Chloramide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 july 2013 To 27 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study, according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Chloramide

EC Number:

234-217-9

EC Name:

Chloramide

Cas Number:

10599-90-3

Molecular formula:

ClH2N

IUPAC Name:

chloranamine

Test material form:

other: solution

Details on test material:

- aqueous solution of Monochloramine at 1 %
- Due to the instability of the Monochloramine, the solution is prepared extemporaneously.
- Used within 12-18 hours of preparation
- Appearance: colourless liquid
- Storage conditions: 4°c, protected from light.

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine, 50 µg/mL gentamicin, 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

other: Thymidine kinase heterozygous cells

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at ca -80°C.

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.25, 0.5, 1, 2, 4, 8, 16, 32.1, 64.2 and 128.3 µg/ml
Mutation tests:
- without S9 mix (3 hours): 1, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75 and 4 µg/mL
- with S9 (3 hours): 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7 and 8µg/mL
- without S9 mix (24 hours): 1, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75 and 4 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Evaluation criteria:

Acceptance criteria for test substance:
The highest concentration tested was one that allowed the maximum exposure up to 5000 µg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (ie. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxicsubstance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Acceptance criteria for vehicle controls:
The mean vehicle control value for mutant frequency was between 50 to 170 x 10-6. The mean cloning efficiency was between 65 to 120 %. The mean suspension growth was between 8 to 32 on Day 2 following 3-hour treatments and between 32 to 180 on Day 2 following a 24-hour treatment. Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.

Acceptance criteria for positive controls:
Positive controls showed an absolute increase in mean total MF above the mean concurrent vehicle control MF of at least 300 x 10-6. At least 40% of this was due to the number of small mutant colonies. Mean RTG’s for the positive controls were greater than 10%. There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity. There was not excessive heterogeneity between replicate cultures.

Assays that did not fulfil the required criteria were rejected and therefore are not reported.
This decision was at the discretion of the Study Director.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989). Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend (p<0.05).

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: No precipitate was observed by eye at the end of treatment. Exposure to Monochloramine at concentrations from 0.25 to 128.3 g/mL in the absence and presence of S9 mix (3-hour exposure) resulted in relative suspension growth (RSG) values from 113 to 0% and from 98 to 0% respectively.
Following a continuous exposure for 24 hours in the absence of S9 mix, no precipitate was observed by eye at the end of treatment. Exposure to concentrations from 0.25 to 128.3 g/mL resulted in RSG values from 129 to 0%.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Main mutation test - 3-hour treatment in the absence of S9 mix:

Cultures were exposed to Monochloramine at concentrations from 1 to 4 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Monochloramine at concentrations from 1 to 2.5 µg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 103 to 15% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity.

The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Main mutation test - 3-hour treatment in the presence of S9 mix:

Cultures were exposed to Monochloramine at concentrations from 1 to 8 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Monochloramine at concentrations from 1 to 4.5 µg/mL were assessed for determination of mutation frequency. RTG values from 120 to 13% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.

The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency

and an acceptable increase in the number of small colony mutants.

The results obtained in response to the exposure of cultures to Monochloramine in the presence of S9 mix did not demonstrate mutagenic potential. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. There were no clear increases in the mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05). Therefore it was considered not to be beneficial to perform a direct repeat of the assay.

Main mutation test - 24-hour treatment in the absence of S9 mix:

Cultures were exposed to Monochloramine at concentrations from 1 to 4 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Monochloramine at concentrations from 1 to 2.25 µg/mL were assessed for determination of mutation frequency. RTG values from 120 to 22% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.

Although this assay did not reach the required 10-20% RTG it is considered valid in accordance with Moore et al. (2002) which states that a chemical can be considered nonmutagenic when there is no culture showing an RTG value between 10-20% if there is no evidence of mutagenicity in a series of data points within 100% to 20% RTG and there is at least one negative data point between 20% and 25% RTG.

The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions of this study, test substance did not demonstrate mutagenic potential in this in vitro cell mutation assay.

Executive summary:

Monochloramine was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. Monochloramine was freshly prepared and analysed by the department of Formulation and Inhalation Analysis at Huntingdon Research Centre, Huntingdon, Cambridgeshire, PE28 4HS prior to use on this study. A final concentration of 128.3 g/mL, dosed at 10%v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up a concentration which altered the pH of the medium by less than 1 unit in comparison to vehicle control. Toxicity was observed in the preliminary toxicity test. Following a 3-hour exposure to Monochloramine at concentrations from 0.25 to 128.3 g/mL, relative suspension growth (RSG) was reduced from 113 to 0% and from 98 to 0% in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 129 to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG). Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix were 2.5 and 4.5 g/mL respectively. In the absence and presence of S9 mix RTG was reduced to 15 and 13% respectively.

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 2.25 g/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The RTG was reduced to 22%.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

It was concluded that Monochloramine did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.