Reaction products of N-methyldiethanolamine and Boric Acid (1:1.5)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6 February to 27 February, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of N-methyldiethanolamine and Boric Acid (1:1.5)

EC Number:

947-350-9

Molecular formula:

N.A. (UVCB)

IUPAC Name:

Reaction products of N-methyldiethanolamine and Boric Acid (1:1.5)

Test material form:

liquid: viscous

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction

Test concentrations with justification for top dose:

5000, 2500, 1250, 625 and 313 µg/plate, based on results obtained in the preliminary toxicity test

Vehicle / solvent:

water

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

- Bacterial strains
In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall and increases permeability to certain classes of chemicals. All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens. TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system.
Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.
Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
Histidine requirement: No Growth onMinimal plates+Biotin. Growth onMinimal plates+Biotin+Histidine.
Tryptophan requirement: No Growth onMinimal agar plates. Growth onMinimal plates+Tryptophan.
uvrA, uvrB: Sensitivity to UV irradiation.
rfa: Sensitivity to Crystal Violet.
pKM101: Resistance to Ampicillin.
Bacterial cultures in liquid and on agar were clearly identified with their identity.
- Media
The following growth media were used:
Nutrient Broth
Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
Nutrient Agar
Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
Minimal Agar
Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-BonnerMedium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
Top Agar
"Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use, 10mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5mMtryptophan) was added to the top agar (100 mL).
- S9 tissue homogenate
One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics:
Species Rat
Strain Sprague Dawley
Tissue Liver
Inducing Agents Phenobarbital – 5,6-Benzoflavone
Producer MOLTOX,Molecular Toxicology, Inc.
Batch Number 3878
The production and quality control certificate can be found in Addendum 1 of this report.
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each
10 mL):
S9 tissue fraction 1.0mL
NADP (100 mM) 0.4mL
G-6-P (100 mM) 0.5mL
KCl (330 mM) 1.0mL
MgCl2 (100 mM) 0.8mL
Phosphate buffer (pH 7.4, 200 mM) 5.0mL
DistilledWater 1.3mL
10.0mL
- Preliminary toxicity test
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in theMain Assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.
- Main Assays
TwoMain Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
The firstMain Assay was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.
The overlay mixture was composed as follows:
Overlay agar (held at 45°C) 2.0mL
Test or control item solution 0.1mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
Bacterial suspension 0.1mL
The secondMain Assay was performed using a pre-incubation method. The components were added in turn to an empty test-tube:
Bacterial suspension 0.1mL
Test item solution or control item solution 0.1mL
or control item solution 0.05mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
The incubate was vortexed and placed at 37°C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.
- Incubation and scoring
The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates from Main Assays were held at 4°C for approximately 24 hours before scoring, while plates from preliminary toxicity test were immediately scored by counting the number of revertant colonies on each plate.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showin increasing numbers of mutant colonies with increasing dose levels.

Statistics:

- Structure of Main Assay Tables
These tables show, for each Salmonella typhimurium or Escherichia coli tester strain, the individual plate counts obtained for the negative and positive controls, and at each dose level of the test item. The mean number of revertant colonies and standard error of the mean are also presented. The "untreated" plates received no treatment. The titre of the bacterial cultures is given (million cells/plate).
- Regression line
The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as:
y = a +bx
where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test results

Species / strain:

other: all tester strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test item does not induce reverse mutations in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The potential for the test item to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy, was evaluated in an experimental study according to the OECD Guideline 471 (1997). The following five tester strains were used: TA1535, TA1537, TA98, TA100 and WP2 uvrA. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone. In a preliminary toxicity test, the test item was assayed at 50.0, 158, 500, 1580 and 5000 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration; neither toxicity nor relevant increases in revertant numbers were observed in any tester strain, at any dose level, neither in the absence nor presence of S9 metabolism.

On the basis of the results obtained in the preliminary toxicity test, the test item was assayed at 313, 625, 1250, 2500 and 5000 µg/plate with all tester strains in Main Assay I, using the plate incorporation method. No toxicity was observed at any dose level with any tester strain, neither in the absence nor presence of S9 metabolic activation; no relevant increase in revertant numbers was observed at any concentration tested. Main Assay II was performed using the same concentrations and included an additional pre-incubation step for all treatments. Neither toxicity nor relevant increase in the number of revertant colonies was observed in the pre-incubation assay, at any dose level, with any tester strain, neither in the absence nor presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period, at any concentration, in any experiment. The test item did not induce the necessary two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism, therefore, it can be concluded that the test item does not induce reverse mutations in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.