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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
HPRT, OECD 476 and GLP: negative Ames, OECD 471 and GLP: positive with TA98 in the presence and absence of S9 Ames II with highly purified test material: negative In vivo micronucleus study in hamsters: negative
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: modified OECD 471 protocol (Ames II), non-GLP
Principles of method if other than guideline:
Liquid fluctuation Ames II test with and without liver S9 mix from induced male Wistar rats.
The test method is used to evaluate the mutagenic potential based on the ability to induce point mutations in selected loci of several strains of Salmonella typhimurium in a modified version of the Ames test (OECD 471), designated Ames II Assay (microtiter version), both with and without the addition of a metabolizing system (S9 mix) obtained from liver from induced rats.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
other: TA 98, TA Mix (TA 7001 - TA 7006)
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix
Test concentrations with justification for top dose:
O; 4; 20; 100; 500; 2500 and 5000 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (with S9) and 2-nitrofluorene plus 4-nitroquinoline- N-oxide (without S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Bacteria from overnight cultures showing an optical density of at least 2.0 (measured at a wavelength of 600 nm) were used. 5 ml of the overnight cultures were added to tubes containing 25 ml Ames II Exposure medium and were gently mixed. After thorough pipetting bactiera suspension, test substance (or vehicle or control) and S9 mix (if applicable) were added. The 24-well plates were incubated at 37°C with shaking at 250 rpm for about 90 minutes. After this incubation period, 2.8 ml Ames II Reversion indicator medium (containing bromocresol purple) was pipetted to each well of the 24-well plate. The contents of each well of the 24-well plates were distributed in 50 μl aliquots over 48 wells of a 348-well Revertant Colony Selection plate (RCSP). The plates were sealed in plastic bags and incubated at 37°C in the dark. After 48 hours incubation each 48-well section of the RCSP were scored and the number of positive wells (yellow= high number of his+ revertants) were counted.

NUMBER OF REPLICATIONS: 48 wells in triplicate plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: beckground lawn, decrease in the number of yellow wells
Evaluation criteria:
Evaluation was performed by the following comparisons/calculation:
- An increase in the mean number of positive wells in dose groups was compared to the mean value of the concurrent negative control (Evaluation factor 1 F).
- An increase in the mean of revertant wells in dose groups was calculated on the basis of the baseline data of the actual experiment (Evaluation factor 2 F). The baseline was derived from the mean spontaneous revertant number plus the value of standard deviation (mean+ SD) from the distribution of spontaneous data.
- An increase in the mean of revertant wells in dose groups was calculated on the basis of the baseline data of an experimental run (Evaluation factor 3 F). A run consists of a variable number of experiments generally testing different test substances together each using the same vehicle control. This leads to an accumulation of replicates for negative controls which was used to calculate the mean spontaneous reversion number for each run.
A test substance is considered mutagenic in this test system if more than a doubling of Evaluation factor 3 F is observed in at least one test group. This finding should be dose-dependent and/or reproducible.
Species / strain:
other: TA 98, TA Mix (TA 7001 - TA 7006)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
An increase in the number of positive wells (his+ revertants) was not observed either without S9 mix or after the addition of a metabolizing system.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No bacteriotoxic effect (clearing of the background lawn, decrease in the number of yellow wells) was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 500 μg/ml and above
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

SUMMARY OF RESULTS

TA98 without S9 mix

DOSE [µg/ml] REM RCSP 1 RCSP 2 RCSP 3 MEAN MEANC SD 1F 2F 3F
DMSO   0 0 1 0.3 1.0 0.47 1.0 0.7 0.5
4 1 0 1 0.7 0.47 0.7 0.5 0.3
20 0 1 0 0.3 0.47 0.3 0.2 0.2
100 2 1 1 1.3 0.47 1.3 0.9 0.6
500 p 3 4 2 3.0 0.82 3.0 2.0 1.4
2500 p 1 1 2 1.3 0.47 1.3 0.9 0.6
5000 p 3 1 2 2.0 0.82 2.0 1.4 1.0
4-NQO+2-NF 43 41 47 43.7 2.49 43.7 29.7 20.9

TA98 with S9 mix

DOSE [µg/ml] REM RCSP 1 RCSP 2 RCSP 3 MEAN MEANC SD 1F 2F 3F
DMSO 0 1 1 0.7 1.0 0.47 1.0 0.7 0.4
4 3 1 1 1.7 0.94 1.7 1.1 0.6
20 0 2 3 1.7 1.25 1.7 1.1 0.6
100 0 5 1 2.0 2.16 2.0 1.4 0.8
500 p 5 3 1 3.0 1.63 3.0 2.0 1.1
2500 p 5 1 1 2.3 1.89 2.3 1.6 0.9
5000 p 4 1 2 2.3 1.25 2.3 1.6 0.9
2-AA 48 48 48 48.0 0.00 48.0 32.6 18.4

TA Mix without S9

DOSE [µg/ml] REM RCSP 1 RCSP 2 RCSP 3 MEAN MEANC SD 1F 2F 3F
DMSO 1 1 0 0.7 1.0 0.47 1.0 0.7 0.8
4 0 0 0 0.0 0.00 0.0 0.0 0.0
20 1 0 2 1.0 0.82 1.0 0.7 0.8
100 0 0 0 0.0 0.00 0.0 0.0 0.0
500 p 0 0 1 0.3 0.47 0.3 0.2 0.3
2500 p 1 0 0 0.3 0.47 0.3 0.2 0.3
5000 p 0 1 0 0.3 0.47 0.3 0.2 0.3
4-NQO+2-NF 46 47 46 46.3 0.47 46.30 31.50 37.4

TA Mix with S9

DOSE [µg/ml] REM RCSP 1 RCSP 2 RCSP 3 MEAN MEANC SD 1F 2F 3F
DMSO   0 1 2 1 1 0.82 1.0 0.6 0.8
4 0 0 0 0.0 0.00 0.0 0.0 0.0
20 1 0 0 0.3 0.47 0.3 0.2 0.3
100 2 2 1 1.7 0.47 1.7 0.9 1.3
500 p 0 1 0 0.3 0.47 0.3 0.2 0.3
2500 p 0 0 0 0.0 0.00 0.0 0.0 0.0
5000 p 2 0 0 0.7 0.94 0.7 0.4 0.5
2-AA 19 20 23 20.7 1.70 20.7 11.4 15.5

REM = Remarks

P = Precipitation

RCSP = Revertant Colony Selection Plate (384-Well)

SD = Standard Deviation

MEAN = Mean of Replicates

MEANC = Mean corrected : < 1 = 1

F = Factor

1F = Based on Meanc

2F = Baseline Data I Based on Meanc + SD

3F = Baseline Data I Based on Mean + SD of a Run

4-NQO + 2-NF = 4-nitroquinoline: 5 μg/ml + 2-nitroflourene 100 μg/ml

2-AA = 2-aminoanthracene: 125 μg/ml

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions of this study, the test substance is not a mutagenic substance in the Ames II Assay (Salmonella typhimurium reverse mutation assay) in the absence and the presence of metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

AMES-Test

In a GLP compliant study following OECD guideline 471, the test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium TA 1535, TA100, TA1537, TA 98 and Escherichia coli WP2 uvrA, in a reverse mutation assay at dose levels of 33 μg - 5000 μg/plate. A standard plate test (SPT) was performed with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix. No bacteriotoxic effect was observed under all test conditions. According to the results of the present study, the test substance led to a dose dependent increase in the number of his+ revertants with the strain TA 98 both with and without S9 mix. The increase of revertants was reproducible in two experiments carried out independently of each other. Based on the recent assessment criteria the test substance has to be considered positive. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data. Thus, under the experimental conditions chosen here, it is concluded that the test item is a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation (BASF, 2015).

This finding was confirmed in an older supporting study. This study was performed with the following concentrations with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 mL. In the experiments without and with microsomal activation, the number of back-mutant colonies of Strain TA 98 was significantly greater after treatment with the test substance than in the controls. A slight increase in the number of back-mutant colonies was also observed in the experiments without microsomal activation on Strain TA 1535 and TA 1537. The test item thus exerted a mutagenic potential in this test system (Ciba-Geigy, 1979).

A third Ames test is available yielding negative results. In this study, the test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies (RCC, 2007).

The positive results described above are most likely triggered by an unknown impurity, since the third available study using test material from a different batch produce a clear negative result. To confirm this hypothesis, the test article batch that caused a positive result in the latest Ames test was further purified and a follow-up Ames II study was performed with this highly purified test material. To ensure comparability of the Ames II findings, the test material of the standard purity (before the additional purification step) was also investigated in an Ames II study.

In the Ames II study with the standard purity test material, two vehicles were used (ultrapure water and DMSO) in order to investigate if the positive finding observed before could be related to chemical interaction of the unknown impurity present in the test item with DMSO. According to the results of this study, the test item led to an evident and partly dose-dependent increase in the number of revertant wells with the strain TA 98 with and without S9 mix using vehicle DMSO and only with S9 mix using vehicle ultrapure water. Thus, under the experimental conditions of this study, the test substance is a mutagenic substance in the Ames II Assay (Salmonella typhimurium reverse mutation assay) in the absence and the presence of metabolic activation. The magnitude of the positive finding in tester strain TA98 with S9 mix was similar for both vehicles, therefore the positive reaction observed is apparently independent from the vehicle used (BASF, 2015).

In the follow-up Ames II study using the highly purified test material of the same batch, only DMSO was used as vehicle. According to the results of this study, an increase in the number of positive wells (his+ revertants) was not observed either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the purified test substance is not a mutagenic substance in the Ames II Assay (Salmonella typhimurium reverse mutation assay) in the absence and the presence of metabolic activation. It can therefore be concluded, that the positive results obtained previously were indeed due to an unknown impurity. The additional purification step removed this unknown impurity and a positive result was no longer obtained (BASF 2016).

This purification step will be implemented into production and all future batches produced will be characterized by this increased purity that does not lead to a positive result in the Ames test.

HPRT-Test

A GLP-compliant study following OECD guideline 476 was performed to assess the test item's potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. In both experiments the treatment period was 4 hours with and without metabolic activation. The main experiments were evaluated at concentration range of 0.41 - 830 µg/ml. Precipitation of the test item was observed in the first experiment at 11.1 µg/mL and above with and without metabolic activation. In experiment II precipitation was noted at 80 and 830 µg/mL with and without metabolic activation. No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures was observed up to the highest concentration of both experiments with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutant frequency generally did not exceed the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of the first experiment without metabolic activation at 1.2 and 3.7 µg/mL. This effect however, was based upon a rather low solvent control of 6.0 mutant colonies/106 cells and thus, biologically irrelevant. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of the second experiment without metabolic activation. However, the trend was judged as biologically irrelevant as the mutation frequency did not exceed the threshold described above or the historical range of solvent controls. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.0 up to 28.6 mutants per 106 cells; the range of the groups treated with the test item was from 0.0 up to 39.5 mutants per 106 cells. EMS (150 µg/mL) and DMBA (2.2 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay (Harlan, 2015).

Genetic toxicity in vivo

The potential of the test item to cause genotoxicity was observed in two in vivo nucleus anomaly tests. In the key study the test item was administered by gavage thrice weekly for twelve weeks to Chinese hamsters at dose levels of 250, 500, 1000 and 2000 mg/kg. The animals were sacrificed six hours after the last application. From the bone marrow smears were made. The bone marrow smears from animals treated with various doses of the test item showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item (Ciba-Geigy, 1981)

In the supporting study Chinese hamsters were treated by gavage with one daily application on 2 consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made. The bone marrow smears from animals treated with various doses of the test item showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 9.4% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (PEG 400) alone (Ciba-Geigy, 1980).

Two sister chromatid exchange assays are available for the test article. In the first assay (Ciba-Geigy, 1980), a positive result was reported in the highest dose group. However, this positive result was mainly triggered by the result obtained in one animal; with the other animals of this dose group the responses were similar to the control group. Therefore, this one finding might have been a chance finding and a confirmation experiment was performed with more dose groups and double animal numbers. This repeat assay clearly demonstrated that oral treatment with the test item does not trigger an increase in sister chromatid exchanges (Ciba-Geigy, 1983).


Justification for selection of genetic toxicity endpoint
The most recent Ames study with highly purified test material was selected, showing that the previously reported positive findings were due to an unknow impurity that is no longer present following an additional purification step.

Justification for classification or non-classification

Positive findings in two Ames studies were initially available for the test material, contradicted by a negative result in an Ames test using a different test material batch. It could be shown that these positive findings were due to an unknown impurity. After further purification of the positive tested batch, the positive finding could no longer be reproduced. It can therefore be concluded that the additional purification step has removed the unknown impurity responsible for the positive findings. Since this additional pusrification step will be implemented into the standard production of the test article, there is no longer any concern regarding the positive Ames findings previously reported.

The test article was further negative in an HPRT study, negative in two in vivo micronucleus assays, and negative when tested for sister chromatid exchanges. Based on the available data, classification for genotoxicity is not warranted.