Phosphoric acid, mono- and di-C11-14 (linear and branched) alkyl esters

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2009.1.26 to 2009.4.2

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance potassium salt of alkyl (C=9-15) phosphate. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts

IUPAC Name:

Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts

Constituent 2

Reference substance name:

97468-13-8 (Phosphoric acid, C9-11 branched and linear alkyl esters, potassium salts) and 91844-80-3 (Phosphoric acid, C13-15 branched and linear alkyl ester, potassium salts); 50/50

IUPAC Name:

97468-13-8 (Phosphoric acid, C9-11 branched and linear alkyl esters, potassium salts) and 91844-80-3 (Phosphoric acid, C13-15 branched and linear alkyl ester, potassium salts); 50/50

Test material form:

liquid: viscous

Details on test material:

- Analytical purity: 33.7%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan Inc., Tsukuba Breeding Center
- Age at study initiation: 8 weeks old
- Weight at study initiation: 302 to 329 g
- Assigned to test groups randomly: yes, under following basis: based on their body weights measured on the day of animalallocation(February2,2009) by stratification based on their body weights to give homogeneous distributions of body weights among the groups.
- Fasting period before study: no fasting
- Housing: Polycarbonate cages (TR-PC-200, 265Wx426Dx200H mm, Tokiwa Kagaku Kikai Co., Ltd. ) sterilized by autoclave were used and exchanged at animal allocation to treatment groups.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): acceptable range: 19 to 25 degrees (actual range: 22 to 23.4 degree)
- Humidity (%): acceptable range: 35 to 75 % (actual range: 46.5 to 60.9 %)
- Air changes (per hr): lOt o 30 times per hour with filtered fresh air
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hars light (7:00 to 19:00)

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water for injection (Otsuka Pharmaceutical Factory Inc.)
- Concentration of test material in vehicle: 200, 100, 50, 25 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no. (if required): K7L87

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test substance suspensions were prepared just before each administration according to the followlng procedures.
The weighing amounts were corrected for purity of the test substace (33.67%).

(1)The test substance was weighed according to the tables below (see "any other information on materials and methods incl. tables")
(2)The vehicle (water for injection) was added, and the test substance was dispersed homogenously. The vehicle was further added to make the prescribed concentration.
(3)The test substance formulations (solutions) were stirred with a magnetic stirrer.

Duration of treatment / exposure:

Dosing fequency was twice at a 24hours interval. Bone marrow cells were collected at 24 hours after the final dosing.

Frequency of treatment:

twice at a 24hours interval

Post exposure period:

Bone marrow cells were collected at 24 hours after the final dosing.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CP)
- Justification for choice of positive control(s):
This substance is commonly used as a positive control in the micronucleus tests and is recommended in the Guidelines on Genotoxicity Tests of Pharmaceuticals. In addition, there is abundant of historical data at the testing facility.
- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a study conducted previously at the testlng facility, entitled “Micronucleus Test of i20081503 using Rats (Study number: B081258; i20081503 was the previous name of Phosphoric acid, C13-15 branched and linear alkyl ester, potassium salts (50/50)” that was not compliant with GLP regulations, the test substance was administered to male rats at 500, 1000 and 2000 mg/kg. As a result, mortality was not observed at any doses; however, decrease in bodyweight, salivation, prone position, moderate decrease in locomotor activity and soiled perineal regeon were noted at 2000mg/kg, and salivation was noted at 1000 mg/kg. Therefore, the highest dose for
this study was set at 2000 mg/kg, and the lower doses of 1000, 500 and 250 mg/kg were set with a common ratio of 2. Furthermore, the negative andpositive control groups treated with the vehicle (water for irtjection) and 20mg/kg of CP, respectively were also set. The dose volume was set at 10mL/kg of body weight in all the groups. The dosing volume was calculated for each animal based on the body weight measured just before each administration.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Dosing fequency was twice at a 24hours interval. Bone marrow cells were collected at 24 hours after the final dosing.

DETAILS OF SLIDE PREPARATION:
1) The animals were anesthetized with sodium thiopental (RAVONAL⑧, Mitsubishi Tanabe Pharma Corporation, manufacturlng number 87004, concentration 50 mg/mL) administered intraperitonea11y. The abdominal cavity was opened, and the animals were euthanized by exsangulnation from the abdomlnal aorta.
2) The bone marrow cells were collected by washing the cavity with approximately 0.5 mL of 10% neutral buffbred formalin (Muto Pure Chemicals Co., Ltd., Lot. No.: 080709).
3) The cell suspension was mixed further with 0.5 mL of 10% neutral buffbred formalin and then left to stand 5 minutes to obtain the supernatant.
4) The supernatant was mixed with l mL of 10% neutral buffbred formalin and centrifuged at about 170×g (1000rpm) for5mlnutes. The supernatant was discarded.
5) The precipitate was resuspended in a small amount of 10% neutral buffbred formalin and the resulting cell suspension was stored in a serum tube at roomtemperature.
6) The cell suspension was stained with the same volume of 500ug/mL aqueous solution of acridine orange just before the microscopic observation, and was spread on a slide.

METHOD OF ANALYSIS:
Bone marrow cell specimens were observed under a fluorescence microscope withB excitation filter in a blind manner.
One thousand erythrocytes per animal (500 ce11s per area x2 areas of view), including both immature and mature erythrocytes, were examined to determine the percentage of immature erythrocytes (IMEs) among total erythrocytes. A total of 2000 IMEs in 2 areas of view under the microscope(1000 IMEs per area) were examined for the number of micronucleated ce11s (NMIMEs).
Micronuclei in the cytoplasm and the types of erythrocytes (immature or mature) were identified according to the methods of Hayashi et al. Erythrocytes stained with red fluorescence in the cytoplasm were recognized as mature erythrocytes. Small bodies with yellowish green fluorescence in the cytoplasm were recognized as micronuclei.

OTHER:

Evaluation criteria:

The ability of the test substance to induce MNIMEs was judged positive when the test
substance significantly increased the number of MNIMEs compared to the negative
control, with a dose-dependency.

Statistics:

The number of MNIMEs, percentage of IMEs among total erythrocytes and body weight of animals were statistically analyzed. Among the tests listed below requiring the significance levels of both 5% and 1%, the significance level of 5% was applied at first. When there was no signincant diffbrence in the level of 5%, the significance level of 1% was not applied.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Body weight

Treatment group	Dose level (mg/kg) Frequency Route	Animal Number	Body weight (g)
			Just before the first dosing	Just before the second dosing	Before preparation of specimens
Negative control (Water for injection)	0   Twice* p.o.	10101 10102 10103 10104 10105 Mean±SD	308 327 322 308 309 314.8±9.0	314 334 327 311 320 321.2±9.4	326 348 338 326 330 333.6±9.4
Test Substance	250   Twice* p.o.	10201 10202 10203 10204 10205 Mean±SD	309 306 308 320 323 313.2±7.7	322 316 316 328 339 324.2±9.7	329 321 328 338 354 334.0±12.7
	500   Twice* p.o.	10301 10302 10303 10304 10305 Mean±SD	322 315 306 309 328 316.0±9.1	331 322 314 319 341 325.4±10.7	348 331 323 327 348 335.4±11.8
	1000   Twice* p.o.	10401 10402 10403 10404 10405 Mean±SD	307 308 326 318 318 315.4±7.9	311 319 331 330 326 323.4±8.4	324 325 345 339 342 335.0±9.8
	2000   Twice* p.o.	10501 10502 10503 10504 10505 Mean±SD	312 314 302 326 320 314.8±9.0	318 320 299 335 323 319.0±13.0	324 329 290 347 329 323.8±20.8
Positive control (CP)	20   Twice* p.o.	10601 10602 10603 10604 10605 Mean±SD	307 320 309 314 329 315.8±8.9	306 322 314 317 330 317.8±9.0	315 328 329 325 348 329.0±12.0

CP:Cyclophosphamidemonohydrate,  

*ina 24 hr interval

 

 

Table 2.　 Clinical signs

Treatment group	Dose level (mg/kg) Frequency Route	Animal number	First dosing			Second dosing			Before preparation of specimens
			Before	0-1 hr	3 hr	Before	0-1 hr	3 hr
Negative control (Water for injection)	0   Twice* p.o.	10101 10102 10103 10104 10105	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -
Test Substance	250   Twice* p.o.	10201 10202 10203 10204 10205	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -
	500   Twice* p.o.	10301 10302 10303 10304 10305	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -
	1000   Twice* p.o.	10401 10402 10403 10404 10405	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -
	2000   Twice* p.o.	10501 10502 10503 10504 10505	- - - - -	- - Di - -	- - - - -	- - - - -	- - D(+) S(+) -	- - D(+),So - -	- - - - -
Positive control (CP)	20   Twice* p.o.	10601 10602 10603 10604 10605	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -	- - - - -

CP:Cyclophosphamidemonohydrate,  

*ina 24 hr interval

-: Noabnormarily, D: Decrease inlocomotoractivity, S: Salivation, So: Soiledpernealregion, Di: Diarrhea

 

 

Table 3. Results of micronucleus test

Treatment group	Dose level (mg/kg) Frequency Route	Animal number	MNMEs			IME ratio among total erythrocytes
			Number ofIMEsscored	Number	Incidence (%)	Number of erythrocytes scored	IMEs/ (IMEs+MEs) (%)
Negative control (Water for injection)	0   Twice* p.o.   Total /	10101 10102 10103 10104 10105 Mean±SD	2000 2000 2000 2000 2000 10000	4 4 1 1 3 13	0.20 0.20 0.05 0.05 0.15 0.13±0.08	1000 1000 1000 1000 1000	56.5 54.9 54.3 50.5 55.0 54.2±2.2
Test Substance	500   Twice* p.o.   Total /	10301 10302 10303 10304 10305 Mean±SD	2000 2000 2000 2000 2000 10000	1 2 3 4 3 13	0.05 0.10 0.15 0.20 0.15 0.13±0.06	1000 1000 1000 1000 1000	50.2 52.1 49.7 49.4 53.5 51.0±1.8
	1000   Twice* p.o.   Total /	10401 10402 10403 10404 10405 Mean±SD	2000 2000 2000 2000 2000 10000	6 1 1 3 4 15	0.30 0.05 0.05 0.15 0.20 0.15±0.11	1000 1000 1000 1000 1000	50.6 51.4 55.4 56.2 51.7 53.1±2.5
	2000   Twice* p.o.   Total /	10501 10502 10503 10504 10505 Mean±SD	2000 2000 2000 2000 2000 10000	2 1 4 1 2 10	0.10 0.05 0.20 0.05 0.10 0.10±0.06	1000 1000 1000 1000 1000	50.5 49.3 49.6 55.0 54.6 51.8±2.8
Positive control (CP)	20   Twice* p.o.   Total /	10601 10602 10603 10604 10605 Mean±SD	2000 2000 2000 2000 2000 10000	78 97 82 118 104 479##	3.90 4.85 4.10 5.90 5.20 4.79±0.82	1000 1000 1000 1000 1000	23.9 28.6 21.9 31.5 24.3 26.0±3.9**

IME: Immature erythrocyte, MNIMEs:MicronucleatedIMEs, MEs: Mature erythrocyte,

CP:Cyclophosphamidemonohydrate,  

*ina 24 hr interval

##: Significantly (p<0.01) different from the negative control byKastenbaum& Bowman’s method

**: Significantly (p<0.01) different from the negative control by Student’s t-test

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It was concluded that Phosphoric acid, C13-15 branched and linear alkyl esters, potassium salts was negative for micronucleus induction in rats bone marrow under the conditions employed in this study, and that the test substance did not have genotoxic potential to induce chromosome aberration on in vivo.

Executive summary:

Phosphoric acid, C13-15 branched and linear alkyl esters, potassium salts was tested in an in vivo micronucleus test with male Crl: CD(SD) rats (8 weeks old at dosing) to examine its ability to induce micronucleated cells in the bone marrow.

The animals (5 animals per group) received oral gavage administration of the negative control (water for injection), the test substance at 250, 500, 1000 or 2000 mg/kg or the positive control (cyclophosphamide monohydrate 20mg/kg) twice at a 24 hr interval. Bone marrow cells were collected 24 hr after the final administration. Bone marrow cell specimens were prepared to examine the incidence of micronucleated immature erythrocytes (MNIMEs) and percentage of immature erythrocytes (IMEs).

No statistically significant difference was detected in the numbers of MNIMEs per 10000 IMEs (2000 cells/animal x 5 animals/group) between the test substance groups and the negative control group. No significant decrease was detected in the percentage of IMEs in any of the test substance groups compared to the negative control group, indicating no bone marrow toxiclty. In contrast, the number of MNIMEs signincantly increased, and the percentage of IMEs significantly decreased in the positive control group compared to the negative control group.

The incidences of MNIMEs in the negative control groups were within the range of the historical control data of the testing facility. Significant increases were detected in the incidence of MNIMEs in the positive control group. These results confirmed the validity of this study.

It was concluded that Phosphoric acid, C13-15 branched and linear alkyl esters, potassium salts did not induce micronucleated erythrocytes in the rat bone marrow cells under the conditions employed in this study.