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Key value for chemical safety assessment

Additional information

Valid experimental data were available to assess the genetic toxicity in vitro and in vivo.

In vitro:

Gene mutation in bacteria:

Tripropylene glycol diacrylate was tested for mutagenicity in an Ames test with and without metabolic activation, at 0, 20, 100, 500, 2500 and 5000 µg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98 and TA 100 and in a second experiment at 0, 1000, 2000, 3000, 4000 and 5000 µg/plate in TA 1535 only; (acc. OECD 471; BASF AG 2003). An increase in the number of mutant colonies that lacked dose dependency was observed only in the strain S. typhimurium TA1535 after addition of S9 mix from about 500 µg - 1000 µg/plate (factor 1.7 - 1.8) onward with a maximum response at 2500 µg - 3000 µg/plate (factor 2.5 - 2.6) and a reduced mutation rate (factor 2 - 2.1) at higher concentrations. Cytotoxicity was observed at 500 µg/plate and higher, depending on test strain.

 

In another standard plate Ames test with and without metabolic activation (tested at 0, 20, 100, 500, 2500 and 5000 µg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98 and TA 100, tripropylene glycol diacrylate was not mutagenic. Metabolic activation was from S9 fraction from the liver of rats, treated with Aroclor 1254 (acc. OECD 471; BASF AG 1989).A slight decrease in the number of his+ revertants was observed in the standard plate test only with TA 98 at doses > 100 µg/plate. In the preincubation assay a weakly bacteriotoxic effect was found depending on the strain and test conditions at doses > 2500 µg/plate (TA 100, TA 1535) or at doses > 500 µg/plate (TA 98).

 

A third Ames test also provided negative results for tripropylene glycol diacrylate with or without metabolic activation, tested in S. typhimurium strains TA 1535, TA 1537, 1538, TA 98 and TA 100, tested at concentrations of 0.005, 0.01, 0.1, 1.0, 5.0, 10.0, 25.0, 50.0 µl per plate (Litton Bionetics Inc. 1980, Val. 2). The test material was toxic to the strains TA-1537 and TA-1538 at 10 µl per plate and to TA-100 at 25 µl and 50 µl per plate. The test material was also slightly toxic at 10 µl per plate for TA-1535 and at 25 µl and 50 µl per plate for the strain TA-98.

 

Gene mutation in mammalian cells:

The test substance was tested positive for genotoxicity in a mouse lymphoma assay using L5178Y cells at levels up to 12.5 nl/ml without metabolic activation and up to 75 nl/ml with metabolic activation (Litton Bionetics Inc.1980, Val. 2).The test material induced toxicity-related increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells with and without rat liver S9 microsomal activation. Only highly toxic treatments (survival rate 13% to 34%) in the 3 to 10 nl/ml concentration range induced approximately 2-to 4-fold incresses in the mutant frequency in the absence of S9 activation. With activation, similar increases were observed for moderately to highly toxic treatments (survival rate 20% to 60%) in the 25 to 75 nl/ml concentration range. No colony sizing was performed to differentiate between gene mutations and clastogenic activity. Multifunctional acrylates frequently cause positive results in vitro (but not in vivo) only in clastogenicity assays presumably due to their high cytotoxicity (Mutagenicity assessment of acrylate and methacrylate compounds and implications for regulatory toxicology requirements, Johannsen et al, Regulatory Toxicology and Pharmacology 50 (2008) 322–335). The relevant data are also summarized in the attached document.

To fully exclude that Tripropylene glycol diacrylate has the potential to cause gene mutations in mammalian cells an additional HPRT assay according to OECD and GLP guidelines was performed (BASF 2015). Chinese hamster ovary cells were treated with up to 30µg/mL without S9 mix and up to 140µg/mL after the addition of S9. Due to cytotoxicity, only concentrations up to 8.74µg/mL without S9 and 60µg/mL with S9 could be evaluated. No biologically relevant increase in the mutant frequency was observed in two repeat experiments. Tripropylene glycol diacrylate is thus considered non-mutagenic in mammalian cells.

 

In vivo:

Tripropylene glycol diacrylate showed no mutagenic activity in in-vivo assays with rodents. In a mouse micronucleus assay on polychromatic erythrocytestripropylene glycol diacrylateled to a negative result after single oral administration of 87.5, 175, 350 mg/kg bw. Sampling times were 24 and 48 h (BASF AG 2004, Val. 1).As a negative control, male mice were administered merely the vehicle, olive oil,by the same route, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity. The administration of the test substance led to clinical signs. According to the results of the present study, the single intraperitoneal administration of tripropylene glycol diacrylate did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. A dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected from about of 87.5 mg/kg body weight onward.

 

Another in vivo micronucleus test was also negative after oral administration of tripropylene glycol diacrylate. In this test doses of 2000, 1000 and 500 mg/kg bw were administered. Sampling time was 24 and 48 h after administration. 5 males per dose and harvest time point were used (Covance 2007, Val. 1). All animals in all the dose groups and controls appeared normal immediately after dosing and remained healthy until the appropriate harvest timepoint. TPGDA did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (500, 1000, or 2000 mg/kg). TPGDA was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose of the test article analyzed. The vehicle control group had approximately0.09% micronucleated PCEs and the group mean was within the historical control range. The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control.

 

In another study the test substance was applied dermally to Tg.AC mice (3 times a week for 20 weeks). Peripheral blood leukocytes were evaluated for DNA damage (single-strand breaks, alkali labile sites, DNA crosslinking) at weeks 4, 8, 12, 16, and 20 by using the alkaline (pH > 13) single cell gel (SCG) assay. Peripheral blood polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were evaluated for the presence of micronuclei at week 20 (Tice 1997, Val. 2). The extent of DNA migration in leukocytes of mice treated by dermal application with TPGDA at 1, 5, or 10 μmol per mouse was not significantly different, either by trend test analysis or by a pairwise comparison of each treatment dose against the concurrent vehicle control, at any sample time. TPA (0.002 μmol per mouse), the positive control for the tumorigenicity studies, also failed to significantly alter the extent of DNA migration or its intercellular dispersion in leukocytes of mice treated by dermal application.After 20 weeks of treatment, the frequency of micronucleated PCE and NCE in blood were not increased in the mice treated with TPGDA or TPA. The percentage of PCE was increased in mice treated with TPGDA. This increase was highly significant. By a pairwise comparison, the lowest effective dose of TPGDA inducing a significant increase in percentage of PCE was 10 μmol per mouse. TPA, at 0.002 μmol per mouse also induced a marginally nonsignificant increase in the percentage of PCE. This observed increase in the rate of erythropoiesis may reflect bone marrow/blood toxicity, a homeostatic mechanism in response to the treatment-induced tumor burden, and/or a hematopoietic response to epidermal keratinocyte cytokines induced by tissue injury.

 


Short description of key information:
GENETIC TOXICITY IN VITRO:
Gene mutation in bacteria
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2 uvrA, with and without metabolic activation (Ames test): weakly positive in S. typhimurium TA 1535 (acc. OECD 471; BASF AG 2003)
- S typhimurium TA1535, TA 1537, TA 98 and TA 100, with and without metabolic activation (Ames test): negative (acc. OECD 471; BASF AG 1989)
- S typhimurium TA 1535, TA 1537, 1538, TA 98 and TA 100, with and without metabolic activation (Ames test): negative (Litton Bionetics 1980, Val. 2)
Gene mutation in mammalian cells
Mouse L5178Y cells with and without metabolic activation (Mouse Lymphoma Assay): positive (Litton Bionetics 1980, Val. 2)
HPRT with and without metabolic activation: negative (OECD 476, BASF 2015)

GENETIC TOXICITY IN VIVO:
- Mouse, MNT in-vivo, i.p.: negative (acc. OECD 474, BASF AG 2004)
- Mouse, MNT in-vivo, oral: negative (acc. OECD 474, Covance 2007)
- Mouse, Single cell gel assay and micronucleus assay, dermal: negative (Tice 1997, Val. 2)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance was weakly positive in one of three Ames tests in a single tester strain (TA1353) with metabolic activation. The increase in revertant colonies was at most a factor of 2.6 above concurrent negative controls and lacked a dose response relationship. In combination with two further Ames assays tested at equally high or even higher concentrations, which showed no positive response in all tester strains with or without metabolic activation, the test substance is not considered to cause gene mutations in bacteria.

Tripropylene glycol diacrylate was also non-mutagenic in mammalian cells in an HPRT assay, but caused an increase in colony number in a mouse lymphoma assay at cytotoxic concentrations only. As discussed above, based on experience with other multifunctional acrylates and on the fact that the test substance did not cause gene mutations in bacteria or mammalian cells, this positive response is due to clastogenicity as a result of high cytotoxicity.

The potential to cause chromosome damage in vivo was clarified by three micronucleus tests and a single cell gel assay in mice, in which no increase in micronucleated cells was observed.

Overall, Tripropylene glycol diacrylate is considered to be non-mutagenic to bacteria and mammalian cells and non-clastogenic in vivo. No classification for mutagenicity is required.