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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
The data from the bacterial mutation assays indicated no genotoxic potential of 6PPD (Hatano Research Institute 1999, Monsanto Co. 1976, 1991b, Zeiger 1987). This finding is confirmed by the results from mammalian cell mutation assays in CHO cells (Monsanto Co 1987b, and OECD SIDS 2005) and in the Mouse Lymphoma Assay (Monsanto Co. 1977). An in vitro chromosome aberration assay revealed clastogenic effects of 6PPD (Tanaka et al., 1999). In addition, an ambigous result for clastogenicity was noted in CHO cells treated with 6PPD (Monsanto Co. 1987c). No clastogenic effects were noted in another in vitro chromosome aberration assay using CHO cells (NTP, 1987). 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 6PPD did not significant increase the frequency of chromosome aberrations (Monsanto Co. 1988). In addition, 6PPD showed no clastogenic activity in the cytogenetic assay in vivo or the micronucleus test in vivo (George and Kuttan, 1996; OECD SIDS 2005). In conclusion, based on the findings discussed above, the weight of evidence indicates a low or even a non-genotoxic potential of 6PPD in vitro and no genotoxicity in vivo and thus no classification is required. This conclusion is in line with the OECD SIDS report (2005).
Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
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: comparable to guideline study
Principles of method if other than guideline:
other: in vivo bone marrow cytogenetics rat metaphase analysis
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: gavage
Duration of treatment / exposure:
once
Frequency of treatment:
once
Post exposure period:
6, 18 and 30 h
Remarks:
Doses / Concentrations:
preliminary study: 900, 1300, 1790 mg/kg bw, main experiment: 1000 mg/kg bw
Basis:

No. of animals per sex per dose:
pre-test: 1 per dose and sex, main test: 5 per dose and sex and sampling time
Control animals:
yes, concurrent vehicle
Tissues and cell types examined:
bone marrow from femur
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid

Range-finding experiment:

Clinical signs and necropsy

1790 mg/kg:

Male: diarrhea, decreased body tone and activity, tremor, abnormal gait and stance, piloerection and redish-brown discoloration around the nasal and oral regions, net weight loss 10 grams, dead by day 2

Necropsy: hemorrhagic areas on the thymus and stomach lining, stomach and intestines were distended and fluid filled

Female: piloerection, decreased body tone and decreased activity, net weight loss 11 grams, dead by day 3

Necropsy: dark red lungs and adrenals, and distended fluid-filled stomach and intestines

1300 mg/kg bw:

Male and female: prostrate and moribund by day 1, by day 2 both rats were dead

necropsy: grossly distended stomach filled with reddish discolored material

900 mg/kg:

Male/female: decreased body tone, abnormal gait and stance, piloerection and diarrhea on day 1. Male also exhibited decreased activity; on day day, the male exhibited poor grooming and decreased body tone, the female exhibited an abnormal gait and stance. no signs were observed on day 7 prior to study termination.

Main experiment

Clinical signs 1000 mg/kg

No pharmacotoxic signs of treatment were observed in animals administered Santoflex 13 immediately after dosing. Prior to colchicine, however, signs observed in all groups treated with Santoflex 13 were as follows:

6 Hour Group: (observed approximately 4 hours after dosing); all rats had abnormal gait and decreased body tone with most of the rats also exhibiting decreased activity and abnormal stance. One male and one female exhibited piloerection and one male had body drop (skeletal muscle weakness demonstrated by lowered body position)

18 Hour Group (observed approximately 16 hours after dosing); all rats had abnormal gait and stance and most of the rats also had decreased body tone and activity. Two males had diarrhea and one female had lacrimation and a yellow discoloration at the anal-genital region. Two females also exhibited tremors and piloerection at this time.

30 Hour Group (observed approximately 28 hours after dosing); all rats had diarrhea and decreased body tone, with most of the rats also exhibiting piloerection, decreased activity and/or abnormal gait and stance. Signs observed in three or less animals included body drop, lacrimation, ataxia, tremors and poor grooming around the oral, nasal region.

No pharmacotoxic signs were observed in rats administered the vehicle or positive controls with the exception of one male in the 6 hour corn oil group which exhibited diarrhea prior to colchicine administration.

Summary of Metaphases Scored:

A total of 500 metaphases/group (50/rat) were analyzed in each group, except for the CP group in which only a total of 435

metaphases were analyzed.

Proportion of Cells with Aberrations:

The proportion of cells with one or more aberrations (1 aberration or greater) was analyzed by Chi-square analysis by group comparing the number of cells with aberrations versus the number of normal cells. Each time of sacrifice was analyzed separately and groups within each sacrifice time were compared individually to the vehicle control. Cells with gaps only were not considered aberrant for this analysis. A statistically significant increase (p 0.01) was detected with the positive control CP group at the 18 hour sacrifice. No other significant differences were noted.

Analysis of Aberrations per Cell:

The mean number of aberrations per cell per animal were analyzed for statistically significant increases by a one way

ANOVA for each time interval. Data reflect the total number of aberrations seen in all cells scored per group. No statistically significant differences (p 0.05 or p 0.01) from the vehicle controls were detected by this analysis in animals treated with Santoflex 13.

Santoflex 13 treated groups were also analyzed by one way ANOVA for time related differences in aberrations/cell/animal when compared to the vehicle control groups. No statistically significant differences were observed.

Animals treated with CP gave a statistically significant (p 0.01) increase in the number of aberrations.

All rats dosed with Santoflex 13 (1000 mg/kg) exhibited from mild to severe pharmacotoxic signs at all time intervals evaluated. These observations suggest that Santoflex 13 was tested near the maximum tolerated dose. Statistical analysis of the data indicated that Santoflex 13 (1000 mg/kg) did not produce significant increases in the number of aberrations or in the

number of aberrant metaphases at any of the three sacrifice times evaluated.

The authors concluded that Santoflex 13 (1000 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.

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

Additional information

Additional information from genetic toxicity in vivo:

In vitro data

The mutagenic potential in bacteria of the test substance 6PPD was evaluated in a GLP and OECD guideline study (TG 471). Here, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA were used. Treatment by the pre-incubation method was done for doses up to 5000 µg/ plate or even lower if toxicity was indicated. The tester strains were evaluated with and without metabolic activation. In this study no biologically relevant and dose dependent increases in revertants was observed in any of the tester strains evaluated with and without metabolic activation (Hatano Research Institute 1999). In addition, in several other bacterial mutation assays the test substance also indicated a non-mutagenic potential (Monsanto Co. 1976, 1991b, Zeiger 1987). This is confirmed by a considerable number of additional negative Ames Test reports for 6PPD (details not given).

The negative findings from the bacterial mutation assays were confirmed in mammalian cell mutation assays. The test substance 6PPD was negative in HPRT tests on CHO cells (Monsanto Co. 1987b, and OECD SIDS 1985) and in a mouse lymphoma assay with L5178Y cells, with S-9 mix up to a dose of 16 µg /ml and without S9-mix up to a dose of 4 µg/ ml; toxic effects were found with and without S-9 mix in high doses (8 µg/ml without S9, 33 µg/ml with S9) (Monsanto Co 1977).

A chromosomal aberration test with Chinese hamster lung cells (CHL/IU) was performed with harvest times of 6 hours (short-term treatment) both with and without metabolic activation (S9-mix) and with harvest times of 24 and 48 hours (continuous treatment) in the absence of a metabolic activation system. No increase of aberrations was observed for the short-term exposure with test concentrations of 0.00063-0.0025 mg/ml without S9-mix (cytotoxicity from 0.0050 mg/ml), and of 0.0038-0.015 mg/ml with S9-mix (cytotoxicity from 0.030 mg/ml). For the continuous exposure with test concentrations of 0.0025-0.010 mg/ml (cytotoxicity from 0.020 mg/ml) a significant increase in the number of cells with aberrations was reported (24 hour harvest: from 0.005 mg/ml dose-dependently; 48 hour harvest: at 0.01 mg/ml) out of the cytotoxic range. The study was performed according to OECD TG 473 and the Japanese Guidelines for Screening Toxicity Testing of Chemicals (Tanaka et al., 1999).

In addition, a low clastogenic response was noted in CHO cells treated with 6PPD (Monsanto Co. 1987c). A significant increase (p<0.05) in cells with chromosomal aberrations were noted at the 24 hours harvest time at concentration of 10 µg/ml with metabolic activation (6.0 % vs. 1.0% control) and at 10 µg/ml and 12.5 µg/ml without metabolic activation (3.0%, 3.3% vs. 0 % control). Because of the low magnitude of response, the authors conclude that the biological significance of the findings is questionable. No clastogenic effects were noted in another in vitro chromosome aberration assay using CHO cells (NTP, 1987) and in several in vivo assays (see below).

Two in vitro tests for induction of Unscheduled DNA Synthesis (UDS Test) in primary rat hepatocytes showed no genotoxic potential for 6PPD (OECD SIDS 2005, Monsanto 1986).

In vivo data

The genotoxic potential of 6PPD was evaluated in an in vivo bone marrow chromosome aberration assay. Male and female Sprague-Dawley rats were administered with 1000 mg/kg bw test substance once via gavage. Groups of 5 males and 5 females (treatment group and vehicle control each) were sacrificed at 6, 18 and 30 hours following test substance administration. The body weight and clinical observations were recorded during the study. The bone marrow cells were prepared and at least 50 mitotic cells per animal were analysed for cytogenetic aberrations. Clinical signs were observed for all 1000 mg/kg treated animals at 6 and 18 and 30 hours after application. The clinical signs observed were abnormal gait and decreased body tone with most of the rats also exhibiting decreased activity and abnormal stance. Several animals also exhibited piloerection, diarrhea, lacrimation, tremor, body drop, staining of the anal-genital region and poor grooming around the oral, nasal region. No pharmacotoxic signs were observed in rats administered the vehicle or positive controls with the exception of one male in the 6 hour corn oil group which exhibited diarrhea prior to colchicine administration. Results from the 6, 18 and 30 hours sacrifice data show that no statistically significant increase in the frequency of chromosome aberrations compared to control values was seen in the groups treated with 6PPD. A statistically significant increase (p 0.01) was detected with the positive control. The authors concluded that Santoflex 13 (1000 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 Co. 1988).

In addition, no mutagenic effects were noted in a Chromosomal Aberration Test on Swiss mice treated with 6PPD. Neither a statistically significant increase of aberrant cells nor an increase in miconucleated cells in the bone marrow of treated mice was observed (George and Kuttan, 1996).

6PPD was shown to be negative in two mouse bone marrow micronucleus assays (OECD SIDS 2005, George and Kuttan, 1996).


Justification for selection of genetic toxicity endpoint
see discussion; in vitro and in vivo experiments are available.

Justification for classification or non-classification

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