1-Butanamine, N-butyl-, reaction products with polyethylene-polypropylene glycol ether with trimethylolpropane (3:1) acrylate

Endpoint summary

Additional information

For the test stubstance are a gene mutation assay in bacteria (Ames) according to OECD 471 available.

Gene mutation in bacteria

In a gene mutation assay in bacteria (Ames) according to OECD 471 and GLP (BASF 1993) Propylidynetrimethanol, ethoxylated, propoxylated, esters with acrylicacid, reaction products with 1 -Butanamine, N-butyl- (>1<6.5) did not lead to an increase in the number of his + or trp+ revertants in both, the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system. Concentrations up to 5000µg/plate were tested in Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli 1412 uvrA. S9 fraction was prepared from aroclor induced rat liver.

Read across

The test item is Propylidynetrimethanol, ethoxylated, propoxylated, esters with acrylicacid, reaction products with 1-butanamine, n-butyl (Cas:173011-06-8). The concentration of EO/PO is >1 <6.5. The chosen read-across substances is Propylidynetrimethanol, ethoxylated, esters with acrylicacid, reaction products with 1-butanamine, n-butyl (Cas: 195008-76-5) concentration of. EO>1 <6.5.

In general basic physicochemical properties such as water solubility, vapour pressure and the partition coefficient (LogPow) are quite similar. The water solubility was 0.9 g/l the vapour pressure 2.3 hPa and the logPow 4.2 and 0.8 g/l, 1.6 hPa and the logPow 3.5 for the read-across substance.

Both molecules show no acute toxicity after oral or dermal exposure and did not induce gene mutations in bacteria. Both showed no skin irritation and Cas: 195008-76-5 was slightly eye irritating. Due to the very similar structure, physicochemical properties and almost identical results in the toxicological studies mentioned above, comparable results are also expected for the endpoints for which read across was performed.

Gene mutation in mammalian cells

The test substance was also not mutagenic in an HPRT assay with mammalian cells according to OECD 476 and GLP (BASF 2012). Two independent experiments were carried out, both with and without the addition of phenobarbital and β-naphthoflavone induced rat liver S9 mix. The maximum doses assessed were limited by the cytotoxic properties of the test substance:

• Without S9 mix (4h and 24h exposure) 0.16; 0.31; 0.63; 1.25; 2.50μg/mL
• With S9 mix (4h exposure) 6.25; 12.5; 25; 50μg/mL (1st experiment); 4.38; 8.75; 17.5; 35; 70μg/mL (2nd experiment).

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations.

Micronucleus test in vitro

An in vitro study according to OECD 487 and GLP was performed to assess the potential of propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). The cells were incubated with the test substance at varying concentrations in DMSO for 4h in the presence or absence of phenobarbital and β-naphthoflavone induced rat liver S9 and harvested after 24h after the start of the experiment. Due to ambigous results and low cell quality (slides not scorable), three independent experiments were performed with the following doses (concentrations used were limited by the high cytotoxicity of the test substance):

1st Experiment

• 4 hours exposure; 24 hours harvest time; without S9 mix: not scorable
• 4 hours exposure, 24 hours harvest time, with S9 mix: 0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 and 100.00 μg/mL

2nd Experiment

• 4 hours exposure, 24 hours harvest time, without S9 mix: 0; 0.13; 0.25; 0.50; 1.00; 2.00 and 4.00 μg/mL
• 4 hours exposure, 24 hours harvest time, with S9 mix: 0; 7.50; 15.00; 30.00; 60.00 and 100.00 μg/mL

3rd Experiment

• 4 hours exposure, 24 hours harvest time, without S9 mix: 0; 0.16; 0.31; 0.63; 1.25; 2.50 and 5.00 μg/mL
• 4 hours exposure, 24 hours harvest time, with S9 mix: 0; 5.00; 10.00; 20.00; 40.00 and 80.00 μg/mL

At least 1 000 cells of each culture were analyzed for micronuclei, i.e. at least 2 000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei. PH and osmolarity were not affected and no precipitation occured at the concentrations used.

Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC) or low cell quality was observed at least at the highest applied test substance concentration in all experimental parts at all test conditions. Without metabolic activation, the test substance induced a slight, but significant increase in 2 independent experiments only in the highest dose scorable due to cytotoxicity. With metabolic activation, only one out of three experiments led to an increase in micronuclei in the highest dose above historical control values. Thus the test substance is considered to have a clastogenic effect in vitro at or close to cytotoxic concentrations without metabolic activation. Results obtained after addition of S9 remain ambiguous.

Micronucleus test in vivo

7 male NMRI mice were orally exposed via gavage to a single dose of 500, 1000, and 2000mg/kg b.w. of propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- in corn oil. At least 2000 bone marrow polychromatic erythrocytes were evaluated for micronuclei 24 and 48h (only 2000mg/kg b.w.) after exposure. No increase in micronuclei was observed, the ratio of polychromatic to normochromatic erythrocytes was unaltered, and the number of polychromatic erythrocytes was not decreased compared to vehicle control animals. Animals exposed to the positive control of 40mg/kg bw.w cyclophosphamide showed a substantial increase in micronuclei.

Short description of key information:
Ames: negative (OECD 471, GLP, BASF 1993)
HPRT: negative (OECD 476, GLP, BASF 2012)
MNT in vitro: positive (OECD 487, GLP, BASF 2012)
MNT in vivo: negative (OECD 474, GLP, BASF 2013)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Propylidynetrimethanol, ethoxylated, propoxylated esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- was not mutagenic in two in vitro assays, one in bacteria and one in mammalian cells. It did lead to chromosomal damage in an in vitro micronucleus test, but only at or very close to cytotoxic concentration. Dose dependency was also not always observed and only 1 out of 3 experiments using metabolic activation led to an increase in micronuclei above historical control values at the highest dose used. In addition, this positive result was not confirmed in vivo. Thus, classification for mutagnicity according to 67/548/EEC and CLP/EU-GHS is not required.