Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Physical state: pale yellowish solid at 20 °C
Batch No.: K8 4309 L481
Expiry date of batch: 09 March 2017
Purity: 100 % (UVCB)
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light - Species / strain / cell type:
- lymphocytes: Human
- Details on mammalian cell type (if applicable):
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.
Preliminary Toxicity Test: male, aged 35 years
Main Experiment: male, aged 29 years - Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Based on the precipitate observations in the solubility test, the maximum dose level selected for the Preliminary Toxicity Test was 320 µg/mL. Since the OECD 473 guideline requires only one precipitating dose level to be tested, irrespective of toxicity it was decided to reduce the maximum dose for the Preliminary Toxicity Test to a level where the lowest precipitating dose level could more easily be identified and exclude the majority of dose levels where precipitate was seen.
The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. - Vehicle / solvent:
- Vehicle and positive controls were used in parallel with the test item.
The vehicle control used was Minimal Essential Medium (Sigma, Batch No. RNBD4261).
- Untreated negative controls:
- yes
- Remarks:
- The vehicle control used was Minimal Essential Medium.
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Evaluation criteria:
- Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1. The number of cells with structural chromosome aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2. No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3. There is no concentration-related increase at any dose level.
A test item can be classified as genotoxic if:
1. The number of induced structural chromosome aberrations is outside the range of the laboratory historical control data.
2. At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3. The observed increase in the frequency of cells with structural aberrations is considered to be dose-related.
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes. - Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day,
orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames
test.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%. - Conclusions:
- The test item was considered to be non-clastogenic to human lymphocytes in vitro.
- Executive summary:
The test item was assayed in an in vitro chromosome aberration test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix).
The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure.
The test item was completely dissolved in Minimal Essential Medium. Minimal Essential Medium served as the vehicle control. The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item concentrations of 1.25 to 320 µg/mL medium were employed.
The maximum dose was reduced to 320 µg/mL based on the precipitate observations in the solubility test and the requirement to test the lowest precipitating dose level.
Hence, 80.0 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.
In the absence of S9, mitomycin C (MMC) was used as positive control at 0.2 and 0.05 µg/mL for 4(20)-hour cultures and 24-hour continuous exposure cultures, respectively. It was dissolved in Minimal Essential Medium.
In the presence of S9, cyclophosphamide (CP) was used as positive control at 2 µg/mL. It was dissolved in dimethyl sulphoxide.
All vehicle (MEM) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells
with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item demonstrated no-marked toxicity in the preliminary toxicity test and the maximum mdose level selected for the main test was based on the lowest precipitating dose level. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose
level.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- other: read across from analogue substance
- Adequacy of study:
- key study
- Study period:
- 07 October 2015 - 03 November 2015
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell transformation assay
- Specific details on test material used for the study:
- Test item without emulsifier was investigated.
Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Physical state: pale yellowish solid at 20 °C
Batch No.: K8 4309 L481
Expiry date of batch: 09 March 2017
Purity: 100 % (UVCB)
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light - Target gene:
- TK +/- locus of the L5178Y mouse lymphoma cell line.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- L5178Y TK+/- 3.7.2c mouse lymphoma cell line
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.
- Test concentrations with justification for top dose:
- There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups, with the most marked reductions observed in the 24-hour exposure group. Precipitate of the test item was observed at and above 40 µg/mL in all three exposure groups, which was used as the limiting factor for dose level selection in the mutagenicity test.
Two replicate cultures A,B were used for each concentration in all three exposure groups including positive and negative control. - Vehicle / solvent:
- Solvent RPMI 1640 (R0) exposure groups were used as the vehicle controls.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- RPMI 1640 (R0)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Following solubility checks performed in-house, the test item was accurately weighed and formulated in R0 culture media prior to serial dilutions being prepared. The test item is a UVCB compound therefore no molecular weight or purity correction was made. The test item was considered to be a complex mixture and therefore treated at the maximum recommended dose level of 5000 µg/mL initially. Solvent (R0) exposure groups were used as the vehicle controls.
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures
were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at six dose levels of the test item (2.5 to 40 µg/mL in all three exposure groups), vehicle and positive controls. To each universal was added 2 mL of S9-mix if required, 2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group). The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.
At the end of the exposure period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in humidified air and subcultured every 24 hours for the
expression period of two days, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.
RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10) and without serum (R0), are used during the course of the study. - Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director’s discretion. - Statistics:
- The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in any exposure group.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in humidified air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al, 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
PB/BNF S9 was prepared in-house on 01 March 2015 from the livers of male Sprague-Dawley rats weighing approximately 250g. These had each received, orally, three consecutive daily doses of phenobarbital/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day. This procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Envigo Research Limited, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. The conduct of the procedure may be reviewed, as part of the Envigo Research Limited, Shardlow,
UK Ethical Review Process. The S9 was stored at approximately -196 °C in a liquid nitrogen freezer.
S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.
20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test. - Remarks on result:
- other:
- Remarks:
- 4 h exposure
- Conclusions:
- The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.
- Executive summary:
The test item was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (L5178Y TK+/- 3.7.2c mouse lymphoma cell line) both in the presence and absence of metabolic activation. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix. The test item was completely dissolved in RPMI 1640 (R0). The concentrations employed were chosen based on the results of a cytotoxicity study. The dose range used in the preliminary toxicity test was 1.25 to 320 µg/mL for all three of the exposure groups, due to the excessive precipitate that was noted in the solubility test.
There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups, with the most marked reductions observed in the 24-hour exposure group. Precipitate of the test item was observed at and above 40 µg/mL in all three exposure groups, which was used as the limiting factor for dose level selection in the mutagenicity test.
Six concentrations 2.5, 5, 10, 20, 30, 40 µg test item/mL were selected for the experiments without and with metabolic activation, respectively.
The maximum dose level used in the Mutagenicity Test was limited by precipitate of the test item. The vehicle controls had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive controls induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at
any of the dose levels in the main test, in any of the three exposure groups.
Referenceopen allclose all
Exemplary results of Ames-Test: Plate incorporation - number of revertants (mean) +/- SD for all tested doses with and without metabolic activation.
Experiment 1 – Without Metabolic Activation
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD | ||||
|
Base-pair substitution strains |
Frameshift strains |
||||
|
|
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
Solvent Control (Water) |
119 107 119 (115) 6.9# |
11 17 15 (14) 3.1 |
21 21 21 (21) 0.0 |
19 12 17 (16) 3.6 |
7 15 8 (10) 4.4 |
|
1,5 µg |
93 103 81 (92) 11.0 |
16 12 9 (12) 3.5 |
25 19 32 (25) 6.5 |
23 19 21 (21) 2.0 |
15 7 17 (13) 5.3 |
|
5 µg |
97 97 114 (103) 9.8 |
14 10 9 (11) 2.6 |
27 28 15 (23) 7.2 |
15 12 19 (15) 3.5 |
12 7 16 (12) 4.5 |
|
15 µg |
94 126 110 (110) 16.0 |
14 12 13 (13) 1.0 |
19 24 32 (25) 6.6 |
28 11 11 (17) 9.8 |
9 15 7 (10) 4.2 |
|
50 µg |
117 129 114 (120) 7.9 |
9 9 10 (9) 0.6 |
17 21 19 (19) 2.0 |
17 20 23 (20) 3.0 |
11 11 11 (11) 0.0 |
|
150 µg |
94 112 118 (108) 12.5 |
14 11 10 (12) 2.1 |
16 21 24 (20) 4.0 |
13 12 27 (17) 8.4 |
7 9 12 (9) 2.5 |
|
500 µg |
103 95 107 (102) 6.1 |
8 10 16 (11) 4.2 |
32 36 17 (28) 10.0 |
21 25 12 (19) 6.7 |
9 12 15 (12) 3.0 |
|
1500 µg |
109 91 110 (103) 10.7 |
6 12 10 (9) 3.1 |
23 13 25 (20) 6.4 |
11 8 24 (14) 8.5 |
5 9 12 (9) 3.5 |
|
5000 µg |
94 P 115 P 124 P (111) 15.4 |
9 P 12 P 5 P (9) 3.5 |
29 P 36 P 26 P (30) 5.1 |
13 P 13 P 23 P (16) 5.8 |
8 P 4 P 11 P (8) 3.5 |
Positive controls S9-Mix (-) |
Name |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|
Dose Level |
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
|
No. of Revertants |
386 392 444 (407) 31.9 |
298 358 372 (343) 39.3 |
579 521 476 (525) 51.6 |
192 210 178 (193) 16.0 |
964 869 671 (835) 149.5 |
† Experimental procedure repeated at a later date due to poor colony growth in the original test
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
P Test item precipitate
# Standard deviation
Test Results: Experiment 1 – With Metabolic Activation
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD | ||||
|
Base-pair substitution strains |
Frameshift strains |
||||
|
|
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
Solvent Control (Water) |
110 115 124 (116) 7.1# |
12 11 8 (10) 2.1 |
20 28 29 (26) 4.9 |
16 20 16 (17) 2.3 |
8 9 13 (10) 2.6 |
|
1,5 µg |
117 125 119 (120) 4.2 |
8 12 10 (10) 2.0 |
23 32 25 (27) 4.7 |
15 15 23 (18) 4.6 |
7 17 12 (12) 5.0 |
|
5 µg |
118 106 126 (117) 10.1 |
13 9 5 (9) 4.0 |
17 36 25 (26) 9.5 |
11 29 19 (20) 9.0 |
15 7 15 (12) 4.6 |
|
15 µg |
114 129 121 (121) 7.5 |
17 9 9 (12) 4.6 |
29 29 16 (25) 7.5 |
16 28 29 (24) 7.2 |
8 11 7 (9) 2.1 |
|
50 µg |
82 122 119 (108) 22.3 |
8 8 8 (8) 0.0 |
31 23 29 (28) 4.2 |
23 19 15 (19) 4.0 |
9 13 13 (12) 2.3 |
|
150 µg |
91 97 94 (94) 3.0 |
11 11 12 (11) 0.6 |
28 20 23 (24) 4.0 |
28 27 19 (25) 4.9 |
17 19 17 (18) 1.2 |
|
500 µg |
106 106 99 (104) 4.0 |
8 7 12 (9) 2.6 |
33 39 27 (33) 6.0 |
20 21 25 (22) 2.6 |
13 8 7 (9) 3.2 |
|
1500 µg |
117 97 90 (101) 14.0 |
10 11 7 (9) 2.1 |
27 27 17 (24) 5.8 |
28 17 20 (22) 5.7 |
5 13 11 (10) 4.2 |
|
5000 µg |
102 P 98 P 97 P (99) 2.6 |
11 P 7 P 5 P (8) 3.1 |
25 P 28 P 20 P (24) 4.0 |
16 P 32 P 33 P (27) 9.5 |
16 P 15 P 9 P (13) 3.8 |
Positive controls S9-Mix (+) |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
|
Dose Level |
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
|
No. of Revertants |
719 647 540 (635) 90.1 |
243 221 215 (226) 14.7 |
372 351 405 (376) 27.2 |
195 183 207 (195) 12.0 |
342 376 341 (353) 19.9 |
† Experimental procedure repeated at a later date due to poor colony growth in the original test
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
P Test item precipitate
# Standard deviation
Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure Without Metabolic Activation (S9)
Treatment Group |
Replicate |
Mitotic Index (%) |
Number of Cells Scored |
Total Number of Aberrations |
Frequency of Aberrant Cells (%) |
||
|
|
|
|
(+ Gaps) |
(- Gaps) |
(+ Gaps) |
(- Gaps) |
Vehicle Control (MEM) |
A |
3.40 |
150 |
2 |
1 |
2 |
1 |
|
B |
2.90 |
150 |
0 |
0 |
0 |
0 |
|
Total |
(100) |
300 |
2 |
1 |
2 (0.7) |
1 (0.3) |
10 µg/ml |
A |
2.20 |
150 |
0 |
0 |
0 |
0 |
|
B |
2.30 |
150 |
0 |
0 |
0 |
0 |
|
Total | (71) | 300 | 0 | 0 | 0 (0.0) | 0 (0.0) |
20 µg/ml |
A | 3.40 | 150 | 2 | 2 | 2 | 2 |
| B | 2.25 | 150 | 3 | 3 | 3 | 3 | |
| Total | (90) | 300 | 5 | 5 | 5 (1.7) | 5 (1.7) | |
40 µg/ml |
A | 2.70 | 150 | 2 | 1 | 2 | 1 |
| B | 1.45 | 150 | 1 | 1 | 1 | 1 | |
| Total | (66) | 300 | 3 | 2 | 3 (1.0) | 2 (0.7) | |
80 µg/ml |
A | 1.50 | 150 | 1 | 1 | 1 | 1 |
| B | 5.00 | 150 | 1 | 1 | 1 | 1 | |
| Total | (103) | 300 | 2 | 2 | 2 (0.7) | 2 (0.7) | |
| Mitomycin C 0.2 µg/ml | A | 1.85 | 35* | 21 | 18 | 18 | 15 |
| B | 1.65 | 150 | 20 | 20 | 12 | 12 | |
| Total | (56) | 185 | 41 | 38 | 30 (16.2) | 27 (14.6)** | |
* Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed
** P < 0.001
Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure With Metabolic Activation (S9)
Treatment Group |
Replicate |
Mitotic Index (%) |
Number of Cells Scored |
Total Number of Aberrations |
Frequency of Aberrant Cells (%) |
||
|
|
|
|
(+ Gaps) |
(- Gaps) |
(+ Gaps) |
(- Gaps) |
Vehicle Control (MEM) |
A |
5.45 |
150 |
1 |
1 |
1 |
1 |
|
B |
4.75 |
150 |
1 |
0 |
1 |
0 |
|
Total |
(100) |
300 |
2 |
1 |
2 (0.7) |
1 (0.3) |
10 µg/ml |
A |
2.40 |
150 |
1 |
1 |
1 |
1 |
|
B |
3.00 |
150 |
1 |
1 |
1 |
1 |
|
Total |
(53) |
300 |
2 |
2 |
2 (0.7) |
2 (0.7) |
20 µg/ml |
A |
4.60 |
150 |
1 |
1 |
1 |
1 |
|
B |
2.10 |
150 |
2 |
1 |
2 |
1 |
|
Total |
(66) |
300 |
3 |
2 |
3 (1.0) |
2 (0.7) |
40 µg/ml |
A |
4.95 |
150 |
2 |
2 |
2 |
2 |
|
B |
2.75 |
150 |
0 |
0 |
0 |
0 |
|
Total |
(75) |
300 |
2 |
2 |
2 (0 .7) |
2 (0.7) |
80 µg/ml |
A |
3.25 |
150 |
1 |
0 |
1 |
0 |
|
B |
5.35 |
150 |
1 |
0 |
1 |
0 |
|
Total |
(84) |
300 |
2 |
0 |
2 (0.7) |
0 (0.0) |
Cyclophosphamide 2.0 µg/ml |
A |
3.75 |
67* |
16 |
16 |
16 |
15 |
|
B |
0.80 |
69* |
15 |
15 |
15 |
15 |
|
Total |
(45) |
136 |
31 |
31 |
31 (22.1) |
30 (22.1)** |
* Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed
** P < 0.001
Results of Chromosome Aberration Test – Main Experiment 24-hour Continuous Exposure Without Metabolic Activation (S9)
Treatment Group |
Replicate |
Mitotic Index (%) |
Number of Cells Scored |
Total Number of Aberrations |
Frequency of Aberrant Cells (%) |
||
|
|
|
(+ Gaps) |
(- Gaps) |
(+ Gaps) |
(- Gaps) |
|
Vehicle Control (MEM) |
A |
8.00 |
150 |
0 |
0 |
0 |
0 |
|
B |
7.40 |
150 |
0 |
0 |
0 |
0 |
|
Total |
(100) |
300 |
0 |
0 |
0 (0.0) |
0 (0.0) |
10 µg/ml |
A |
4.40 |
150 |
4 |
2 |
4 |
2 |
|
B |
3.95 |
150 |
0 |
0 |
0 |
0 |
|
Total |
(100) |
300 |
4 |
2 |
4 (1.3) |
2 (0.7) |
20 µg/ml |
A |
6.80 |
150 |
2 |
0 |
2 |
0 |
|
B |
3.60 |
150 |
1 |
1 |
1 |
1 |
|
Total |
(68) |
300 |
3 |
1 |
3 (1.0) |
1 (0.3) |
40 µg/ml |
A |
6.70 |
150 |
0 |
0 |
0 |
0 |
|
B |
4.15 |
150 |
0 |
0 |
0 |
0 |
|
Total |
(70) |
300 |
0 |
0 |
0 (0.0) |
0 (0.0) |
80 µg/ml |
A |
7.60 |
150 |
0 |
0 |
0 |
0 |
|
B |
6.25 |
150 |
0 |
0 |
0 |
0 |
|
Total |
(90) |
300 |
0 |
0 |
0 (0.0) |
0 (0.0) |
Mitomycin C 0.2 µg/ml |
A |
3.25 |
82* |
17 |
16 |
15 |
15 |
|
B |
2.45 |
150 |
16 |
14 |
15 |
13 |
|
Total |
(37) |
232 |
33 |
30 |
30 (12.9) |
28 (12.1)** |
* Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed
** P < 0.001
Frequency of polyploid cells was 0% in treatment groups ofthe test item.
Historical Aberration Ranges for Vehicle Control Cultures
1. 4(20)-hour exposure without S9
% cells with aberrations (-gaps): 0.48 (mean)
Standard Deviation: 0.56
% cells with polyploids: 0.05 (mean)
Standard Deviation: 0.17
2. 4(20)-hour exposure with S9 (1%)
% cells with aberrations (-gaps): 0.40 (mean)
Standard Deviation: 0.48
% cells with polyploids: 0.02 (mean)
Standard Deviation: 0.11
3. 24-hour exposure without S9
% cells with aberrations (-gaps): 0.44 (mean)
Standard Deviation: 0.52
% cells with polyploids: 0.07 (mean)
Standard Deviation: 0.22
4. 4(20)-hour exposure with S9 (2%)
% cells with aberrations (-gaps): 0.53 (mean)
Standard Deviation: 0.56
% cells with polyploids: 0.09 (mean)
Standard Deviation: 0.24
Historical Aberration Range for Positive Control Cultures
1. 4(20)-hour exposure without S9 (Mitomycin C)
% cells with aberrations (-gaps): 39.86 (mean)
Standard Deviation: 14.04
% cells with polyploids: 0.02 (mean)
Standard Deviation: 0.13
2. 4(20)-hour exposure with S9 (1%) (Cyclophosphamide)
% cells with aberrations (-gaps): 30.66 (mean)
Standard Deviation: 9.65
% cells with polyploids: 0.02 (mean)
Standard Deviation: 0.12
3. 24-hour exposure without S9 (Mitomycin C)
% cells with aberrations (-gaps): 39.09 (mean)
Standard Deviation: 14.57
% cells with polyploids: 0.02 (mean)
Standard Deviation: 0.013
There was evidence of moderate toxicity following exposure to the test item in 4-hour and 24-hour exposure group in the absence of metabolic activation only, as indicated by the %RSG and RTG values. There was no evidence of reductions in viability in any of the exposure groups, indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with the positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in any exposure group. The GEF was not exceeded at any test item dose level, and all values observed were within the acceptable range for a vehicle control. Precipitate of the test item was observed at 30 and 40 µg/mL in all three exposure groups.
Table 1: Summary of Results - main experiment 4-hours exposure
Treatment (µg/ml) |
4-hours-S-9 | Treatment (µg/ml) |
4-hours+S-9 | ||||
| %RSG | RTG | MF§ | %RSG | RTG | MF§ | ||
| 0 | 100 | 1.00 | 118.56 | 0 | 100 | 1.00 | 117.72 |
| 2.5 | 98 | 1.11 | 104.43 | 2.5 | 109 | 0.96 | 133.41 |
| 5 | 93 | 1.03 | 98.00 | 5 | 106 | 0.99 | 140.40 |
| 10 | 83 | 0.96 | 111.51 | 10 | 100 | 0.94 | 110.14 |
| 20 | 91 | 0.89 | 117.68 | 20 | 97 | 0.93 | 114.17 |
| 30 | 90 | 0.92 | 122.24 | 30 | 105 | 0.94 | 134.81 |
| 40 | 80 | 0.99 | 84.23 | 40 | 119 | 0.99 | 124.79 |
| Linear trend | NS | Linear trend | NS | ||||
| Ethylmethanesulphonate | Cyclophosphamide | ||||||
| 400 | 75 | 0.48 | 1303.80 | 1.5 | 71 | 0.34 | 1139.70 |
§ = Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis
Test for linear trend (4-hours-S-9)
Slope: -4.400E-007
Variance: 9.960E-014
b²/Sb: 1.944
Test for linear trend (4-hours+S-9)
Slope: 8.855E-008
Variance: 1.770E-013
b²/Sb: 0.044
Table 2: Summary of Results - main experiment 24-hours exposure
Treatment (µg/ml) |
24-hours-S-9 | ||
| %RSG | RTG | MF§ | |
| 0 | 100 | 1.00 | 135.04 |
| 2.5 | 102 | 1.12 | 127.88 |
| 5 | 91 | 1.09 | 140.68 |
| 10 | 98 | 1.18 | 107.01 |
| 20 | 81 | 0.90 | 114.38 |
| 30 | 76 | 0.98 | 137.10 |
| 40 | 76 | 0.82 | 154.02 |
| Linear trend | NS | ||
| Ethylmethanesulphonate | |||
| 150 | 42 | 0.47 | 840.12 |
Test for linear trend (24-hours-S-9)
Slope: 2.937E-007
Variance: 1.721E-013
b²/Sb: 0.502
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
based on the specific rules for adaptation set in Column 2 of Annex VIII of the REACH Regulation no further in vivo testing are required since only negative results were determined during the in vitro testing covering the tonnage of registration
Justification for classification or non-classification
According to the CLP Regulation (EC n. 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:
- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans
- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.
No evidence of positive results were determined in the three in-vitro tests covering mammalian gene mutation, mammalian (human) chromosome aberration and in vitro gene mutation study in bacteria. Therefore the substance is not classified for germ cell mutagenicity under Regulation 1272/2008.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
