H112323

Endpoint summary

Additional information

Mutagenicity:

A study was conducted to determine the mutagenic potential of test substance according to OECD Guideline 471, 472 and UK Department of Health Guidelines (DoH, 1989).

Four strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and two strains of E. coli (WP2P and WP2P uvrA were used in the bacterial mutagenicity assay.

In two separate experiments, the test substance did not induce any significant, reproducible increases in the observed numbers of revertant colonies in strains TA1537, TA98, TA100, WP2P and WP2P uvrA in the absence of an auxiliary metabolising system (S9), or in the four Salmonella strains in the presence of S9. With both E. coli strains in the presence of S9 and with TA1535 in the absence of S9, small but reproducible statistically significant increases in colony numbers were observed.

In each experiment, the positive controls responded as expected indicating that the assay was performing satisfactorily.

Test substance gave negative (non-mutagenic), response with S. typhimurium strains TA1537, TA98, TA100 in the presence and absence of an auxiliary metabolising system (S9), with strain TA1535(+S9), and with E. coli strains WP2P and WP2P uvrA (-S9). With strains TA1535 (-S9) and WP2P and WP2P uvrA (+S9), the test substance gave a weak positive i.e. mutagenic response (Callander RD, 1991).

In order to clarify the weak mutagenic effect in bacteria for humany, a study was conducted to determine the ability of test substance to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay equivalent or similar to OECD Guideline 486. The autoradiographic technique was used in the assay.

Male Alderley Park (Alpk:APfSD) rats were given a single oral dose of test substance by gavage at dose levels of 1250 or 2000 mg/kg bodyweight. The highest dose level tested was the limit dose for a non-toxic agent in this assay. Two sampling times were used, 2 h and 16 h after dosing. Two independent experiments were carried out at each time point, validated by concurrent control treatments of rats dosed with dried corn oil (the vehicle for test) and with the genotoxin dimethylhydrazine dihydrochloride.

In all four UDS experiments colouration of the GI tract was observed in all animals treated with test substance. Colouration of the urine, kidneys, liver and testes was observed in animals treated at both dose levels of test substance in both 16 hour UDS experiments. Colouration of the urine was not observed in either of the 2 h experiments but colouration of the testes was observed in one 2 hour experiment and colouration of the kidneys, liver and testes was observed in the other 2 h experiment.

These observations clearly indicate that test substance has been absorbed following administration via the oral route. Hepatocytes from treated rats were assessed for the induction of UDS. Examination of the mean net nuclear grain count and percentage of cells in repair showed that test substance did not induce DNA repair, as measured by UDS, at either dose level or time point investigated.

The sensitivity of the test system was confirmed by the detection of an induction of UDS following treatment of animals with the genotoxin dimethylhydrazine dihydrochloride. The test substance did not induce DNA repair in rat liver in vivo up to a limit dose of 2000 mg/kg bw (Barber G and Mackay JM, 1994) Hence, the test substance is not mutagenic in mammalians.

 

Clastogenicity:

A study was conducted to investigate the clastogenic potential of test substance in human lymphocytes according to OECD Guideline 473 and UKEMS recommended procedures for basic mutagenicity tests.

Human lymphocytes from two donors (one male and one female), were treated in vitro with a range of concentrations of test substance both in the presence and absence of S9-mix. Cultures from both donors were harvested at the standard time of 72 h after culture initiation and, in addition, cultures from the female donor were also harvested at the later time of 96 h after culture initiation.

A preliminary cytotoxicity test was conducted using test substance concentrations from 2.8 to 8720 µg/mL. No significant reductions in mitotic activity were observed in cultures from either donor with 8720 µg/mL being the maximum concentration selected on the basis of it being equivalent to the limit dose for the assay. Cultures from both donors treated with test substance at concentrations of 872, 4360 and 8720 µg/mL, in the presence and absence of S9-mix, were selected for chromosomal aberration analysis at the 72 h sampling time and cultures from the female donor treated at 4360 µg/mL only, were selected for analysis at the 96 h sampling time.

No statistically or biologically significant increases in percentage of aberrant cells, compared to the medium control values, were seen at any of the test substance concentrations tested, in the presence or absence of S9-mix, at either of the sampling times investigated.

The sensitivity of the test system, and the metabolic activity of the S9-mix employed, was clearly demonstrated by the increases in the frequencies of chromosomal aberrations induced by the positive control agents, mitomycin C and cyclophosphamide. Test substance is not clastogenic to cultured human lymphocytes in vitro (Jones and Mackay JM, 1991).

A study was conducted to assess the potential of the test substance to induce micronucleated polychromatic erythrocytes in the bone marrow of CD-1 mice performed according to OECD Guideline 474.

A single oral dose was given to groups of 5 male and 5 female mice at a dose level of 5600 mg/kg (equivalent to 5000 mg/kg corrected for a water content of 10.5 % w/w), this being the limit dose level for the assay. Bone marrow samples were taken 24 and 48 h after dosing.

No statistically or biologically significant, increases in the incidence of micronucleated PCE, over the vehicle control values, were seen in either sex at either of the sampling times investigated. Comparison of the percentage of PCE showed, no statistically significant decreases, compared to the vehicle control values, in either sex at either of the sampling times. However, colouration of the urine, internal tissues and skin (subcutaneous fat) was observed in the animals treated with test substance, indicating that the test substance was absorbed following administration via the oral route.

The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen. Test substance is not clastogenic in the mouse micronucleus assay (Mackay JM, 1992).

 

 

 

Short description of key information:
In the bacterial reverse mutation test, test substance showed negative, response with S. typhimurium strains TA1537, TA98, TA100 in the presence and absence of S9-mix, with strain TA1535(+S9), and with E. coli strains WP2P and WP2P uvrA (-S9). With strains TA1535 (-S9) and WP2P and WP2P uvrA (+S9), the test substance gave a weak positive results.Hence, the results were equivocal in the Ames test.
The test substance was found to be non-genotoxic both in in-vitro chromosomal aberration and in-vivo micronucleus test and unscheduled DNA synthesis tests. Consequently H112323 was evaluated to be not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance was found to be non-genotoxic both in in-vitro chromosomal aberration and in-vivo micronucleus test and unscheduled DNA synthesis tests. However, the results were equivocal in Ames test. Test substance was negative in response with S. typhimurium strains TA1537, TA98, TA100 in the presence and absence of S9-mix, with strain TA1535(+S9), and with E. coli strains WP2P and WP2P uvrA (-S9). With strains TA1535 (-S9) and WP2P and WP2P uvrA (+S9), the test substance gave a weak positive result.

Based on the overall weight of evidence test substance, is expected not to have any genotoxic potential. Therefore no classification is required for genotoxicity according to EC criteria (67/548/EEC) and according to CLP criteria (EC 1272/2008).