Registration Dossier

Diss Factsheets

Toxicological information

Endpoint summary

Currently viewing:

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation assay; OECD 471; Dreher, D. (2018); Positive (mutagenic)

Mammalian cell cytogeneticity assay; OECD 473; Lyoyd, M. (2018); Positive (clastogenic)

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo Micronucleus screen OECD 474 incorporated into OECD 442; N Barraclough (2018) - Negative

In vivo Comet assay in the rat OECD 489 (2021): study ongoing, decision pending completion.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned
Remarks:
An OECD 489 in vivo Comet assay in the rat is ongoing - a justification provided from the laboratory for issues with study timeline below.
Study period:
04 January 2021 -
Justification for type of information:
When the testing proposal decision for the OECD 489 (TPE-D-2114467612-47-01/F) was received from ECHA an appropriate testing laboratory was found to conduct the test and the test procedures (study plans, live phase etc.) were initiated without delay. The OECD 489 study is ongoing but has been subject to delays in completion due to i) problems encountered in the bio-analytical phase and ii) the requirement for additional generation of data through histopathological analysis in order to confirm study conclusions. Data therefore remains outstanding which is key to determining the outcome. Attached is a letter from the laboratory outlining the status of the study, and a proposed timeline for completion where possible. Reporting is expected 4-6 weeks after completion of laboratory work. As soon as the study is complete, a spontaneous dossier update will be undertaken. The Contract Research Organisation (CRO) has provided further detail on the delays and expected completion. This can be found in the "attached justification" section.
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29 July 2016
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Based on the in vivo OECD 474 data, all measured parameters produced negative results and as such the test item is not considered clastogenic. However, data on mutagenicity is currently inconclusive and as such the overall mode of action for genotoxicity and its relevance to human cannot be confirmed. As such, an OECD489 in vivo Comet assay in the rat is currently ongoing to provide further evidence.

Additional information

in vitro genotoxicity data

 

OECD 471 (2018) - In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimuriumwere exposed to Reaction mass of N,N,N’,N-tetrabutylmethylenediamine and dibutylamine in acetone at concentrations of 5, 16, 50, 160, 500, 1600 and 5000 µg/plate a screening experiment followed by concentrations of 31.25, 62.5, 125, 250, 500, 100 and 200 µg/plate (TA98 also tested at 400 and 750 µg/plate) in a secondary experiment. Both tests were conducted in the presence and absence of S9 mix.

 

The test item was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

 

It was concluded that the test article induced mutation in histidine-requiring strains TA98 and TA100, of Salmonella typhimurium when tested under the conditions of this study.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

OECD 473 (2018) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine at concentrations of 0, 15, 20, 25, 30, 35, 45, 50, 70 and 90 µg/mL for 3 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested without metabolic activation at test item concentrations of 0, 5, 15, 27.5 and 35 µg/mL.

 

Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine was tested up to precipitating and cytotoxic concentrations of 55.99 and 93.31 µg/mLfor the 3 h exposure, in the absence and presence f S9 mix, respectively and 93.31 µg/mL for the 20 h exposure with S9 mix. Positive controls induced the appropriate response.

 

There was evidence that the test item induced structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested for 3+17 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 20+0 hours in the absence of S-9 and is therefore considered to have clastogenic potential in this chromosome aberration test. Sporadic increases in the frequencies of cells with numerical aberrations, particularly polyploidy, which marginally exceeded the concurrent controls and the normal ranges, were observed under all treatment conditions, but this assay is not specifically designed for the quantitative evaluation of polyploidy.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.

 

 

In vivo genotoxicity data

 

OECD 474 (incorporated into OECD 422 study design) - The genotoxicity potential of the Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was investigated in an  Oral (Gavage) Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422, adopted 29 July 2016). Covance study 8359422. Four groups of 10 male and 10 female sexually mature Crl:WI(Han) rats were administered 0 (control article [vehicle: corn oil]), 30, 50, or 100 mg/kg/day test item once a day to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation). The control article (vehicle) was corn oil, and formulations were administered at a dose volume of 5 mL/kg. The positive control slides were prepared under the Covance Positive control Slide Bank Study (Covance study 8360181) from animals that had received two daily oral gavage doses of 10 mg/kg/day cyclophosphamide (dissolved in saline). Sampling of these animals was on Day 3 (approximately 24 hours post the last dose administration).

 

Animals treated with the test item at all doses exhibited group mean %PCE values that were either similar to or slightly higher than the value for the concurrent vehicle control group but which fell within the historical vehicle control range. As such, there was no evidence of bone marrow toxicity. Treatment with the test item exhibited group mean MN PCE frequencies that were similar to and not significantly (p≤0.05) higher than those observed in the concurrent vehicle control group for all doses. Individual MN PCE values were consistent with historical vehicle control distribution ranges and similar to values observed in the concurrent vehicle control group.

 

Based on the conditions of the study, it can be stated that the test item did not induce micronuclei in the polychromatic erythrocytes (PCE) of the bone marrow of male and female Han-Wistar rats treated at doses of 30, 50 or 100 mg/kg/day. The test item was considered non clastogenic and does not meet the criteria for classification in accordance with GHS and Regulation (EC) No 1272/2008 (CLP).

 

OECD 489 Comet assay in the rat - Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was further investigated via an OECD 489 study, commencing in January 2021. Due to issues with bioanalysis and the requirement for additional histopathological analysis in order to confirm outcome, this is currently ongoing. Further information is provided within 7.6.2. 

Justification for classification or non-classification

OECD 422 study design. The reproductive and developmental screening test (OECD 422) incorporated with micronuclus screen OECD 474; the test item was considered non-clastogenic and does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008 (CLP). However, data on mutagenicity is currently inconclusive based on the positive in vitro gene mutation and as such the overall classification on genotoxicity is also considered inconclusive. An OECD 489 Comet assay in vivo is ongoing in order to provide further information to the weight-of-evidence analysis for clasification or non-classification.