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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: A registrant-specific bacterial reverse mutation assay (OECD 471) showed that the substance was not mutagenic.

 

Chromosome aberration study: Under the experimental conditions reported, a similar multi-constituent test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Hordaphos CC MS is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration (OECD 473).

 

In vitro gene mutation assay: Under the experimental conditions reported a similar multi-constituent test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Hordaphos CC MS is considered to be non-mutagenic in an HPRT assay (OECD 476).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: chromosome aberration
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium. Properly maintained: yes.
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000.0 µg/mL
Experiment II: 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000.0 µg/mL

Without metabolic activation:
Experiment I: 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000.0 µg/mL
Experiment II: 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.

METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

The test item, dissolved in deionised water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after the start of treatment with the test item. In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 5000.0 µg/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. No visible precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity was observed. The pH was adjusted to physiological values. In this study, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices could be observed.  In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 – 1.5 % aberrant cells, excluding gaps) were within the range of the solvent control values (0.0 – 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.  No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. In both experiments, either EMS (550 or 770 µg/mL) or CPA (7.5 or 20.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:
Under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Hordaphos CC MS is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.
Executive summary:

The test item, dissolved in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations. The highest applied concentration in this study (5000.0 µg/mL of the test item) was chosen with regard to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.


Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
other: mammalian gene mutation assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Type and identity of media: MEM. Periodically checked for Mycoplasma contamination: yes. Periodically checked for karyotype stability: yes. Periodically "cleansed" against high spontaneous background: yes.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 156.3; 312.5; 625.0; 1250; 2500; 5000 µg/mL
with metabolic activation: 156.3; 312.5; 625.0; 1250; 2500; 5000 µg/mL
Experiment II:
without metabolic activation: 156.3; 312.5; 625.0; 1250; 2500; 5000 µg/mL
with metabolic activation. 156.3; 312.5; 625.0; 1250; 2500; 5000 µg/mL
In both main experiments the cultures at the lowest concentration in the presence and absence of metabolic activation were not continued since a minimum of only four analys-able concentrations is required by the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5x10exp. 6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
experimental group: p-value:
experiment I, culture I without S9 mix 0.831
experiment I, culture II without S9 mix 0.564
experiment I, culture I with S9 mix 0.553
experiment I, culture II with S9 mix 0.308
experiment II, culture I without S9 mix 0.871
experiment II, culture II without S9 mix 0.773
experiment II, culture I with S9 mix 0.373
experiment II, culture II with S9 mix 0.789
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
In the pre-experiment the pH at concentrations of 156.3 µg/mL and above was adjusted to neutral with 2 N sodium hydroxide. In the main experiments the pH at concentrations of 312.5 µg/mL and above was adjusted as described above.

- Effects of osmolality: Not effected
- Evaporation from medium: Not examined
- Water solubility: Not indicated

- Precipitation:
The test medium was checked for precipitation or phase separation at the end of each treatment period prior to removal of the test item. No precipitation occurred up to the maximum concentration in the presence and absence of metabolic activation following 4 and 24 hours treatment.

- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four ana-lysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20 % relative survival or cell density at subcultivation and the ana-lysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evalu-ated at the end of treatment by the unaided eye.

In the range finding pre-experiment test item concentrations between 39.1 and 5000 µg/mL according to OECD guideline were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
Relevant cytotoxic effects indicated by a relative suspension growth below 50% were noted at 2500 µg/mL and above without metabolic activation following 4 hours treatment.

The test medium was checked for precipitation or phase separation at the end of each treatment period prior to removal of the test item. No precipitation occurred up to the maximum concentration in the presence and absence of metabolic activation following 4 and 24 hours treatment.
In the pre-experiment the pH at concentrations of 156.3 µg/mL and above was adjusted to neutral with 2 N sodium hydroxide. In the main experiments the pH at concentrations of 312.5 µg/mL and above was adjusted as described above.
Based on the results of the pre-experiment, the maximum concentration of the main ex-periments was again 5000 µg/mL. The individual concentrations were spaced by a factor of 2.

In both main experiments the cultures at the lowest concentration in the presence and absence of metabolic activation were not continued since a minimum of only four analys-able concentrations is required by the guidelines.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Main experiments:
No relevant cytotoxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
Conclusions:
Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Hordaphos CC MS is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The test item Hordaphos CC MS was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

No relevant cytotoxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration.

In the first culture of experiment I the induction factor exceeded the threshold of three times the mutation frequency of the corresponding solvent control at 2500 µg/mL in the presence of metabolic activation. The historical range of solvent controls was also exceeded at this experimental point (44.0 versus 36.6 mutant colonies per 106cells, see Historical Data, Annex II). However, the effect was not reproduced in the parallel culture under identical experimental conditions, and was therefore, judged as biologically irrelevant.

In the second culture of the second experiment the historical range of solvent controls was also exceeded at 312.5 µg/mL with metabolic activation (40.4 versus 36.6 mutant colonies per 106cells).The increase was not dose dependent and was again not reproduced in the parallel culture under indentical experimental conditions and was therefore, judged as biologically irrelevant.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies.No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 10.8 up to 21.7 mutants per 106cells; the range of the groups treated with the test item was from 9.4 up to 44.0 mutant colonies per 106cells.

EMS(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 May 2018 to 29 October 2018
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch 34448
Purity 67.9 - 71.2%
Expiry 30 March 2022
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium, other: TA 97a
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0.08, 0.16, 031, 0.63, 1.25, 2.5 and 5 µL/plate based on the results of the first experiment.
Vehicle / solvent:
Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-Amino-Antracene & 4-Nitro-1, 2-phenylene Diamine
Details on test system and experimental conditions:
Preparation
On the day of the start of the first and the second experiment, a stock solution containing 50 mL/L of the test item in DMSO was prepared. The test item solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for the first experiment: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate

The following nominal concentrations were prepared for the second experiment: 5 µL/plate, 2.5 µL/plate, 1.25 µL/plate, 0.63 µL/plate, 0.31 µL/plate, 0.16 µL/plate and 0.08 µL/plate.

Test System
Specification Species
Salmonella typhimurium LT2 Strains TA97a, TA98, TA100, TA102 and TA1535

Mutations of the strains are listed in the table below.

Origin and Culture
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilizates in the refrigerator at 2-8 °C. The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.

Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Culture of Bacteria
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutrient broth. After incubation for eight hours at 37 ±1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Conduct of Experiment
Preparations Different media and solutions were prepared preliminary (exact production dates are documented in the raw data). On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table surface was disinfected. The S9 mix was freshly prepared and stored at 0 °C.

Experimental Parameters
First Experiment
Date of treatment 07. May 2018
Concentrations tested 5 / 1.5 / 0.5 / 0.15 / 0.05 µL/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method plate incorporation method

Second Experiment
Date of treatment 16. May 2018
Concentrations tested 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 / 0.08 µL/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method pre-incubation method

Description of the Method
General preparation
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used. For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:

-100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-500 µL S9 mix or phosphate buffer (for test without metabolic activation).
-100 µL bacteria suspension
-2000 µL overlay agar (top agar) The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:

-100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-500 µL S9 mix or phosphate buffer (for test without metabolic activation).
-100 µL bacteria suspension

After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate. The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.



Evaluation criteria:
Evaluation
The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
Statistics:
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In both experiments, no precipitation of the test item Methyl dihydrogen phosphate was observed at any of the tested concentrations up to 5 µL/plate. In the first experiment, the test item caused no cytotoxicity towards all bacteria strains In the second experiment, the test item caused cytotoxicity towards all bacteria strains in the highest concentrations (5 µL/plate), only. The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL. Nearly all of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Since all criteria for acceptability have been met, the study is considered valid.
Conclusions:
Based on the results of this study it is concluded that Methyl dihydrogen phosphate is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
Executive summary:

Based on the results of this study it is concluded that Methyl dihydrogen phosphate is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

No evidence of genetic toxicity has been reported during in vitro studies involving a similar multi-constituent substance, further investigation is not required and classification under the criteria given by Regulation (EC) No. 1272/2008 is unnecessary.