Reaction mass of biphenyl-2-yl diphenyl phosphate and triphenyl phosphate and bis(biphenyl-2-yl) phenyl phosphate

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:

experimental study

Adequacy of study:

key study

Study period:

2008-12-11 - 2009-01-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study

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

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of biphenyl-2-yl diphenyl phosphate and triphenyl phosphate and bis(biphenyl-2-yl) phenyl phosphate

EC Number:

700-393-5

IUPAC Name:

Reaction mass of biphenyl-2-yl diphenyl phosphate and triphenyl phosphate and bis(biphenyl-2-yl) phenyl phosphate

Details on test material:

- Name of test material (as cited in study report): #5 Biphenyl diphenyl phosphate (mixture of the phosphates of ortho-Phenyl Phenol and Phenol and containing 25 % of Triphenyl Phosphate)
- Substance type: organic
- Physical state:Colorless to light yellowish clear liquid
- Analytical purity: > 99 %
- Impurities (identity and concentrations): no data
- Lot/batch No.: F01003
- Expiration date of the lot/batch: 2009-03-15
- Stability under test conditions: no data
- Storage condition of test material: room temperature; store in a cool, dry, well-ventilated area; keep containers tightly closed when not in use.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BioLASCO Taiwan Co. Ltd.
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation: 29.4 g ~ 32.3 g for males and 22.8 g ~ 25.3 g for females
- Assigned to test groups randomly: [no/yes, under following basis: ] no data
- Fasting period before study: no data
- Housing: polycarbonate cage; cage feeder was composed of stainless steel
- Diet (e.g. ad libitum): no data (autoclaved laboratory rodent diet 5010, LabDiet, PMI Nutrition - International, Brentwood MO, USA
- Water (e.g. ad libitum): ad libitum (autoclaved RO water)
- Acclimation period: 7 days in the testing room before dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 60 ± 20%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

none

Duration of treatment / exposure:

single dosing, dosing day is defined as Day 1

Frequency of treatment:

not applicable

Post exposure period:

5 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.5 g/kg b.w.
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1 g/kg b.w.
Basis:
nominal in water

Doses / concentrations 3

Remarks:

Doses / Concentrations:
2 g/kg b.w.
Basis:
nominal in water

No. of animals per sex per dose:

5 male / 5 female

Control animals:

yes, historical

Examinations

Tissues and cell types examined:

- the percentage of PCE (polychromatic erythrocytes) in erythrocytes (PCE %)
- micronucleus frequency in thousand PCE (MN%o PCE)

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

RESULTS

1) Mortality

Mortalities of all groups were 0 %. There was no abnormal clinical sign observed in the study. The mortality was summarized in Table 1:

Table 1. In vivo Mammalian Erythrocyte Micronucleus Test - Mortality

Groups	Dose	Day 0	Day 1	Day 2	Day 3	Day 4	Mortality^*
		Animals found dead
Negative Control	Sterile water	0	0	0	0	0	0/10
Positive Control Cyclophosphamide	80 mg/kg b.w.	0	0	0	0	0	0/10
	0.5 g/kg b.w.	0	0	0	0	0	0/10
#5 Biphenyl diphenyl phosphate (CAS number: 132-29-6)	1 g/kg b.w.	0	0	0	0	0	0/10
	2 g/kg b.w.	0	0	0	0	0	0/10

Note: No animal death occurred.

2) Body Weight

The mean body weight was summarized in Table 2:

Table 2. In vivo Marrrmaliari Erythrocytre Micronucleus Test - Body Weight

Groups		Animal Body Weight (g, mean + SD, n=5)
	Dose	Male		Female
		Day 1	Day 5	Day 1	Day 5
Negative Control	Sterile water	30.7 ± 1.1	31.8 ± 1.1	24.0 ± 0.7	24.9 ± 0.6
Positive Control Cyclophosphamide	80 mg/kg b.w.	30.7 ± 1.1	31.7 ± 1.0	24.0 ± 0.7	24.9 ± 0.6
#5 Biphenyl diphenyl phosphate (CAS number: 132-29-6)	0.5 g/kg b.w.	30.6 ± 1.0	31.6 ± 1.1	23.9 ± 0.6	24.6 ± 0.5
	1 g/kg b.w.	30.5 ± 1.0	31.5 ± 1.1	23.9 ± 0.8	24.7 ± 0.8
	2 g/kg b.w.	30.7 ± 1.1	31.7 ± 1.1	24.1 ± 0.8	24.9 ± 0.7

Note: Body weights were presented in mean + standard deviation. After analyzing with t-est, there were no significant differences between all groups.

3) Percentage of PCE in Erythrocytes

The PCE percentage of negative control group at 24, 48 and 72 hours was 3.92 ± 0.56 %, 3.51 ± 0.81 % and 3.85 ± 0.49 %, respectively. The PCE percentage of positive control group was decreased with time and 20 % lower than the PCE percentage of negative control group, indicated that cyclophosphamide inhibited erythropoiesis. All testing groups were not significantly different from negative control group, indicated that "#5 Biphenyl diphenyl phosphate" did not affect erythropoiesis. The percentage of PCE was summarized in Table 3:

Table 3. In vivo Micronucleus Test Hours after Dosing

Groups	Dose	PCE%, mean ± SD, n=10
		24 hours	48 hours	72 hours
Negative Control	Sterile water	3.92 ± 0.56	3.51 ± 0.81	3.85 ± 0.49
Positive Control Cyclophosphamide	80 mg/kg b.w.	3.12 ± 0.63	1.52 ± 0.21	0.61 ± 0.14
#5 Biphenyl diphenyl phosphate (CAS number: 132-29-6)	0.5 g/kg b.w.	3.74 ± 0.35	3.70 ± 0.53	3.62 ± 0.63
	1 g/kg b.w.	3.76 ± 0.43	4.01 ± 0.45	3.75 ± 0.38
	2 g/kg b.w.	3.66 ± 0.48	4.02 ± 0.35	3.59 ± 0.43

Note:

1. The PCE % was presented in Mean ± SD.

2. Positive control group represented inhibition on erythropoiesis

4) Micronucleus frequency

The micronucleus frequencies in PCE were examined with fluorescent microscope and summarized in Table 4:

Table 4. In vivo Mammalian Erythrocyte Micronucleus Test - Micronucleus Frequency in PCE at 24, 48 and 72 hours after dosing

Groups	Dose	PCE%, mean ± SD, n=10
		24 hours	48 hours	72 hours
Negative Control	Sterile water	1.50 ± 0.71	1.20 ± 1.14	1.10 ± 0.57
Positive Control Cyclophosphamide	80 mg/kg b.w.	6.00 ± 1.33*	20.60 ± 3.84*	9.80 ± 1.81*
#5 Biphenyl diphenyl phosphate (CAS number: 132-29-6)	0.5 g/kg b.w.	1.30 ± 0.48	1.30 ± 1.06	0.80 ± 0.63
	1 g/kg b.w.	1.50 ± 1.18	1.30 ± 0.48	1.10 ± 0.99
	2 g/kg b.w.	1.60 ± 0.70	2.00 ± 0.82	1.40 ± 0.70

Note:

1. Micronucleus frequency (%o PCE) was presented in Mean ± SD.

2. The results were analyzed with Poisson distribution, and the star symbol (*) indicated that it was significant different (p<0.05) from negative control group. Positive control group represented genotoxicity at 24 ~ 72 hours.

The micronucleus frequencies in PCE of negative control group at 24, 48 and 72 hours (1.50 ± 0.71, 1.20 ± 1.14 and 1.10 ± 0.57 %OPCE) and positive control group at 24, 48 and 72 hours (6.00 ± 1.33, 20.60 ± 3.84 and 9.80 ± 1.81 %o PCE) were both conformed to the criteria.

After statistic analyzing, all three testing groups were not significantly different from negative control group.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The animals which were administrated orally up to the dosage of 2 g/kg of "#5 Biphenyl diphenyl phosphate" presented negative result in micronucleus test. The test substance had neither genotoxicity in mice nor inhibition in erythropoiesis.

Executive summary:

This study was to evaluate the genotoxicity of "#5 Biphenyl diphenyl phosphate" with mammalian peripheral blood micronucleus test. The experimental design was based upon OECD Guideline 474.

The test article, "#5 Biphenyl diphenyl phosphate", was suspended in sterile water to 0.5, 1, and 2 g/kg for the three testing groups. Including sterile water as negative control, each material was administrated orally. Cyclophosphamide (80 mg/kg) was as positive control and injected intraperitoneally. After 24, 48 and 72 hours, collected blood samples from tail vein, smeared on acridine orange-coated slide, and examined the percentage of polychromatic erythrocytes (PCE%) and micronucleus frequency ( %o PCE) with fluorescent microscope.

The results indicated that all three testing groups were not statistically different from negative group in both PCE percentage and micronucleus frequency.

In conclusion, "#5 Biphenyl diphenyl phosphate" presented negative result in micronucleus test. The test substance had neither genotoxicity in mice nor inhibition in erythropoiesis.