Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The data from the bacterial mutation assays indicated no genotoxic potential of 7PPD (WTR 1990, Monsanto Co. 1976, 1977a, 1977b). This negative finding is confirmed by the results from a mammalian cell mutation assays (Monsanto Co. 1988). An in vitro chromosome aberration assay with CHO cells revealed weak clastogenic effects of 7PPD (Monsanto Co. 1989). Under in vivo conditions the potential clastogenic activity in vitro could not be confirmed. In an in vivo bone marrow chromosome aberration assay with Sprague-Dawley rats 7PPD did not significant increase the frequency of chromosome aberrations (Monsanto Co. 1989). In conclusion, based on the findings discussed above, the weight of evidence indicates a low or even a non-genotoxic potential of 7PPD in vitro and no genotoxicity in vivo and thus no classification is required.
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
no mitotic index determined but MTD reached; 3 times 50 instead of 2 times 100 cells scored
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromosome aberrations
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix, S9 from Aroclor 1254-induced rat liver homogenate (S9)
Test concentrations with justification for top dose:
pre-test I(+/-S9): 5, 20, 50, 100, 200, 500, 1000, 2000, 5000 µg/ml; pre-test II (+/-S9): 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 µg/ml,
main experiment I (+/-S9): 0, 7.5, 10, 15 µg/ml, main experiment II (-S9): 7.5, 10 µg/ml;
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: -S9: methylmethanesulfonate, +S9: cyclophosphamide
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
other: solvent aceton induced a relative high number of aberrant cells in main experiment I
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: Chinese hamster Ovary (CHO)
Remarks:
Migrated from field 'Test system'.

1. Range-Finding Experiments:

CHO cells were treated with 5 to 5000 µg/ml of SANTOFLEX 14 both in the presence and absence of activation. The cells

were scored for both mitotic index and average cell generation time and compared to the solvent (acetone) control. The average cell generation time for the solvent control was approximately 12 hours for both with and without activation, with a mitotic index of 5

to 8 %. Cytotoxic effects were observed at 20 µg/ml both in the absence and presence of activation as indicated by a sharp reduction in mitotic index and an extended average generation time.

The cytotoxicity of SANTOFLEX 14 was further defined in a second range-finding experiment where concentrations ranged

from 1 to 20 µg/ml with and without activation. The cells were scored for both mitotic index and average cell generation time. Cytotoxic effects were observed at 12.5 µg/ml and higher concentrations in the absence and presence of activation as indicated by an extended average generation time (-S9 20.3 h at 12.5 µg/ml vs. 12.0 control; +S9: at 12.5 µg/ml 15.2 h vs. 12.7 control).

Based on the range-finding experiments, the highest concentrations of SANTOFLEX 14 tested were 15 µg/ml with and without activation. The harvest times were 12 and 24 hours for 1.5, 5, 7.5 and 10 µg/ml with and without activation, 18 and 36 hours for 15 µg/ml with activation and 24 and 48 hours for 15 µg/ml without activation. The harvest times were

chosen to represent approximately 1X and 2X that of the cell generation times.

2. Cytogenetics Studies:

A. Without activation: The SANTOFLEX 14 levels of 7.5, 10 and 15 µg/ml were chosen as the highest three scorable doses.

Statistically significant increases in number of cells with structural aberrations (12 cells with aberrations vs. 3 cells with aberrations solvent control) and average structural aberrations per cell (0.060 vs. 0.015 solvent control) were observed at the 15 ug/ml dose level for the 48 hour harvest time ( % aberrant cells: 6 % vs. 1.5% solvent control) and for average structural aberrations per cell for the 24 hour harvest time (0.120 vs. 0.045 solvent control). A significant dose-response was not observed.

B. With activation: The SANTOFLEX 14 dose levels of 7.5, 10 and 15 µg/ml were selected as the three highest scorable

doses. Statistically significant increases in the number of cells with structural aberrations (at 10 µg/ml 39 vs. 15 solvent control) and average structural aberrations per cell (10 µg/ml: 0.383 vs. 0.090 solvent control) were observed for the 10 µg/ml dose level (% aberrant cells: 19.9 % vs. 7.5% solvent control) and for the number of cells with aberrations at the 7.5 µg/ml dose level for the 12 hour harvest time (at 7.5 µg/ml 29 cells with aberrations vs. 15 cells with aberration solvent control). A dose-related response was not observed.

C. Retest: Because of the relatively high aberration levels (-S9: 10%, +S9: 7.5%) for the 12 hour harvest time in the solvent control, the experiment was repeated using dose levels of 7.5 and 10 µg/ml with and without activation. The 24 and 48 hour harvests were not repeated as the solvent background was low and therefore the statistically significant response of the 15 ug/ml dose level without activation was concluded to be treatment induced. The 10 µg/ml dose level with activation was again statistically significant for the number of cells with aberrations ( 17 cells with aberrations vs. 6 cells solvent control) and for structural aberrations per cell (0.090 vs. 0.030 solvent control) A dose-response relationship was not observed.

Conclusion:

Without activation, a. statistically significant increase in aberration frequency was observed for the 15 µg/ml dose level. In the presence of activation, a statistically significant response was observed at the 10 µg/ml dose level for the 12 hour harvest.

A significant dose-response relationship was not observed. The retest experiment confirmed the statistically significant

response of the 10 µg/ml dose level in the presence of activation. A significant dose-response was not observed.

The authors suggested that the observed effects might be secondary because the effects were observed in the range of cytotoxicity indicated by an increased generation time (10 µg/ml +S9: pre-experiment: 13.7h vs. 12.7 h solvent control, 15 µg/ml -S9: 22.0 h vs. 12.1 h solvent control) and in addition because of a lack of a dose response relationship.

Conclusions:
Interpretation of results (migrated information):
positive
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro data : The mutagenic potential in bacteria of the test substance 7PPD was evaluated in a GLP study (WTR 1990). Here, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 were used. Treatment by the pour-plate method was done. Cytotoxicity was determinate in a preliminary toxicity test using Salmonella typhimurium tester strain TA 98. The lowest level of 7PPD causing visible thinning of the background lawn of non-revertant cells was 250 µg/ml; based on this finding 250 µg/plate was selected as the top exposure level in the main experiment. No increases in revertant colony numbers over control counts were obtained with any tester strains following exposure to 7PPD at concentration levels from 2.5 µg to 250µg/plate. Inhibition of growth, observed as thinning of the background lawn of non-revertant cells occurred in all strains following exposure to 7PPD at 250µg/plate, and in strain TA 1535 only with 7PPD at 79 µg/plate. The authors concluded, that 7PPD was negative in the Ames assay, under the experimental conditions used. In addition, in several other earlier bacterial mutation assays the test substance also indicated a non-mutagenic potential (Monsanto Co. 1976, 1977a, 1977b). The negative findings from the bacterial mutation assays are confirmed in a mammalian cell mutation assay. The test substance 7PPD was negative in a HGPRT assay, done with CHO cells (Monsanto Co. 1988). Initial cytotoxicity experiments were conducted with 7PPD in CHO cells at different S9 concentrations. The concentrations that induced significant cytotoxicity were 7µg/ml, 10 µg/ml, 30 µg/ml, and 70 µg/ml in the absence of S9 and in the presence of 1%, 2%, 5% and 10%, S9, respectively. An initial experiment to determine the potential mutagenicity of the test material was conducted using a range of S9 concentrations. In this experiment, 7PPD was significantly cytotoxic to the CHO cells at levels of 5µg/ml and greater in the absence of exogenous metabolic activation. In the presence of S9 activation, significant cytotoxicity was demonstrated for treatment levels of 7 µg/ml, >10 µg/ml, 20 µg/ml and 70 µg/ml in the presence of 1%, 2%, 5% and 10% S9, respectively. No statistically significant increases in mutation frequency were observed in any of the 7PPD treated cultures. The non-mutagenicity of 7PPD was confirmed by a subsequent experiment. In this experiment, 7PPD was tested at 1, 3, 5, 7, and 10 µg/ml in the absence of S9 and at 10, 15, 20, 25, and 30 µg/ml in the presence of 5 % S9 activation. 7PPD was observed to be significantly cytotoxic at levels of 5 µg/ml and greater in the absence of S9. In the presence of 5% S9, 7PPD was not cytotoxic at 30µg/ml. No statistically significant increases in mutant frequency were observed in this experiment. A chromosomal aberration test with CHO cells was performed to determine the clastogenic potential of 7PPD (Monsanto 1989). In a range-finding experiment CHO cells were treated with 5 to 5000 µg/ml of 7PPD both in the presence and absence of activation. The cells were scored for both mitotic index and average cell generation time and compared to the solvent (acetone) control. The average cell generation time for the solvent control was approximately 12 hours for both with and without activation, with a mitotic index of 5 to 8 %. Cytotoxic effects were observed at 20µg/ml both in the absence and presence of activation indicated by a sharp reduction in mitotic index and an extended average generation time. The cytotoxicity of 7PPD was further defined in a second range-finding experiment where concentrations ranged from 1 to 20 µg/ml with and without activation. The cells were scored for both mitotic index and average cell generation time. Cytotoxic effects were observed at 12.5 µg/ml and higher concentrations in the absence and presence of activation as indicated by an extended average generation time (-S9 20.3 h at 12.5 µg/ml vs. 12.0 control; +S9: at 12.5 µg/ml 15.2 h vs. 12.7 control). Based on the range-finding experiments, the highest concentrations of 7PPD tested were 15µg/ml with and without activation. The harvest times were 12 and 24 hours for 1.5, 5, 7.5 and 10 µg/ml with and without activation, 18 and 36 hours for 15 µg/ml with activation and 24 and 48 hours for 15µg/ml without activation. In the main experiment without metabolic activation 7PPD levels of 7.5, 10 and 15 µg/ml were chosen as the highest three scorable doses. Statistically significant increases in number of cells with structural aberrations (12 cells with aberrations vs. 3 cells with aberrations solvent control) and average structural aberrations per cell (0.060 vs. 0.015 solvent control) were observed at the 15 µg/ml dose level for the 48 hour harvest time (% aberrant cells: 6 % vs. 1.5% solvent control) and for average structural aberrations per cell for the 24 hour harvest time (0.120 vs. 0.045 solvent control). A significant dose-response was not observed. In the main experiment with metabolic activation 7PPD dose levels of 7.5, 10 and 15 µg/ml were selected as the three highest scorable doses. Statistically significant increases in the number of cells with structural aberrations (at 10 µg/ml 39 vs. 15 solvent control) and average structural aberrations per cell (10 µg/ml: 0.383 vs. 0.090 solvent control) were observed for the 10 µg/ml dose level (% aberrant cells: 19.9 % vs. 7.5% solvent control) and for the number of cells with aberrations at the 7.5 µg/ml dose level for the 12 hour harvest time (at 7.5 µg/ml 29 cells with aberrations vs. 15 cells with aberration solvent control). A dose-related response was not observed. Because of the relatively high aberration levels (-S9: 10%, +S9: 7.5%) for the 12 hour harvest time in the solvent control, the main experiment was repeated using dose levels of 7.5 and 10 µg/ml with and without activation. The 24 and 48 hour harvests were not repeated as the solvent background was low and therefore the statistically significant response of the 15 µg/ml dose level without activation was concluded to be treatment induced. The 10 µg/ml dose level with activation was again statistically significant for the number of cells with aberrations (17 cells with aberrations vs. 6 cells solvent control) and for structural aberrations per cell (0.090 vs. 0.030 solvent control) A dose-response relationship was not observed. The authors concluded that 7PPD had a weak clastogenicity in CHO cells with and without metabolic activation. Because the significant increases in aberrant cells were observed in the range of cytotoxicity secondary effects could not be excluded. In vivo data The genotoxic potential of 7PPD was evaluated in an in vivo bone marrow chromosome aberration assay with Sprague-Dawley rats (Monsanto 1989). A dose range finding experiment was performed to select the maximum tolerated dose. Rats were dosed per gavage with 1050, 1100, 1200, 1500 and 2000 mg/kg bw. Mortality was observed in animals treated with 1500 and 2000 mg/kg test substance. Animals dosed with 1100 and 1200 mg/kg bw exhibited moderate to severe clinical signs. No clinical signs were noted in animals which were dosed with 1050 mg/kg bw. Based on these findings, a dose level of 1100 mg/kg was chosen for the main experiment. In the main experiment male and female rats (five per sex and treatment) were administered single oral with the 1100 mg/kg bw. The treated animals were sacrificed 6, 18 and 30 hours after dosing. Concurrent solvent controls were also included for all preparation time points. An extra group of rats was dosed with the positive control cyclophasphamid and sacrificed 18 hours later. Approximately two hours prior to each sacrifice, animals were administered colchicine to arrest cells in metaphase. At the appropriate time, animals were sacrificed and both femurs were removed from each animal and metaphase slides were prepared. A total of 50 metaphase cells were analyzed for each animal. The occurrences of clinical signs were observed during the study. No clinical signs were observed in animals administered 7PPD immediately after dosing; whereas clinical signs were noted prior colchicine administration in all treatment groups. In the 6 hour group all males and females had decreased body tone. In addition all females had vocalization on touch and two females had arched back and abnormal stance. In the 18 hour group two males had decreased body tone. One male had vocalization when touched, abnormal stance and arched back. Another male had piloerection and diarrhea. All females had decreased body tone with four females exhibiting piloerection and abnormal stance. Two females made vocalization when toughed. One female had also arched back. In the 30 hour group all treated rats had decreased body tone and piloerection. Three males and two females made vocalizations when touched. One male and one female had an arched back. In addition, one female had abnormal gait. Based on these findings the authors suggested that rats dosed with 1100 mg/kg 7PPD exhibited moderate to severe clinical signs indicating that these animals were dosed at or near the maximum tolerated dose. One male of the positive control group exhibited decreased body tone. No clinical signs were observed in animals from the solvent control. No statistically significant increase in number of aberrations or in the number of aberrant metaphases were noted in 7PPD treated animals in any of the three sacrifice times compared to the corresponding solvent control; whereas significant increased were noted in the positive control. Based on these findings the authors concluded that 7PPD (1100 mg/kg) was negative in its ability to induce structural chromosomal aberrations to the hemopoietic cells of the rat bone marrow under the experimental conditions of this assay (Monsanto 1989)

Justification for classification or non-classification

No classification is required according to the classification criteria 67/548/EWG and regulation no. 1272/2008 (GHS).