Urea, reaction products with diethylene glycol, formaldehyde and glyoxal

Endpoint summary

Additional information

Genetic toxicity in vitro

Ames-Test

The test substance Fixapret PC, vor Magnesiumchlorid-Zugabe 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 and Escherichia coli, in a reverse mutation assay according to OECD 471 guideline and GLP.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 10 000 μg/plate (SPT) 33 μg - 10 000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No Precipitation of the test substance was found with and without S9 mix.

TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 1 000 μg/plate onward.

MUTAGENICITY: A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Thus, under the experimental conditions of this study, the test substance Fixapret PC, vor Magnesiumchlorid-Zugabe is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

HPRT-Test

The study was performed to investigate the potential of Fixapret PC, vor Magnesiumchlorid-Zugabe to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD 476 guideline and GLP (Harlan, 2012). The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. On request of the sponsor the highest concentration applied in the pre-experiment was 10000 ug/mL. The concentration range of the main experiments was limited by cytotoxic effects. The test item was dissolved in deionised water.

Relevant cytotoxic effects indicated by a relative cloning efficiency I below 50% in both parallel cultures were observed in the first experiment at 2500 ug/mL and above in the presence and absence of metabolic activation following 4 hours treatment. In the second

experiment cytotoxic effects as described above occurred at 234.4 ug/mL and above in the absence and at 1875 ug/mL and above in the presence of metabolic activation. The recommended cytotoxic range of approximately 10-20% relative coning efficiency I was

covered with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. An increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the first culture of the first experiment without metabolic activation at 312.5 and 3750 ug/mL. In the second culture of the second experiment without metabolic activation the induction factor exceeded the threshold at 234.4 and 312.5 ug/mL. However, these increases were not reproduced in the parallel culture under identical experimental conditions. The absolute values of the mutation frequency remained within the historical range of solvent controls. Therefore, these increases of the induction factor were judged as biologically irrelevant fluctuation.

In the second experiment the mutant colonies/10^6 cells exceeded the range of the historical solvent control data at 1250 ug/mL and above. However, the threshold of three times the mutation frequency of the corresponding solvent control was not reached or

exceeded. Furthermore, the number of mutant colonies per 10^6 cells numbers in the parallel culture remained well within the historical range of solvent controls. The maximum mutation frequency of 67.4 colonies per 10^6 cells was generated at a cytotoxic level reducing the relative cloning efficiency I below the lower limit of 10% (6.2%) and should therefore, be interpreted with care. No increase occurred in the parallel culture at the same concentration at a relative cloning efficiency I of 11.2%.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A p-value below 0.05 was detected in culture II of the second experiment without metabolic activation and in culture II of the second

experiment with metabolic activation. These trends however, were not reproduced in the parallel cultures under identical conditions.

EMS (150 ug/mL) and DMBA (1.1 ug/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusion

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. Therefore, Fixapret PC, vor Magnesiumchlorid-Zugabe is considered to be non-mutagenic in this HPRT assay.

Chromosom aberration Test

The test item Fixapret PC, vor Magnesiumchlorid-Zugabe, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in one experiment according to OECD guideline 473 and GLP. The following study design was performed:

Without and without S9 mix: Exposure period 4 hrs; Recovery 14 hrs; Preparation interval 18 hrs.

In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the highest evaluated concentration without metabolic activation, where only 50 metaphases were evaluated.

The highest applied concentration (10000.0 ug/mL) was chosen at request of the sponsor.

Dose selection for the cytogenetic experiments was performed considering the toxicity data. The doses selected for evaluation were: 625.0, 1250.0 and 2500.0 ug/mL.

In the absence of S9 mix clear cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.

In the absence of S9 mix statistically significant increases above the range of the laboratory

historical control data (0.0 - 4.0 % aberrant cells, excluding gaps) were observed after treatment with 1250.0 and 2500.0 ug/mL (22.0 and 45.0 % aberrant cells, excluding gaps).

In the presence of S9 mix one statistically significant increase above the range of the laboratory historical control data was observed after treatment with 2500.0 ug/mL (20.0 % aberrant cells, excluding gaps). In addition, the number of exchanges was increased in the

absence and presence of S9 mix at least in the highest evaluated concentration.

No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

No relevant increase in endomitotic metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens (EMS and CPA) were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.

Therefore, Fixapret PC, vor Magnesiumchlorid-Zugabe is considered to be clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to cytotoxic or the highest evaluable concentration.

Genetic toxicity in vivo

In vivo micronucleus assay:

The substance Fixapret PC, vor Magnesiumchlorid-Zugabe was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method according to OECD 474 guideline and GLP (BASF, 2012) . For this purpose, the test substance, dissolved in deionized water, was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight refering to active ingredients in a

volume of 10 mL/kg body weight in each case.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2 000 mg/kg body weight and in the vehicle controls. In the test groups of 1 000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

As vehicle control, male mice were administered merely the vehicle, deionized water, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range.

Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

According to the results of the present study, the single oral administration of Fixapret PC, vor Magnesiumchlorid-Zugabe did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei.

The rate of micronuclei was close to the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data. Thus, under the experimental conditions of this study, the test substance Fixapret PC, vor Magnesiumchlorid-Zugabe does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

Short description of key information:
In vitro:
Ames-Test: negative (BASF, 2012)
HPRT-Test: negative (Harlan, 2012)
in vivo:
in vivo Micronucleus Assay: negative (BASF, 2012)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Negative results were obtained with an Ames-Test and with a HPRT-Test. The positive in vitro results in a chromosome aberration test could not be confirmed in an in vivo Micronucleus Assay. Threfore no classification for mutagenicity is required according to EU directive 67/548/EEC and EU classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.