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Toxicological information

Genetic toxicity: in vivo

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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
only a single dose was tested despite the toxicity noted in the preliminary study. Only 1000 polychromatic erythrocytes per dose were scored for the incidence of micronucleated immature erythrocytes.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Tris chloroisopropyl phosphate
Purity: 97.85% including all isomers

name: tris-chloroisopropylphosphate
manufacturer: Bayer AG (now LANXESS)
sample no: 002871/1989
content: 97.85% analytical date 8 Aug 1990
expiry date: 13 Dec 1991
appearance: clear liquid
storage: room temperature
MW: 327.6
Molecular formula:C9H18Cl3O4P
CAS no: 13674-84-5
intended use: industrial chemical
Specific details on test material used for the study:
TS-Freetext:
Tris chloroisopropyl phosphate.
Purity: 97.85% including all isomers.

name: tris-chloroisopropylphosphate
manufacturer: Bayer AG
sample no: 002871/1989
content: 97.85% analytical date 8 Aug 1990
expiry date: 13 Dec 1991
appearance: clear liquid
storage: room temperature
MW: 327.6
Molecular formula:C9H18Cl3O4P
CAS no: 13674-84-5
intended use: industrial chemical

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: F Winkelmann Borchen strain Bor: NMRI (SFP Han)
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 29-42 g
- Assigned to test groups randomly: yes
- Fasting period before study: n/a
- Housing:groups up to 3 animals in makrolon type 1 cages
- Diet (e.g. ad libitum): Altromin 1324 standard diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period:at least 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-22.5C
- Humidity (%): 48-50% mean
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle


IN-LIFE DATES: From:25 Sept 19900 To: O9 Oct 1990

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
peanutoil for TCPP
The positive control Cyclophosphamide was dissolved in deionized water.
Details on exposure:
Young adult male and female NMRI mice received a single intraperitoneal injection of 350 mg/kg test compound in peanut oil. This was based on the outcome of a preliminary toxicity study. In this pilot study groups of five animals including males and females were administered TCPP intraperitoneally at doses of 250, 300, 325, 350, 375 and 500 mg/kg. Apathy, staggering gait, lateral position, spasm, extension spasm, leaping spasm, twitching, shivering and difficulty in breathing were noted in animals up to 48 hours. 2/5 animals died in the 375 mg/kg dose group and 3/5 animals died in the 500 mg/kg group.
In the main study, the dose of 350 mg/kg was administered to four groups of 5 male and 5 female mice ¿ a replacement group and a 16, 24 and 48-hour sacrifice group.
Duration of treatment / exposure:
single injection
Frequency of treatment:
single injection
Post exposure period:
16, 24 or 48 hrs prior to sacrifice
Doses / concentrations
Remarks:
Doses / Concentrations:
0 or 350mg/kg
Basis:

No. of animals per sex per dose:
5/sex/dose/timepoint
Control animals:
yes
Positive control(s):
positive control: cyclophosphamide, 20 mg/kg bw sacrificed at 24hrs post dosing.

Examinations

Tissues and cell types examined:
Bone marrow erythroblasts.
Details of tissue and slide preparation:
Bone marrow smears were prepared by Schmid´s method
Evaluation criteria:
1000 polychromatic erythrocytes (PCEs) were counted.
A test was considered positive if there was a relevant and significant increase in the number of polychromaitc erythrocytes showing micronuclei in comparison to the negative control, at any interval.
Atest was considered negative if there was no relevent or significnat inrease in the rate of micronucleated polychromatic erythrocytes at any time point. A test was also considered negative if there was a significant increase which was within the range of historical negative controls.
An equivocla result was considerd to be a non-significant increase in the number of micronucleated polychromatic erythrocytes above the historical negative control range.
Statistics:
The treated group with the highest mean and the positive controls were checked by Wilcoxon's non-parametric rank sum test with respect of the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error proability was below 5% and the treatment group figure was higher than the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. The group with the highest mean was compared with the negative control using a one-sided chi-squared test. A variation was considered statistically significant if the error was below 5% and the treatment group figure was higher than that of the negative control.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Treated animals showed signs of toxicity for up to 16 hours after dosing including apathy, staggering gait, spasm, twitching shivering, difficulty breathing and salivation. After 16 hours, their appearance and activity appeared normal. Feeding behaviour throughout the study was normal. 2/40 animals died during the test period and this was related to the acute toxicity of TCPP.

Any other information on results incl. tables

The ratio of PCEs to NCEs was not significantly altered by TCPP treatment compared to the negative control. The incidence of micronucleated PCEs or NCEs was not significantly increased in the treatment groups compared to the negative control. Cyclophosphamide did not induce a significant alteration in the ratio of PCEs to NCEs but did significantly increase the incidence of micronucleated PCEs.

Summary of MN test results after acute i.p. treatment with 350 mg/kg bw

The treatments were as follows:

 Experimental Group  Dose in mg/kg Body Weight  Route of Application andTime of Sacrifice
 negative control  0  i.p.; 24 hrs.
 test substance  350  i.p.; 16 hrs
   test substance  350  i.p.; 24 hrs
   test substance  350  i.p.; 48 hrs
   test substance  350  replacement group
 positive control Cyclophosphamide  20  i.p.; 24 hrs

Summary of Results of Micronucleus test with Tris-chlorisopropylphosphate (TCPP) (after acute intraperitoneal treatment with 350 mg/kg body weight):

          micronucleated Cells per 1000
 experimental groups  number of evaluated polychromatic erytrhrocytes  Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes  normochromatic erythrocytes  polychromatic erythrocytes
 negative control  10,000  1265 + 253  1,3 + 1,2  1,4 + 0,8
 test substance16 hours   10,000  1217 + 486 1,6 + 0,8  2,1 + 1,2
 test substance 24 hours   10,000  1257 + 394  1,3 + 1,2  1,2 + 0,9
 test substance48 hours   10,000  1063 + 307  1,4 + 0,8  1,3 + 1,4
 positive controlCP20 mg/kg   10,000  942 + 182  1,1 + 1,6  17,6* + 5,3

*p< 0,01 in non -parametic Wilcoxon ranking test

Applicant's summary and conclusion

Executive summary:

The micronucleus test was employed to investigate TCPP in male and female mice for a possible clastogenic effect on the chromosomes of bone marrow erythroblasts. The known clastrogen and cytostatic agent, cyclophosphamide, served as positive control.

The treated animals received a single i.p. administration of either TCPP or cyclophosphamide. The femoral bone marrow of groups treated with TCPP was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrified after 24 hours. The doses of TCPP and the positive control cyclophosphamide were 350 and 20 mg/kg body weight, respectively.

Cyclophosphamide, the positive control had a clear clastogenic effect.

The animals treated with TCPP showed symptoms of toxicity after administration. Two of fourty animals died before the end of the test due to the acute intraperitoneal toxicity of 350 mg/kg TCPP.

There was no altered ratio between polychromatic and normochromatic erythrocytes.

No indications of a clastogenic effect of TCPP were found after a single i.p. treatment with 350 mg/kg body weight.