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In vitro mutagenicity

Bacterial gene mutation assays (Ames Test)

DCDPS was tested for mutagenicity in Salmonella typhimuriumstrains TA97, TA98, TA100, and TA1535 according to a protocol comparable to the OECD TGD 471 standards (NIH, 2001). The applied doses were 0, 10, 33, 100, 333, and 1000 µg/plate. Each concentration was tested in triplicates. Concurrent vehicle and positive controls were included. The tests were conducted using the preincubation protocol in the absence and presence of exogenous metabolic activation (liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters). DCDPS was not cytotoxic, but precipitated at concentrations of 100 µg/plate and higher. DCDPS gave a non-mutagenic response under all conditions.

In a further study, DCDPS was tested in Salmonella typhimuriumstrains TA 98, TA 100, TA1535, TA 1537 and TA 1538 in the presence and absence of a metabolic activation system (S9 from induced rat livers) (Callander, 1982). The protocol was equivalent to the plate incorporation protocol of OECD TG 471. The applied concentrations were 1.6, 8, 40, 200, 1000, and 5000 µg/plate. Each concentration was tested in triplicate. Concurrent negative, vehicle (DMSO) and positive controls were included. Two independent experiments were performed. DCDPS was not cytotoxic, but precipitated at 5000 µg/plate. In both the presence and absence of an auxiliary metabolising system, the compound failed to induce any significant increase in the spontaneous mutation rates. Under the conditions of these assays, DCDPS therefore gave an unequivocal negative, i.e. non-mutagenic, response.

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Mammalian cell gene mutation assays

HPRT assay. DCDPS was tested in the CHO/HGPRT mutation assay in the absence and presence of S9 using a protocol comparable to the OECD TGD 476 standards (Jacobson-Kram & Sigler, 1991). The test item was tested beyond its limit of solubility under culture conditions, in the initial assay at dose levels of 2137, 1068.5, 534.25, 267.13, and 133.6 µg/mL in both the absence and the presence of S9 and in the confirmatory assay at dose levels of 2525, 1263, 631.3, 316, and 158 µg/mL in both the absence and presence of S9. Concurrent negative and positive controls were included. DCDPS was negative in this test both in the absence and presence of exogenous metabolic activation.

 

Mouse Lymphoma L5178Y Assay

Riach (1994) reported the results of the mutagenic potential of DCDPS in the mouse lymphoma L5178Y assay both in the presence and absence of the S9 activating system. Four independent mutation assays (two assays in the presence and two assays in the absence of S9, using the agar method) were performed at dose levels of 10, 20, 40, 80, and 160 µg/mL. Concurrent solvent and positive controls were included. Precipitation of the test material was evident at ≥ 80 µg/mL DCDPS. Concentration dependent cytotoxicity occurred in all assays in the absence and presence of S9. In order to reach critical levels of toxicity, the exposure period was increased from 4 to 6 hours.

Under these conditions, in the presence of S9, no significant increases in mutant cell fraction were obtained in one experiment. Marginal increases of 1.6 to 1.7-fold were obtained in some cultures in a second experiment; however, no dose level met the minimum criterion for a positive response set forth in this study, i.e. 1.7 fold and higher increase in mutant cell fraction according the method by McGregor et al (1988). Furthermore, the mutation frequency (MF) in all cultures was less than 90 x 10-6above concurrent control level, which is the criterion for a positive response according to latest recommendations (Kirkland et al, 2006; Moore et al., 2006). The experiments in presence of S9 are classified negative.

In the absence of S9, the mutation frequency (MF) was more than 1.7 fold above the control value at DCDPS concentrations of ≥ 40 µg/mL in the first and second assay. Based on this increase in MF the study author concluded that DCDPS produced a mutagenic response in the mouse lymphoma L5178Y assay in the absence of S9 fraction at concentrations where mean cell survival is low (ranging from 19.5 to 28 %). A re-assessment of the study results applying the latest recommended evaluation criterion for the agar method (Kirkland et al, 2006; Moore et al., 2006) revealed that only the concentration of 40 and 160 µg/mL in the first assay and 160 µg/mL in the second assay caused a significant response as defined as an increased MF of more than 90 x 10-6above concurrent control level (see table below). However, the significant increases in MF were accompanied by strong cytotoxicity, i.e. 22 % relative total growth (RTG) at 40 µg/mL (first assay) and 19.5 % and 20 % RTG at 160 µg/mL (first and second assay, respectively). This indicates that cytotoxicity and not a mutagenic potential of the test substance might have caused the significant increase in MF. The strong influence of cytotoxicity on MF became particularly evident at 40 µg/mL, where a positive response was obtained in the first assay at 22 % RTG, whereas a negative response was seen in the second assay when RTG was 27 %. Furthermore, when a significant difference in RTG was seen between the individual cultures of a single concentration, the culture with the lower RTG value displayed the higher MF (second assay at 80 and 160 µg/mL, see table below). This also demonstrates that MF is linked to cytotoxicity. As positive MLT results are of questionable relevance if there is a clear correlation between cytotoxicity and MF and if the reduction in growth approaches or exceeds 80 % (ICH, 2008; Moore et al, 2006), the MLT results in the absence of S9 are considered as equivocal.

In summary, the re-evaluation of the MLT results applying lately recommended evaluation criteria revealed that DCDPS did not produce a mutagenic response in the presence of S9 and an equivocal response in the absence of S9.

Table. MLT results in the absence of S9.

Concentration [µg/mL]

Precipi-tation

Relative Total Growth [%]

Mutant frequency (MF) x 10E-06

MF x 10E-06 above Control*

 

mean

 

mean

 

First Assay

0 (control)

-

 

100

-

56.25

-

40

no

22

22

177

175.5

119

22

174

80

yes

27

26.5

146

142.5

86

26

139

160

yes

18

19.5

181

179

123

21

177

Second Assay

0 (control)

-

 

100

-

81

-

40

no

27

27

158

153.5

73

27

149

80

yes

23

28

195

166

85

33

137

160

yes

24

20

176

217.5

137

16

259

* MF above control = mean MF(test substance) x 10E-06 – mean MF(control) x 10E-06

Chromosome aberration (ABS) and sister chromatid exchange (SCE) assays

In the Chromosomal Aberration (ABS) test (NIH, 2001) which followed a test protocol comparable to OECD TSG 473 standards CHO cells were incubated with DCDPS continuously in the absence of S9 (harvest time: 15.5 hrs) and for 2 hrs in the presence of S9 (harvest time: 13 hrs). Concurrent vehicle (DMSO) and positive controls were included. DCDPS was tested at concentrations of 94, 201, 432 µg/mL without S9 and at concentrations of 9.4, 20, 930, 2000 µg/mL with S9. The highest concentration was limited by cytotoxicity. A slight, statistically significantly increased chromosomal aberration rate of 3.5% was only seen at 930 µg/mL in the presence of S9. Thus, a second test with S9 was performed using DCDPS concentrations of 750, 1000, 1250, 1500, 2000 µg/mL. In this test no increased number of chromosomal aberrations was found, demonstrating that the isolated finding at 930 µg/mL in the first test was without biological relevance. In conclusion, DCDPS did not induce chromosomal aberrations in CHO cells in the presence or absence of a metabolic activation system.

A study published by the NIH (2001) reports the data of Sister Chromatid Exchange (SCE) testing in CHO cells without and with S9. A first test in absence of S9 was judged to be equivocal, based on the small, dose-related increase in the number of SCEs/cell over the concentration range of 20 to 200 µg/mL, where none of the test concentrations attained the NTP standard protocol evaluation criterion of 20% change. In this test CHO cells were incubated for 26 hours with DCDPS in medium (harvest after 28 hours). The next higher test concentration of 667 µg/mL could not be evaluated due to strong cytotoxicity. A second test without S9 was designed to clarify the equivocal result obtained in the first test using concentrations from 200 to 300 µg/mL and the lengthened incubation time of 33 hours as chemically induced cell cycle delay had occurred in first test. No statistically significant dose-related increase was seen and the SCE frequency was clearly below 20 % for all test concentrations. Consequently, the result of the second test was judged negative. Overall, the NIH scientists weighted data from the first test more than those from the second. Due to the differences in the results of the first and second test, the SCE test without S9 was considered equivocal by the NIH. However, as the second test was explicitly designed to clarify the equivocal result obtained in the first test (i.e. use of concentrations close to the critical concentration of 200 µg/mL and application of a longer incubation time to account for chemically induced cell cycle delay observed in the first test) the negative result obtained in the second trial clearly shows that the equivocal result seen in the first trial is biologically not relevant. This leads to the overall conclusion that DCDPS is negative without S9.

In the SCE test with S9 fraction, cells were incubated with DCDPS concentrations up to 2000 µg/mL for two hours in the presence of S9 (harvesting after 28 hours). Under these test conditions there was no increase in the number of SCEs/cell.

Taking all results into consideration it is concluded that the SCE test is negative in the absence and presence of S9.

 

In conclusion, DCDPS is not considered as an in vitro genotoxic substance.

In vivo mutagenicity

 

Data on in vivo mutagenicity is available from a rat liver unscheduled DNA synthesis (UDS) test (Adams 2000), and two mouse erythrocyte micronucleus assays (Putman 1991, NIH 2001).

 

In the rat liver unscheduled DNA Synthesis (UDS) Test (Adams 2000), DCDPS gave a negative response when tested up to the limit dose of 2000 mg/kg bw.

 

The mouse erythrocyte micronucleus test reported by Putman (1991) was negative. In that study DCDPS was administered once i.p. to groups of 5 males and 5 female ICR mice at concentrations of 196, 980, and 1960 mg/kg bw. The high dose level was calculated to be 80% of the LD50 and caused clinical signs of toxicity. Concurrent vehicle (corn oil) and positive control groups were included. Bone marrow cells were prepared 24 h, 48 h, and 72 h after exposure and analysed for mironuceated PCEs. There was no statistically significant increase in the micronucleus frequency in comparison to controls.

In the second study (published by NIH in 2001, performed in 1992) groups of 5 male B6C3F1 mice were exposed three times every 24 h to DCDPS doses of 200, 400, 600, and 800 mg/kg bw, and analyses were performed 24 h after the last dosing.

While in the first trial the frequency of micronucleated polychromatic erythrocytes was significantly increased in the 400 mg/kg group only, in the second trial a small but statistically significant increase was seen in the 400 and 800 mg/kg groups. Based on these results the overall conclusion in the NTP report is that DCDPS gave a positive response in the micronucleus test in vivo. In both assays a clear dose-response relation could not be established. Furthermore, a comparison with historical control data was not done. Shelby et al. published in 1993 those data for the laboratory which had performed the micronucelus assay. The micronuclei fraction in corn oil treated B6C3F1 control mice ranged from 0.11 % to 0.37 %. This shows that the fraction of 0.38 % micronucleated PCEs at 400 mg/kg in the first trial and of 0.33 % at 400 and 800 mg/kg in the second trial are close to or within the historical control range, so that the biological relevance of the observed increases in micronuclei becomes questionable.

In conclusion, the micronucleus test results in mice are conservatively classified as inconclusive (i.e. one negative test and one positive test with questionable biological relevance). The UDS test in rats is negative.

References

Kirkland D, Aardema M, et al. (2006). Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles. Mutat. Res. 608, 29-42.

Moore MM, Honma M, et al. (2006). Mouse lymphoma thymidine kinase gene mutation assay: follow-up meeting of the international workshop on genotoxicity tests – Aberdeen, Scotland, 2003. Environ. Mol. Mutagen. 47, 1-5.

ICH (2008). Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use, S2(R1).

Shelby MD, Erexson GL et al. (1993). Evaluation of a three-exposure mouse bone marrow micronucleus protocol: results with 49 chemicals. Environ. Mol. Mutagen. 21, 160-179.


Short description of key information:
DCDPS was not mutagenic in bacterial reverse mutation tests with and without S9. It was also negative in the HPRT, chromosome aberration and sister chromatide exchange test in CHO cells in the presence and absence of S9. The mouse lymphoma L5178Y assay was negative in the presence of S9 and equivocal in the absence of S9.
In in vivo test systems, DCDPS did not induce unscheduled DNA synthesis in the rat. In the micronucleus test in mice DCDPS is classified as inconclusive based on one negative test (Putman, 1991) and one positive (NIH, 2001) test. However, in the light of historical control data, the biological relevance of the positive result is questionable. This is further supported by the later negative carcinogenicity studies in B6C3F1 mice (i.e. weighing of evidence), the strain of mice returning the positive micronucleus test result in the NIH study design.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The evaluation of the available in vitro data revealed that DCDPS is not genotoxic in vitro. In vivo, the micronucleus test results in mice were conservatively classified as inconclusive (i.e. one negative test and one positive test with questionable biological relevance), whereas the UDS test in rats was negative.

The synopsis of the data indicates the absence of an unequivocally positive response of DCDPS in any of the relevant mutagnicity assays. Moreover, two-year studies in mice and rats demonstrated the absence of carcinogenicity upon long-term exposure of experimental animals to DCDPS. Thus, it is considered that DCDPS is not mutagenic and accordingly no classification is required.