Alkyl phosphites

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:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was carried out to GLP standard, in accordance with OECD guideline 474. Some details on the test material are missing.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

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 Laboratories, Raleigh North Carolina
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 26.8-38.0 grams, Females 22.0-29.2 grams.
- Assigned to test groups randomly: Yes
- Fasting period before study: Not stated
- Housing: Single housed during the test period, suspended stainless steel and wire mesh with absorbent paper below cages.
- Diet: PMI Feeds Inc, Certified Rodent Diet # 5002 (pellets), ad libitum.
- Water: Automatic watering system, ad libitum.
- Acclimation period: 21 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24.4
- Humidity (%): 40 to 70% relative humidity.
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): 12 hours light (0700 to 1900) 12 hours dark (1900 to 0700).

IN-LIFE DATES: From: To: 1996-06-11 to 1996-06-14

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Carrier: Corn Oil, Best Foods, CPC International, Expiration Date September 1996 and October 1996.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance was diluted with the carrier and administered at a constant volume which did not exceed 1.0 mL/100 grams body weight. Dosing mixtures were prepared fresh for each day of dose administration. Doeses used were 0 (carrier control), 375 (low), 750 (mid) and 1500 mg/kg body weight. Positive control Cyclophosphamide was administered at 20 mg/kg. Three doses were given 24 hours apart via oral gavage. Dosing occured at the same time every day (11:00 ± 2.5 hours).

Duration of treatment / exposure:

72 hours

Frequency of treatment:

3 treatments approximately 24 hours apart (0, 24 and 48 hours).

Post exposure period:

Animals were sacrificed 24 hours after final dose administration.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
375 mg/kg
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
750 mg/kg
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1500 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

Range Finding Test

1st Rangefinder 500, 1000 and 2000 mg/kg, two animals per sex per dose. Second Rangefinder 1500 mg/kg three animals per sex per dose.

Definitive Test

5 animals/sex/dose (except high dose group were 7 animals/sex/dose were used to unsure sufficient survival until sacrifice)

Control animals:

other: yes: concurrent carrier

Positive control(s):

- Cyclophosphamide;
- Justification for choice of positive control(s): Historical Evidence and considered stable during duration of experiment.
- Route of administration: Oral Gavage
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:

Animals were examined for viability twice daily during the dosing period. Detail clinical observations were made as to the nature, onset, severity and duration of toxicological signs after each substance administration.

Body weights were recorded before testing for group allocation purposes and again on the first day of dosing.

A gross necropsy was performed on all animals that succumbed during the study. Animals that expired prior to sacrifice were not utilized for bone marrow collection.

Both femurs from the mice were removed following sacrifice and the bone marrow was aspirated out. Slides were then prepared (see below) and PCEs examined under light microscopy.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Approximately 24 hours after the final dose administration the surviving animals were sacrificed via CO2 asphyxiation. Immediately following sacrifice both femurs were removed from each animal. The bone marrow was aspirated, pooled, flushed in fetal bovine serum and centrifuged. After decanting the supernatant the resulting pellet was resuspended in any remaining supernatant. Smears, 2 slides per animal were prepared from the resuspended pellet. Slides were stained using Acridine Orange and were wet mounted. Slides were labeled with study number and animal number prior to evaluation of PCEs. Since animals were assigned to groups by weight the animal numbers in each group were essentially random and were used as a blind code. Two thousand PCEs from each animal were examined for the presence of micronuclei. The percent of PCEs in the total population of erythrocytes was determined for each animal by counting a total of 1000 polychromatic and normochromatic erythrocyes. PCEs stain fluorescent red/orange, normochromatic erythrocytes are unstained or dull green and micronuclei stain fluorescent bright yellow. Additional criteria for microscopic identification of micronuclei are a circular appearance and a diameter between 1/20 and 1/5 of the cells diameter (Schmid 1975).

Evaluation criteria:

The assay is to be considered valid if the mean incidence of MNE's per 1000 PCE's (calculated from evaluation of 2000 PCE's) does not exceed 4 (or 0.4%) in the carrier control group and if the mean incidence of MNE's for the positive control group is significantly greater (p,0.05) than the carrier control group.

The criteria for a test substance to be considered inducing a positive response when compared to the carrier control were as follows:

1. Dose related statistical increase in the mean number of MNE's, including at least one dose point that is statistically different (p<0.05) from the mean number of MNE's of the carrier control group. This value must also be outside the normal range of the mean number of MNE's in the carrier control group (i.e. greater than 4).

or

2. At least two dose poins that are statisticaly different (p>0.05) fom the mean number of MNE's in the carrier control group and greater than the mean normal range of the mean number of MNE's of the carrier control group (i.e. greater than 4). A dose point was considerd negative if the mean value for a statistically significant increase in MNE's is within the normal range of the carrier control (0-4 micronuclei per 1000 PCE's). This assay is considerd inconclusive if a single dose is statistically different from the carrier control group and there is no dose response.

Statistics:

The following parameters were recorded for each animal during bone marrow cell analysis.
- Ratio of polychromatic to nonchromatic erythrocytes.
- Number of polychromatic erythrocytes with micronuclei.
- Number of polychromatic erythrocytes scored.

Statistical analysis included means and standard deviations of the micronuclei data and test of equality of group means was performed by standard one way analysis of variance (Snedecor and Cochran 1989). If the ANOVA is significant comparisons of vehicle control to dose group means were performed using Duncan's Multiple Range Test (Snedecor and Cochran 1989). A standard regression analysis was performed to test for dose response (Snedcor and Cochran 1989).

Residuals from the ANOVA were anaylsed for nomrality by either Wilk's Criterion (Shapiro and Wilk 1965) or the Kolomgorov-Snirnov Statistics (Hollander and Wolfe 1973). If the residuals are not normally distributed (at 0.01 level significance) in more than 25% of the analyses then nonparametric analyses were performed. The non parametric analyses included the Kruskal-Wallis one wy analysis of variance (Hollander and Wolfe 1973), followed by Dunn's Summed Rank Test (Hollander and Wolfe 1973) if differences were indicated. Dose response was evaluated by Jonkheere's test of ordered response (Hollander and Wolfe 1973). Sexes were analysed seperately.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Toxicity in all doses (increase in severity with each dose). Cytotoxicity was seen in bone marrow at dose levels of 1500 mg/kg.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Initial range finding groups 500, 1000 and 2000 mg/kg, two males and two females per dose group. Second range finding 1500 mg/kg, 3 males and three females per group. Three treatments were adminstered at 0, 24 and 48 hours, animals were then sacrificed 24 hours after final administration.
- Solubility: Soluble in carrier (corn oil)
- Clinical signs of toxicity in test animals: No animals in 2000 mg/kg group survived to terminal sacrifice.
- Evidence of cytotoxicity in tissue analyzed: No observable toxicity was seen in the bone marrow of the 500 or 1000 mg/kg groups. In the 1500 mg/kg goup 1 male died and a substantial decrease in percentage of PCEs was observed when evaluating bone marrow toxicity.
- Harvest times: 24 hours after final administration.


RESULTS OF DEFINITIVE STUDY

Induction of micronuclei (for Micronucleus assay): There were no statistically significant or dose related increases in micronucleus formation at any dose level evaluated. Cytotoxicity was observed in both males and females at the 1500 mg/kg dose level as indicated by statistically significant (p<0.01) decreases in the mean percentage of PCEs when compared with the carrier control.

Clinical Signs: Clinical signs of toxicity were observed in all treatment groups and increased in frequency and severity with increasing dose. Observations included oral discharge, unthrifty coat, anogential staining, soft stool, little sign of stool, dyspnea, hypoactivity and or swollen abdomen. Four males and one female from the 1500 mg/kg group were found dead on days 1, 2 or 3.
Postmortem observations of the animal which succumbed included reddening and or sloughing of the stomach mucosa, gastrointestinal distention and or discoloration of the diaphragm and lungs. All animals in the positive control and carrier controls were within normal limits for the entire study.

Statistical evaluation: No statisctically significant increases in micronucleus formation were observed at any dose level.

Positive control substance (20 mg/kg cyclophosphamide) induced statistically significant (p<0.01) increases in the mean number of MNEs, indicating that this substance was clastogenic and that the test system responded in an appropriate manner. In addition the positive control induced cytotoxicity which was indicated by a statistically significant decrease in PCEs compared with the carrier control.

Carrier Control Group: Since the mean incidence of MNEs per 1000 PCEs did not exceed 4 in the carrier control group and the mean incidence of MNEs for the positive control group was significantly greater than the carrier control, this assay was considered valid.

Analysis of test material: Due to an oversight concentration verification for samples were not conducted until 6 weeks after sampling, these samples were stored at room temperature during this period. Resulting values from analysis showed that samples were 36 to 78% lower than nominal concentrations. Analytical results form another study (EBSI 149270) indicate the test susbtance has limited stability in corn oil. Therefore results for test substance analyses for this study are not thought to be reliable. Note that dosing mixtures were prepared fresh on the day of dosing and the animals responded in a dose dependant manner.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
In conclusion the test substance did not produce an increase in micronuclei formation at any dose level evaluated, but did induce cytotoxicity in the bone marrow oc CD-1 mice at a dose level of 1500 mg/kg.

Executive summary:

The in vivo mammalian bone marrow micronucleus assay is a short term test to evaluate potential clastogenecity of a test substance. Evidence of chromosome breakage or non-disjunction can be readily detected as micronucleated polychromatic erythrocytes (MNEs), a postive results is presumptive evidence of mutagenic potential in mammals. Species tested was the CD-1 mouse and based on rangefinding studies test concentrations were administered at 375, 750 and 1500 mg/kg of test substance via oral gavage to both male and female mice. Carrier control and positve control groups recieved 20mg/kg corn oil and cyclophosphamide respectively. Clinical observations were made after each test substance administration and prior to terminal sacrifice. Body weights were recorded before testing and again on the first day of dosing. A gross necropsy was performed on all animals that sucumbed during the study. All surviving animals were sacrificed approximately 24 hours after recieving the final dose. Immediately after scarifice both femurs were removed and processed. Smears were prepared 2 per animal and stained with Acridine Orange. Two thousand polychromatic erythrocytes were (PCE's) from each animal were examined for the presence of micronuclei and the precentage of PCE's in the total population of erythrocytes was determined. Clinical signs of toxocity were observed in all treatment and these increased in frequency and severity with increasing dose. Four males and one female sucumbed prior to study termination. All animals for the control and positve control groups were within normal limits for the entire study. There were no increases or statistically significant differences in micronuceli formation at any dose level of the test substance evaluated. Cytotoxicity was observed in males and females at the 1500 mg/kg dose level as indicated by statistically significant (p < 0.01) decreases in the mean percentage of PCE's when compared with the carrier control group.

The positive control induced a statistically significant increase in the mean number of MNE's which indicated that the test system responded in the appropriate manner.

In conclusion the test substance did not produce an increase in micronuclei formation at any dose level evaluated, but did induce cytotoxicity in the bone marrow of CD-1 mice at a dose level of 1500 mg/kg.