Dimethylamine-borane (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-06 to 2017-04-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethylamine-borane (1:1)

EC Number:

200-823-7

EC Name:

Dimethylamine-borane (1:1)

Cas Number:

74-94-2

Molecular formula:

C2H10BN

IUPAC Name:

N-Methylmethanamine-borane (1:1)

Test material form:

solid: crystalline

Details on test material:

CAS: 74-94-2
Description: white crystalline solid
Batch No.: 1609024
Purity: 99.33 %
Expiry Date: 14 Sep 2017

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED:
- Source of cells: MOLTOX, INC., NC 28607, USA (TA 98, 1535 and 102), Xenometrix AG, Switzerland (TA 100 and 1537)
MEDIA USED
- Type and identity of media: Nutrient medium: 8 g Nutrient Broth and 5 g NaCl per litre, plus 125 µL ampicillin for TA 98, TA 100, TA 102); Agar Plates: Vogel-Bonner Medium E agar plates contain per litre 15 g Agar Agar, 20 mL Vogel-Bonner salts and 50 mL glucose solution (40%); Overlay Agar: The overlay agar contains per litre: 7.0 g Agar Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H20 and 12.2 mg biotin

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (conditions: see "Any other information on materials and methods"; Results: see "Any other information on results" Table 2). The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:
31.6, 100, 316, 1000, 2500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua dest.
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100 and TA 1535, without S9, 10 µg/plate

Controls 2

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Remarks:

TA 98 (10 µg/plate) and TA 1537 (40 µg/plate), without S9

Controls 3

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 102, 1 µL/plate, without S9

Controls 4

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

TA 98, TA 100, TA 1535, TA 1537, TA102; with S9, 2.5 µg/plate (10 µg/plate for TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, Experiment I), preincubation (Experiment II)
- Cell density at seeding (if applicable): approx. 10^9 cells/mL, 100 µL/plate

EXPERIMENTAL PERFORMANCE
- Experiment I:
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate: 100 μL test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control), 500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation), 100 μL bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain), 2000 μL overlay agar.
- Experiment II:
For the pre-incubation method the following materials were mixed and pre-incubated for 60 min at 37 °C: 100 μL of the test item preparation, 100 µL of the tester strain, 500 µL sterile buffer or the metabolic activation system. Following the pre-incubation, the overlay agar (2000 μL) was added and the mixture was poured onto the surface of a minimal agar plate.

DURATION
- Preincubation period (Experiment II): 60 min at 37 °C
- Exposure duration: 48 h in the dark at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/dose level including the controls
DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

EVALUATION OF MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation). A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control

Evaluation criteria:

A test is considered acceptable if for each strain:
- The bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- The negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2013 -2015) (see “Any other information on material and methods” Table 1)
- Corresponding background growth on negative control, solvent control and test plates is observed
- The positive controls show a distinct enhancement of revertant rates over the control plate
- At least five different concentrations of each tester strain are analysable

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate without S9 in TA 102

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2500 and 5000 µg/plate without S9 in TA 1535 and at 5000 µg/plate with S9 in TA 1537 and TA 102

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test item were noted in some tester strains evaluated in experiment I and II. In experiment I toxic effects of the test item were observed in tester strain TA 102 at a concentration of 5000 µg/plate and higher (without metabolic activation).
In experiment II toxic effects of the test item were noted in tester strain TA 1535 at concentration of 2500 µg/plate and higher (without metabolic activation). In tester strains TA 1537 and TA 102 toxic effects of the test item were observed at a concentration of 5000 µg/plate (without metabolic activation). The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in tester strain TA 1535 at a concentration of 316 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with dimethylmine-borane (DMAB) at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

Remarks on result:

other:

Remarks:

Experiment I

Any other information on results incl. tables

Results of the pre-experiment:

Table 2: Results of the pre-experiment

Substance	Dose (µg/plate)	TA 98 Mutation Factor [toxicity]*		TA 100  Mutation Factor [toxicity]*
		without S9	with S9	without S9	with S9
Solvent Control (A. dest)		1.0	1.0	1.0	1.0
4-NOPD	10.0	19.3	-	-	-
NaN3	10.0	-	-	7.6	-
2 -AA	2.50	-	86.4	-	20.2
Test Item DMAB	3.16	1.0	0.9	0.9	0.9
	10.0	1.1	1.3	1.0	0.8
	31.6	1.0	1.3	0.9	1.0
	100	0.9	1.1	1.0	1.1
	316	0.6	1.1	1.0	1.1
	1000	1.0	1.1	0.9	0.9
	2500	0.7	0.9	1.2	1.2
	5000	1.2	1.1	1.6	1.6

* [toxicity parameter]: B = Background lawn reduced, N = No background lawn

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during under the experimental conditions reported, dimethylamine-borane did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, dimethylamine-borane is considered to be non-mutagenic according to CLP criteria in this bacterial reverse mutation assay .

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471), strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 of Salmonella typhimurium were exposed to dimethylamine-borane (>99 % purity) in water at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation in two independent experiments (plate incorporation test (experiment I) and the pre-incubation test (experiment II).

Dimethylamine-borane was tested up to the limit concentration (5000 µg/plate) in a pre-experiment showing no cytotoxic effects. The positive controls induced the appropriate responses in the corresponding strains. In experiment I toxic effects of the test item were observed in tester strain TA 102 at a concentration of 5000 μg/plate and higher (without metabolic activation). In experiment II toxic effects of the test item were noted in tester strain TA 1535 at concentrations of 2500 μg/plate and higher (without metabolic activation). In tester strains TA 1537 and TA 102 toxic effects of the test item were observed at a concentration of 5000 μg/plate (without metabolic activation). The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in tester strain TA 1535 at a concentration of 316 μg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship. There was no evidence of induced mutant colonies over background in all tester strains and both experiments. Therefore, dimethylamine-borane is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.