Bis(2,4,4-trimethylpentyl)phosphinic acid

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 10 January 10th to May 20th, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP guideline study with some restrictions: no certificate of analysis, only 1000 polychromatic erythrocytes were analysed for micronucleus instead of 2000.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

no certificate of analysis, only 1000 polychromatic erythrocytes were analysed for micronucleus instead of 2000.

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2,4,4-trimethylpentyl)phosphinic acid

EC Number:

280-445-7

EC Name:

Bis(2,4,4-trimethylpentyl)phosphinic acid

Cas Number:

83411-71-6

Molecular formula:

C16H35O2P

IUPAC Name:

bis(2,4,4-trimethylpentyl)phosphinic acid

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent
- Age at study initiation: 35 days
- Weight at study initiation: 22-24 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, overnight prior to dosing
- Housing: housed in groups (sex separated) in plastic disposable cages
- Diet (e.g. ad libitum): free access to pelleted Biosure LAD 1 rodent diet
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: yes, 4-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 °C
- Humidity (%): 31-62 %
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12 hr/12 hr

IN-LIFE DATES: From: To: no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: aqueous 1 % methylcellulose
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: between 9.56 to 250 mg/mL
- Amount of vehicle (if gavage or dermal): 20 ml/ kg bw
- Lot/batch no. (if required): T32398
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Suspensions of the substance were prepared in aqueous 1 % methylcellulose on the morning of the test.

DIET PREPARATION
not applicable as the test substance is administered by gavage.

Duration of treatment / exposure:

A single dose is administered by gavage

Frequency of treatment:

A single dose is administered

Post exposure period:

24, 48 or 72 hrs

Doses / concentrations

Doses / concentrations 1

Remarks:

1080-5000 active ingredient (mg/kg bw)
initial preliminary test phase I

Doses / concentrations 2

Remarks:

191-883 active ingredient (mg/kg bw)
initial preliminary test phase II

Doses / concentrations 3

Remarks:

506-1200 active ingredient (mg/kg bw)
Confirmatory toxicity test

Doses / concentrations 4

Dose / conc.:

900 other: active ingredient (mg/kg bw)

Remarks:

initial micronucleus test

Doses / concentrations 5

Dose / conc.:

1 000 other: active ingredient (mg/kg bw)

Remarks:

Confirmatory micronucleus test

No. of animals per sex per dose:

Preliminary studies: 2/sex/dose
Micronucleus studies: 5/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Justification for choice of positive control(s): no data
- Route of administration: oral (gavage)
- Doses / concentrations: 12 mg/kg w

Examinations

Tissues and cell types examined:

Bone marrow cells:
- polychromatic erythrocytes
- normochromatic erythrocytes
1000 polychromatic erythrocytes were analysed to determine the incidence of micronucleated cells.
1000 erythrocytes were analysed to determine the ratio of polychromatic to normochromatic erythrocytes to assess the test substance toxicity on bon marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: determining of the maximum tolerated dose, ie. the highest dosage which would not induce excessive lethality.

TREATMENT AND SAMPLING TIMES: where possible, 5 males and 5 females from the negative control and test substance groups were sacrificed 24, 48 and 72 hours after dosing. The positive control group was sacrificed 24 hrs after dosing.

DETAILS OF SLIDE PREPARATION: the femurs were cleared of tissue and the proximal epiphysis was removed from each bone. A direct bone marrow smear was made onto a slide after dilution of the marrow with a drop of foetal calf serum. One smear was made from each femur. The prepared smears were fixed in methanol (> 10 min). After air-drying the smears were stained for 10 minutes in 10 % Giemsa (prepared by 1:9 dilution of Gurr's improved R66 Giemsa (BDH) with distilled water). Following rinsing in distilled water and differenciation in buffered distilled water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS: The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal.

Evaluation criteria:

A positive response is normally indicated by a substantial, statistically significant increase (P<0.01) in the incidence of micronucleated polychromatic erythrocytes compared to the incidence for the concurrent vehicle control group for at least one of the sampling times; individual and/or group mean values shoud exceed the laboratory historical control range. A negative result is indicated where individual and group mean incidence of micronucleated polychromatic erythrocytes for animals treated with the test substance are not significantly greater than incidence for the concurrent control group and where these values fall within the historical control range. An equivocal response is obtained when the results cannot be adequately classified using the criteria for a positive or negative response.
Bone marrow cell toxicity (or depression) is normally indicated by a substancial, statistically significant decrease (P<0.01) in the ratio of polychromatic to normochromatic erythrocytes. This decrease would normally be evident at both the 48 and 72 hour sampling points, a decrease at the 24 h time point is not necessarily expected because of the relative long transition time of erythroid cells (late normoblast=>polychromatic erythrocyte (approx. 6h)=>normochromatic erythrocyte (approx. 30h)). A very large decrease in this ratio would be indicative of a cytotoxic effect.

Statistics:

Non-parametric statistical methods, based on rank are chosen for analysis of results.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

No significant increase in the number of micronucleated polychromatic erythrocytes.

Toxicity:

yes

Remarks:

Clinical signs of toxicity were observed such as piloerection, hunched posture, lethargy, ptosis and twitching. Death was observed at the dose of 900 mg/kg bw during the initial main test

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: first preliminary test: 1080-5000 mg/kg bw, second preliminary test: 191-883 mg/kg bw, third preliminary test: 506-1200 mg/kg bw
- Solubility: no data
- Clinical signs of toxicity in test animals: in the first preliminary toxicity test, death is observed at 1080 mg/kg bw and at the higher dose levels. At 3000 and 5000 mg/kg all animals died in the first hours after dosing. The second toxicity test was conducted to refine the results obtained in the first test. No death is observed up to the highest dose level of 883 mg/kg bw. Slight piloerection is observed in the first two hours after dosing in all tested animals.
- Evidence of cytotoxicity in tissue analyzed: the cytoxicity is not examined.
- Rationale for exposure: From the results obtained in the both first test, the maximum tolerated dose was estimated to be approximately 900 mg/kg bw. This dosage was therefore chosen for use in the initial micronucleus test.
- Harvest times: 72h
- High dose with and without activation: not applicable

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay): the test substance did not cause any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes (mnp) at any time point compared with the concurrent control (P>0.01). However slides from the 48h sampling time showed an unusually high incidence of mnp for animals treated with the test substance (the group mean of 2.2 % mnp is at the extreme limit of the historical control range) while the male animal 315 showed a mean value of 8 ‰ which is outside the historical control. Since these results do not meet the criteria for a positive or a negative response, they were regarded as equivocal and a confirmatory micronucleus test was performed only in male rats at the dose level of 1000 mg/kg bw. In this second assay, the results were negative at either sampling time.
- Ratio of PCE/NCE (for Micronucleus assay): No decreases in the ratio of polychromatic to normochromatic erythrocytes were obtained for animals treated with the test substance at any sampling time. In the initial test, the control positive animals were sampling at 24h. In this condition, no decrease of the ratio PCE/NCE was observed. However it should be noted that at the 24 hour sampling time, mitomycin C do not always produce a substancial decrease in this ratio because of the lag caused by erythrocyte maturation. In the confirmatory test, the positive control animals were sampling at 36h and a significant decrease in the ratio PCE/NCE was observed.
- Appropriateness of dose levels and route: the results obtained on the ratio PCE/NCE suggested that the test substance did not reach the target cells (erythrocytes of bone marrow). Therefore, the route of exposure and/or the target tissue seems to be unappropriate.
- Statistical evaluation: no data

Any other information on results incl. tables

Results of the micronucleus tests

Experiment	Sampling time (h)	Treatment	Dose	Ratio PCE/NCE	Incidence mnp	Incidence mnn (total)
I	24	Vehicle	-	0.768	1.0	1.2
		TS	900	0.630^ns	1.0^ns	0.4
		Mitomycin C	12	0.573^ns	29.7**	0.9
	48	Vehicle	-	0.803	0.7	0.5
		TS	900	0.575^ns	2.2^ns	1.0
	72	Vehicle	-	1.125	1.0	1.2
		TS	900	1.416^ns	1.3^ns	0.8
II	36	Vehicle	-	0.870	0.7	0.2
		TS	1000	0.921^ns	1.0^ns	0.7
		Mitomycin C	12	0.479	29.6*	5.5
	48	Vehicle	-	0.821	1.1	1.1
		TS	1000	0.851^ns	1.3^ns	0.5

PCE/NCE : ratio of polychromatic to normochromatic erythrocytes

mnp: Number of micronucleated cells observed per 1000 polychromatic erythrocytes

mnn: Number of micronucleated cells observed per 1000 normochromatic erythrocytes

TS: Test substance

ns: P>0.01

* : P<0.01

** : P<0.001

Mortality data results

Phase	Group	Treatment	Dose (mg/kg bw)	Mortality ratio (Number of dead animals / Number of animal dosed)
				male	female	combined
Preliminary test I	1	TS	1080	0/2	1/2	1/4
	2		1800	1/2	2/2	3/4
	3		3000	2/2	2/2	4/4
	4		5000	2/2	2/2	4/4
Preliminary test II	5	TS	191	0/2	0/2	0/4
	6		318	0/2	0/2	0/4
	7		530	0/2	0/2	0/4
	8		883	0/2	0/2	0/4
Initial main test (24 and 48h sampling)	1	Vehicle	-	0/15	0/15	0/30
	2	TS	900	5/19	10/19	15/38
	3	Mitomycin C	12	0/5	0/5	0/10
Initial main test (72h sampling)	1	Vehicle	-	0/5	0/5	0/10
	2	TS	900	0/10	0/10	0/20
Additional toxicity test	9	TS	506	0/4	No females used
	10		675	0/4
	11		900	0/4
	12		1200	0/4
Confirmatory main test	1	Vehicle	-	0/20	No females used
	2	TS	1000	0/25
	3	Mitomycin C	12	0/5

TS: Test substance

Applicant's summary and conclusion

Conclusions:

Negative
Under the test conditions, the test material did not induce micronucleus in the mouse bone marrow cells.
However, as no decrease of the ratio PCE/NCE was observed in animals treated with the test substance, it can not be assumed with certitude that the test substance reach the target tissue (i.e. bone marrow).

Executive summary:

In an in vivo micronucleus assay, performed according to the OECD No.474 and in compliance with the GLP, the test material diluted in aqueous solution of 1% methylcellulose was administered by gavage to CD1 mice (males and females).

A preliminary test was performed on mice (2/sex/dose) to determine the suitable dose levels for the main micronucleus test. Eight doses from 191 to 5000 mg/kg bw were tested; as death occurred at dose levels higher than 1080 mg/kg bw the maximum tolerated dose was estimated to be approximately 900 mg/kg bw.

Thus, in the initial main test, male and female mice were dosed with 900 mg/kg bw of Bis(2,4,4-trimethylpentyl)phosphinic acid in aqueous solution of 1% methylcellulose. Mice were also treated in the same experimental conditions with the vehicle only (negative control) and other mice were dosed with Mitomycin Cat 12mg/kg bw (positive control). 5 mice per sex were then sacrificed at 24, 48 and 72 h after dosing in order to analyse the number of micronucleated polychromatic erythrocytes. The femurs of each mouse were cleared of tissue and the proximal epiphysis was removed from each bone. A direct bone marrow smear was made onto a slide after dilution of the marrow with a drop of foetal calf serum. One smear was made from each femur. Hunched posture, lethargy, piloerection, ptosis, twitching were observed in the first day after the oral administration of the test substance. Furthermore, five male and ten females died after the treatment. The reasons of this high mortality incidence were not clearly understood. The test substance did not cause any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes (mnp) at any time point compared with the concurrent control. However slides from the 48h sampling time showed an unusually high incidence of mnp for animals treated with the test substance (the group mean of 2.2 ‰ mnp is at the extreme limit of the historical control range) while the male animal 315 showed a mean value of 8 ‰ which is outside the historical control. Since these results did not meet the criteria for a positive or a negative response, they were regarded as equivocal and a confirmatory micronucleus test was performed only in male rats (10 animals for each time point (36 and 48h)) at the dose level of 1000 mg/kg bw. In this second assay, the results were negative at either sampling time. In both experiments, the positive control gave the appropriate response; it induced large, highly significant increase in the frequency of micronucleated polychromatic erythrocytes. In both experiments, no decreases in the ratio of polychromatic to normochromatic erythrocytes (ratio PCE/NCE) were obtained for animals treated with the test substance at any sampling time. In the initial test, the control positive animals were sampling at 24h. In this condition, no decrease of the ratio PCE/NCE was observed. However it should be noted that at the 24 hour sampling time, mitomycin C do not always produce a substancial decrease in this ratio because of the lag caused by erythrocyte maturation. In the confirmatory test, the positive control animals were sampled at 36h and a significant decrease in the ratio PCE/NCE was observed.

 

 In conclusion, under the test conditions, the test substance did not induce micronucleus in the mouse bone marrow cells. However, as no decrease of the ratio PCE/NCE was observed in animals treated with the test substance, it can not be assumed with certitude that the test substance reach the target tissue (i.e. bone marrow).