Phosphoric acid, butyl ester, sodium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-02-07 to 2012-05-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl hydrogen phosphate

EC Number:

203-509-8

EC Name:

Dibutyl hydrogen phosphate

Cas Number:

107-66-4

Molecular formula:

C8H19O4P

IUPAC Name:

dibutyl hydrogen phosphate

Test material form:

liquid

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

other: the cells have a stable karyothype with a modal chromosome number of 22

Metabolic activation:

with and without

Metabolic activation system:

S9 mixPhenobarbital/beta-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 were prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU, weight approx. 220 – 320 g, Harlan Laboratories B.V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin, 22335 Hamburg, Germany) and by peroral administrations of 80 mg/kg b.w. -naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules were kept at –20 °C for up to one week. Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo(a)pyrene. The protein concentration of the S9 preparation was 25.9 mg/mL (Lot. No.: 021211) in the pre-experiment, 22.5 mg/mL (Lot. No.: 020212) in experiment I, and 27.2 mg/mL (Lot. No.: 050412) in experiment II.

Test concentrations with justification for top dose:

137.5, 275.0, 550.0, 1100.0 and 2200.0 µg/mL
A range finding pre-experiment was performed using a concentration range of 17.2 to 2200 μg/mL (≈10 mM) to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation occurred up to the highest concentration with and without metabolic activation following 4 and 24 hours treatment. In the pre-experiment the two highest concentrations were adjusted with 2N NaOH. There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item. Based on the results of the pre-experiment, the maximum concentration of the main experiments was again, 2200 μg/mL or 10 mM. The lower concentrations were spaced by a factor of 2.

Vehicle / solvent:

- solvent used: water

- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

The treatment time was 4 hours in the first experiment with and without metabolic activation. In the second experiment the cells were exposed to the test item for 24 hours without metabolic activation and 4 hours with metabolic activation.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments . 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5*10^2 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
- Harvest time after the end of treatment (sampling/recovery times): 7 days expression time, 8 days cultivation in medium containing 6-TG, afterwards staining of the colonies

FOR GENE MUTATION:
- Expression time: 7 days
- Selection time: 8 days in medium containing 6-thiogunanine (3 - 5x10^5 cells per culture flask)

The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICIY
mutation frequency

Rationale for test conditions:

according to OECD Guideline

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
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 non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concen-trations 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.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Relevant cytotoxic effects were only noted at the maximum concentration of the first experiment without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.
Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Any other information on results incl. tables

Table 1: Results of Experiment I, Culture I

	Conc [µg/mL]	S9-mix	Rel. cloning efficiency I [%]	Rel. cell density [%]	Rel. cloning efficiency II [%]	Mutant colonies/ 10^6 cells	Induction factor
solvent		-	100	100	100	8.9	1
EMS	150	-	89.5	106.9	102	116.8	13.1
Test item	68.8	-	100	Culture was not continued
Test item	137.5	-	102.6	95	77.8	28.6	3.2
Test item	275	-	100.1	85.2	102.3	8.1	0.9
Test item	550	-	94.5	75.9	122.2	17.1	1.9
Test item	1100	-	90.9	92	115.7	11.1	1.2
Test item	2200	-	60.8	18.6	99.1	0	0
solvent		+	100	100	100	11.7	1
EMS	150	+	55.3	83.9	101.7	615.9	52.8
Test item	68.8	+	103.1	Culture was not continued
Test item	137.5	+	99.6	79.8	100.6	12.1	1
Test item	275	+	100.7	78.5	102.8	18.4	1.6
Test item	550	+	100.5	76.1	107.4	10.6	0.9
Test item	1100	+	103.7	78.6	98	31.3	2.7
Test item	2200	+	102.8	76.3	112.8	21.9	1.9

 

Table 2: Results of Experiment I, Culture II

	Conc [µg/mL]	S9-mix	Rel. cloning efficiency I [%]	Rel. cell density [%]	Rel. cloning efficiency II [%]	Mutant colonies/ 10^6 cells	Induction factor
solvent		-	100	100	100	18.5	1
EMS	150	-	91.9	89	83.5	69.9	3.8
Test item	68.8	-	102.7	Culture was not continued
Test item	137.5	-	101.5	89	83.5	21.4	1.2
Test item	275	-	101.5	117.3	105.4	20	1.1
Test item	550	-	101.2	131.8	100.6	18.3	1
Test item	1100	-	100.1	113.2	113.7	11.9	0.6
Test item	2200	-	71.6	24.1	103.6	0	0
solvent		+	100	100	100	10.1	1
DMBA	150	+	52.8	127.4	79	826.2	82
Test item	68.8	+	98.7	Culture was not continued
Test item	137.5	+	100.8	101.4	89.3	6.8	0.7
Test item	275	+	99.4	105.8	101.1	20.2	2
Test item	550	+	99.5	103	88.7	24.3	2.4
Test item	1100	+	102	122.1	91.9	19.3	1.9
Test item	2200	+	98.9	114.1	89.9	14.8	1.5

 

Table 3: Results of Experiment II, Culture I

	Conc [µg/mL]	S9-mix	Rel. cloning efficiency I [%]	Rel. cell density [%]	Rel. cloning efficiency II [%]	Mutant colonies/ 10^6 cells	Induction factor
solvent		-	100	100	100	15	1
EMS	150	-	79.8	96.4	76.9	233.4	15.6
Test item	68.8	-	99.2	Culture was not continued
Test item	137.5	-	103.3	96	97.4	14.2	0.9
Test item	275	-	99.3	98.6	90.7	19.2	1.3
Test item	550	-	90.5	93.4	85.8	20.4	1.4
Test item	1100	-	85.1	84	93.2	18.7	1.3
Test item	2200	-	73.7	80.7	91.6	13.4	0.9
solvent		+	100	100	100	21	1
DMBA	150	+	44.5	141.4	94.2	549	26.1
Test item	68.8	+	100.6	Culture was not continued
Test item	137.5	+	100.1	160.1	93.9	13.7	0.7
Test item	275	+	101.5	168.4	97.7	17.3	0.8
Test item	550	+	100.8	128.1	94.6	20.2	1
Test item	1100	+	99.9	98	93.1	16.5	0.8
Test item	2200	+	103.1	160.4	100.8	15.2	0.7

 

Table 4: Results of Experiment II, Culture II

	Conc [µg/mL]	S9-mix	Rel. cloning efficiency I [%]	Rel. cell density [%]	Rel. cloning efficiency II [%]	Mutant colonies/ 10^6 cells	Induction factor
solvent		-	100	100	100	20	1
EMS	150	-	92	101.8	77.5	377.6	18.9
Test item	68.8	-	96.8	Culture was not continued
Test item	137.5	-	97.8	102.5	104.4	27.9	1.4
Test item	275	-	98.7	106.3	103.4	25	1.3
Test item	550	-	91.6	98.5	110.1	17.7	0.9
Test item	1100	-	86.1	88.9	98	17.7	0.9
Test item	2200	-	78.8	66.6	96.9	27.9	1.4
solvent		+	100	100	100	32.9	1.0
DMBA	150	+	43.8	81.8	91.8	570.6	17.3
Test item	68.8	+	103.6	Culture was not continued
Test item	137.5	+	100.1	100.4	91.7	23.3	0.7
Test item	275	+	103.3	89.5	119.6	19.4	0.6
Test item	550	+	100.9	104.2	113.7	17.6	0.5
Test item	1100	+	104.4	61.2	94.8	22.3	0.7
Test item	2200	+	105.6	109.5	95.6	29.9	0.9

Applicant's summary and conclusion

Conclusions:

The test item was negative in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD guideline 476. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 2200 µg/mL was equal to approximately 10 mM.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item was negative in this HPRT assay.