Reaction product aqueous Phosphoric Acid, 2-Butoxyethanol and 4,4'-Isopropylidenediphenol-Epichlorohydrin

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was tested for in vitro mutagenicity in a bacterial mutation (Ames) test, for the potential for clastogenicity/aneugenicity in an in vitro micronucleus assay and for the mutagenic activity in a mammalian cell gene mutation test (TK Locus in mouse lymphoma cells).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

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 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human blood donations
- Suitability of cells: Considered suitable
- Sex, age and number of blood donors if applicable: 4 donors male and female at 31 to 32 years old. Donors were healthy and non-smokers without any recent exposure to drugs or radiation.

MEDIA USED
The culture medium for the lymphocytes had the following composition:
RPMI 1640 1x (Dutch modification): 500mL
Foetal Calf Serum: 100mL
L-Glutamine (200mM): 6.25mL
Antibiotic solution: 1.25mL
The foetal calf serum was heat-inactivated at 56°C for 20 minutes before use. For the
initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used
in the following proportion: 10mL of PHA was added to 500mL of medium.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of Sprague Dawley rats treated with phenobarbital and benzoflavone

Test concentrations with justification for top dose:

Main phase 1:
3 hour treatment in the absence and presence of S9 mix: 0, 58.5, 87.8, 132, 198, 296, 444, 667, 1000 and 1500 µg/mL.
Continuous treatment in the absence of S9 mix: 0, 39, 58.5, 87.8, 132, 198, 296, 444, 667, 1000 and 1500 µg/mL.

Main phase 2:
3 hour treatment presence of S9 mix: 0, 199, 229, 263, 303, 348, 400, 460, 529, 609 and 700 µg/mL.
Continuous treatment in the absence of S9 mix: 0, 0.206, 0.308, 0.462, 0.694, 1.04, 1.56, 2.34, 3.51, 5.27, 7.90, 11.9 17.8, 26.7 and 40 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Selected during a preliminary solubility test. This solvent was selected since it is compatible with the survival of the cells and the S9 metabolic activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Colchicine in the absence of S9 Mix

Details on test system and experimental conditions:

Formulation procedure:
Solutions of the test item, as received, were prepared immediately before use in DMSO on a weight/volume basis without correction for the displacement due to the volume of the test item. For each Main Assay, a clear solution without any visible precipitation was prepared at 300mg/mL, following vortexing for approximately 5 minutes, and used to prepare serial dilutions. All test item solutions were used within 25 minutes from the initial formulation.

Test culture preparation:
One Main Experiment was performed including solvent and positive controls. Two cultures were prepared at each test point. Lymphocyte cultures were treated fourty-eight hours after they were initiated. Before treatment, cultures were centrifuged for 10 minutes and the culture medium was decanted and replaced with treatment medium. For the short term treatment the composition of the media included the test item or control solution, S9 mix (with and without) and culture medium without PHA. For the continuous treatment this was similar although no S9 mix was included.

For the short term exposure, the treatment media were added to the tubes and the cultures were incubated for 3 hours at 37°C. At the end of treatment time, the cell cultures were centrifuged and washed twice with PBS Solution. Fresh medium was added and the cultures were incubated for a further 28 hours (Recovery Period) before harvesting. At the same time, Cytochalasin-B was added to achieve a final concentration of 6 µg/mL. For the continuous treatment, 3 hours after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 hours before harvesting.

Experimental design:
For the first Main Assay, dose levels were selected on the basis of the solubility of the test item in the culture medium. Since no adequate cytotoxicity levels were obtained in the treatment series in the presence of S9 metabolic activation and using the continuous treatment in its absence, an additional Main Assay was performed. The dose range used was modified to take into account the toxicity observed in the previous experiment. Appropriate negative and positive control cultures were included in both Main Assays. Using the short treatment time, since tests with and without metabolic activation were done concurrently, positive control cultures were treated only with Cyclophosphamide at the dose levels of 20.0 and 15.0 µg/mL. Using the long treatment time, in the absence of S9 metabolism, the positive control cultures were treated with Colchicine at the dose levels of 80 and 40 ng/mL.

Cell harvesting and slide preparation:
The lymphocyte cultures were centrifuged for and the supernatant was removed. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.
A few drops of the cell suspension obtained in this way were dropped onto clean,wet, grease free glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture. The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

Slide evaluation:
The cytokinesis-block proliferation index (CBPI) and % cytotoxicity was calculated. Five hundred cells per cell culture were analysed. At least five dose levels were analysed and when negligible cytotoxicity was observed, scoring was interrupted. The highest dose level for genotoxicity assessment should be selected as a dose which produces a substantial cytotoxicity (approximately 55±5%) compared with the negative control. If the test item does not induce relevant toxicity at any concentration, then the highest treatment level is selected as the highest dose level for scoring. Two lower dose levels are also selected for the scoring of micronuclei. For the three selected doses, for the solvent and positive control Cyclophosphamide, at least 1000 binucleated cells per cell culturewere scored to assess the frequency of micronucleated cells. Concerning cultures treated with Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture were scored.

Rationale for test conditions:

Standard as per OECD guideline

Evaluation criteria:

Positive:
- Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
- The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values.
- There is a significant dose effect relationship.

Negative:
- None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
- There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
- All the results are inside the distribution of the historical control data.

Statistics:

For the statistical analysis, a modified χ2 test was used to compare the number of cells with micronuclei in control and treated cultures. Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

Osmolality and pH effects:
Following treatment with the test item, no relevant variations of pH or osmolality values over the concurrent controls were observed.

Selection of doses for scoring:
Based on the cytotoxicity observed the following doses were selected for micro-nuclei scoring:
First assay: 3 hour treatment (without S9 mix): 132, 198 and 296 µg/mL and with S9 mix: 296, 444 and 667 µg/mL.
Second assay: 3 hour treatment (with S9 mix): 303, 348 and 400 µg/mL.
Second assay: continuous treatment (without S9 mix): 7.9, 11.9 and 17.8 µg/mL.

Result analysis:
Results show that the incidence of micronucleated cells of the negative controls in Main Assay I was within the distribution range of our historical control values. In Main Assay II, the observed values for both treatment series slightly exceeded the 95% control limits of the historical control distribution. However, the incidences were consistent with those reported in literature. In addition, the effect of gender on micronucleated cell (MNC) frequency has been described in literature indicating that females have higher MNC frequency than males. Since our historical data refer mainly to male individuals, this result was not considered critical.

Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed in both Main Assays following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system. The study was accepted as valid.

In the absence of S9 metabolism, no statistically significant increase in the incidence of micronucleated cells over the concurrent negative control was observed at any dose level, nor concentration related increase of cells bearing micronuclei was seen using the short term treatment or the continuous treatment. In the presence of S9 metabolism, a statistically significant increase (p<0.01) of micronucleated cells over the concurrent negative control value was observed in Main Assay I at the highest concentration scored which yielded marked cytotoxicity (67%). A statistically significant linear trend was also indicated. However, a high heterogeneity was observed between the two replicate cultures at the two highest concentrations analysed, therefore an extended analysis to increase the sample size was performed for negative control and for cultures treated at 667 and 444 µg/mL. When small departures from control values are
observed, clarification/confirmation of the results can be obtained by extending the assessment of slides for micronucleated cells, hence one thousand binucleated cells per culture were scored for the presence of micronuclei using slides which had not been examined previously. Data generated in this additional analysis were combined with the data previously generated. Once again, a statistically significant increase of cells bearing micronuclei was observed only at 667 µg/mL, where the cytotoxicity level exceeded the recommended range and a remarkable difference in the incidence of micronucleated cells between replicate cultures was noted. These results have been attributed to an excessive toxic effect. Since there is considerable evidence that excessive toxicity can give rise to 'misleading' positive results that occur only under cytotoxic conditions and not at lower concentrations, Main Assay II was performed using a narrowed dose range.

No statistically significant increase in the incidence of micronucleated cells over the concurrent control was seen at any dose level, although an adequate level of cytotoxicity (53%) was achieved at the top concentration scored. No concentration related increase of cells bearing micronuclei was observed. On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce micronuclei in human lymphocytes after in vitro treatment.

Summary table (Main phase 1, 3 hour treatment)

Treatment	Dose level (µg/mL)	Presence of S9 metabolism			Absence of S9 Metabolism
		% Mn cells	Sig.	%Cytotoxicity	Dose level (µg/mL)	% Mn cells	Sig.	%Cytotoxicity
Solvent	0	0.33	-	-	-	0.3	-	-
Test item	296	0.3	NS	20	132	0.25	NS	26
Test item	444	0.60	NS	30	198	0.35	NS	31
Test item	667	0.98	***	67	296	0.45	NS	47
Cyclophosphamide	20	3.30	***	45	-	-	-	-

Note: results for combined scoring of 2000 cells.

 

Summary table (Main phase 2, 3 hour with S9 mix and continuous (31 hour) treatment without S9 mix)

Treatment	Dose level (µg/mL)	Presence of S9 metabolism			Absence of S9 Metabolism
		% Mn cells	Sig.	%Cytotoxicity	Dose level (µg/mL)	% Mn cells	Sig.	%Cytotoxicity
Solvent	-	0.7	-	-	-	0.95	-	-
Test item	303	1.15	NS	28	7.9	0.5	NS	19
Test item	348	1.25	NS	42	11.9	0.9	NS	33
Test item	400	0.85	NS	53	17.8	0.6	NS	50
Cyclophosphamide	15	3.85	***	50	-	-	-	-
Colchicine	-	-	-	-	0.04	4.5	***	99

Conclusions:

Under the experimental conditions employed, the substance does not induce micronuclei in human lymphocytes after in vitro treatment.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Remarks:

E.Coli WP2 urvA rather than WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix prepared from the livers of Sprague-Dawley rats treated with Phenobarbital and 5,6-Benzoflavone.

Test concentrations with justification for top dose:

Main Phase 1 (Plate incorporation method):
TA1535, TA98 (+/- S9): 313, 625, 1250, 2500 and 5000 µg/plate.
TA1537 (- S9): 313, 625, 1250, 2500 and 5000 µg/plate.
TA1537 (+S9): 156, 313, 625, 1250, 2500 and 5000 µg/plate.
E.Coli WP2 urvA (+/- S9): 156, 313, 625, 1250, 2500 and 5000 µg/plate.
TA100 (+/- S9): 78.1, 156, 313, 625, 1250 and 2500 µg/plate.

Main phase 2 (Pre-incubation method):
TA1535, TA1537, TA98, E.Coli WP2 urvA (+/- S9): 313, 625, 1250, 2500 and 5000 µg/plate.
TA100 (+/- S9): 39.1, 78.1, 156, 313, 625 and 1250 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene (with S9 mix for all strains)

Details on test system and experimental conditions:

Preliminary toxicity test:
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main 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:
Two Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolism 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 first Main 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 second Main Assay was performed using a pre-incubation method.

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 Assay I were held at 4°C for approximately 24 hours before scoring, while plates from the preliminary toxicity test and Main Assay II were immediately scored by counting the number of revertant colonies on each plate.

Rationale for test conditions:

Standard as per relevant OECD guideline

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 showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

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 line does not include the untreated control data, but includes the solvent control data. 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.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight reduction in revertant colonies at the top concentration in the presence of S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

not valid

Positive controls validity:

not valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at the two highest concentrations in the absence and presence of S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight reduction in revertant colonies at the top concentration in the absence of S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item, evident to the unaided eye, was observed at the end of the incubation period at any concentration tested. However, a precipitation of the test item, which interfered with the evaluation of the background lawn, was observed under the microscope at the highest concentration tested (5000 μg/plate). Toxicity, as indicated by reduction in revertant numbers, was observed with TA100 tester strain at the two highest dose levels both in the absence and presence of S9 metabolism. A slight reduction of revertant colonies was also observed at the highest concentration with WP2 uvrA and TA1537 tester strains in the absence and presence of S9 metabolism, respectively. No increase in revertant numbers was observed with any tester strain at any dose level in the absence or presence of S9 metabolism.

The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Conclusions:

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Reference 3

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 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells,

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Standard as per guidelines.
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in liquid nitrogen (-196°C).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: All incubations were carried out in a humid atmosphere (80 - 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C. R10 medium was used (basic growth medium supplemented with heat-inactivated horse serum).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Dose range finder (3 hours treatment): solvent control, 9.8, 19.5, 39, 78, 156 and 33 µg/mL (with and without metabolic activation).
Dose range finder (24 hours treatment): solvent control, 9.8, 19.5, 39, 78, 156 and 33 µg/mL (without metabolic activation).
Main experiment 1 (3 hour treatment): solvent control, 5, 10, 20, 40, 80, 100, 110, 130 µg/mL (without metabolic activation).
Main experiment 1 (3 hour treatment): solvent control, 2.5, 10, 40, 100, 120, 130, 140 and 156 µg/mL (with metabolic activation).
Main experiment 2 (24 hour treatment): solvent control, 1.25, 2.5, 10, 40, 50, 60, 70 and 80 µg/mL (without metabolic activation).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Standard as per OECD guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Test formulation preparation:
A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in dimethyl sulfoxide at 250 mg/mL. The stock solution was treated with ultrasonic waves until the test item had completely dissolved.
To protect the test item from light, amber-colored glassware or tubes wrapped in tin-foil were used for test item preparations. Test item concentrations were used within 2 hours after preparation. The final concentration of the solvent in the exposure medium was 1% (v/v).

Mutagenicity test:
Eight doses of the test item were tested in the mutation assay. The test item was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods. The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

Cell treatment:
Per culture 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate.

In the first experiment, cell cultures were exposed for 3 hours to the test item in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test item in exposure medium for 24 hours in the absence of S9-mix. For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in growth medium (R10-medium).

For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter.

Expression period:
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

Determination of Mutation Frequency (MF):
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. In the second experiment in one of the solvent control groups. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

Determination of mutant colonies:
The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

Rationale for test conditions:

Standard as per OECD guidelines

Evaluation criteria:

Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical
control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

No statistical analysis conducted as results interpreted based on GEF.

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 applicable

Positive controls validity:

valid

Additional information on results:

Solubility:
The test item precipitated in the exposure medium at concentrations of 156 μg/mL and above. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 313 μg/mL.

Dose range finder:
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 9.8 to 313 μg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. In the absence of S9-mix, no toxicity in the suspension growth was observed up to and including the test item concentration of 78 μg/mL compared to the suspension growth of the solvent control. No cell survival was observed at test item concentrations of 156 μg/mL and above.

In the presence of S9-mix, the relative suspension growth was 20% at the test item concentration of 156 μg/mL compared to the relative suspension growth of the solvent controls. No cell survival was observed at the test item concentration of 313 μg/mL. The relative suspension growth was 6% at the test item concentration of 78 μg/mL compared to the relative suspension growth of the solvent controls. No cell survival was observed at test item concentrations of 156 μg/mL and above.

First mutation experiment:
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test in the absence and presence of S9-mix: 2.5, 5, 10, 20, 40, 80, 100, 110, 120, 130, 140 and 156 μg/mL exposure medium.

Toxicity evaluation: In the absence of S9-mix, the dose levels of 2.5 to 40 μg/mL showed no cytotoxicity and 110 and 120 μg/mL showed similar cytotoxicity. Therefore, the dose levels of 2.5 and 120 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 140 and 156 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, the dose levels of 2.5 to 120 μg/mL showed no cytotoxicity. Therefore, the dose levels of 5, 20, 80 and 110 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 5, 10, 20, 40, 80, 100, 110 and 130 μg/mL exposure medium.
With S9-mix: 2.5, 10, 40, 100, 120, 130, 140 and 156 μg/mL exposure medium.
In the absence of S9-mix, the relative total growth of the highest test item concentration (130 μg/mL) was 5% compared to the total growth of the solvent controls, whereas relative total growth of one concentration below (110 μg/mL) was 18% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test item concentration (156 μg/mL) was 8% compared to the total growth of the solvent controls, whereas relative total growth of one concentration below (140 μg/mL) was 27% compared to the total growth of the solvent controls.

Mutagenicity evaluation: No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutation experiment:
To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. Based on the results of the dose-range finding test, the following dose levels were selected for mutagenicity testing: 1.25, 2.5, 5, 10, 20, 40, 50, 60, 70 and 80 μg/mL exposure medium.

Toxicity evaluation:
The dose levels of 1.25 to 20 μg/mL showed no cytotoxicity. Therefore, the dose levels of 5 and 20 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels selected to measure mutation frequencies at the TK-locus were: 1.25, 2.5, 10, 40, 50, 60, 70 and 80 μg/mL exposure medium.

Mutagenicity evaluation:
No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Acceptability criteria of the solvent control:
In the second mutation experiment, the value of one of the cloning efficiency of the solvent controls was not within the range mentioned in the study plan. The value of the cloning efficiency of 128% was not within the limit of the range (65-120%). Normal and sufficient cell growth was observed in the mutation experiment and the suspension growth was within the acceptability criteria, therefore no explanation can be given for the CE value. Since the value of the spontaneous mutation frequency is within the acceptability criteria and clear negative results were obtained, this deviation in the cloning efficiency had no effect on the results of the study.

Experiment 1: Cytotoxic and Mutagenic Response of the Test Item in the Mouse Lymphoma L5178Y Test System

Dose (µg/mL)	RSG (%)	CE day 2 (%)	RCE (%)	RTG (%)	MF per 10^6 survivors
					Total	Small	Large
3 hour treatment without metabolic activation
SC1	100	110	100	100	71	12	58
SC2		101			70	13	56
5	101	99	94	95	83	21	59
10	102	93	88	90	85	22	61
20	100	107	101	101	78	16	60
40	96	89	84	81	79	18	59
80	78	107	101	79	57	7	49
100	50	95	90	45	65	21	42
110	22	86	82	18	54	13	40
130	5	98	93	5	108	26	78
MMS	79	54	51	40	490	246	213

 

Dose (µg/mL)	RSG (%)	CE day 2 (%)	RCE (%)	RTG (%)	MF per 10^6 survivors
					Total	Small	Large
3 hour treatment with metabolic activation
SC1	100	95	100	100	88	21	64
SC2		94			89	31	55
2.5	98	76	80	78	110	27	80
10	99	89	94	93	100	23	74
40	104	99	105	109	73	18	53
100	78	90	95	74	103	19	81
120	81	81	86	70	79	17	60
130	66	77	81	54	84	25	57
140	32	80	85	27	97	18	76
156	9	80	85	8	97	14	80
CP	46	59	63	29	649	315	272

 

Experiment 2: Cytotoxic and Mutagenic Response of the Test Item in the Mouse Lymphoma L5178Y Test System

Dose (µg/mL)	RSG (%)	CE day 2 (%)	RCE (%)	RTG (%)	MF per 10^6 survivors
					Total	Small	Large
24 hour treatment without metabolic activation
SC1	100	95	100	100	90	52	35
SC2		128			53	27	24
1.25	106	98	88	93	82	32	47
2.5	103	98	88	91	89	35	50
10	101	98	88	89	83	31	49
40	71	131	117	84	62	20	40
50	53	89	80	42	57	29	26
60	34	89	80	27	65	36	26
70	30	108	97	30	56	26	29
80	14	85	76	11	61	33	26
MMS	92	66	59	54	869	397	349

Conclusions:

The test substance was not found to be mutagenic in the TK mutation test system under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In all three in vitro genotoxicity tests conducted, the test substance provided a negative response both in the presence and absence of metabolic activation indicating a lack of genotoxicity under the experimental conditions described in the experimental reports.

Justification for classification or non-classification

It was concluded that the test material was not mutagenic or clastogenic based on the conditions employed in the bacterial reverse mutation test, micronucleus test and gene cell mutation test and would not be classified based on the negative results obtained.