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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
Study period:
January to September 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, performed according to standard method.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: the USA EPA, TSCA and FIFRA guidelines and the Japanese Ministry of International Trade and Industry Guidelines for testing of new chemicals.
GLP compliance:
yes (incl. QA statement)
Remarks:
1998-07-21
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
EC Number:
259-709-0
EC Name:
Tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
Cas Number:
55566-30-8
Molecular formula:
C4H12O4P.1/2O4S
IUPAC Name:
tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
Test material form:
other: liquid stored at room temperature
Details on test material:
- Name of test material (as cited in study report): THPS
- Physical state: colourless liquid
- Stability under test conditions: no data (responsability of the sponsor). However, test solutions were freshly prepared.
- Storage condition of test material: stored at room temperature

Test animals

Species:
mouse
Strain:
other: albino Crl:CD-1(ICR)BR
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- sex: Range-finding study: males & females
Main study: males
- Age at study initiation: 5-8 weeks old,
- Weight at study initiation: 22-30 g
- Assigned to test groups randomly: yes
- Housing: caged by up to seven
- Diet and mains water were allowed ad libitum throughout the study
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25°C
- Humidity: 30 to 70%
- Air changes: ca. 15 per hr
- Photoperiod: 12 hr dark / 12 hr light

IN-LIFE DATES: From 12 January to 2 March 2000

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: no toxic effects, no interaction with the test substance and recommanded by the guidelines
- Concentration of test material in vehicle: 9.38, 18.75, 37.5 mg/mL (expressed as main ingredient)
- Total volume applied: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: freshly prepared in sterile water, no other information
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
single administration
Post exposure period:
clinical signs were evaluated 1 hour post dosing and once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
93.8, 187.5, 375 mg/kg bw as main ingredient
Basis:
nominal conc.
equivalent to 122.45, 244.78 and 489.56 mg/kg bw expressed as active substance.
No. of animals per sex per dose:
Vehicle control and test substance: 7 per group
Positive control: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide at 50 mg/kg bw
- Justification for choice of positive controls: known to produce micronuclei under the conditions of the test
- Route of administration: orally

Examinations

Tissues and cell types examined:
erythrocytes from femur bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range-finding toxicity study was carried out in male and female mice at 250, 375 and 500 mg/kg bw, expressed as main ingredient.
In this initial toxicity test, 375 mg/kg bw was established as the maximal tolerated dose. There was no marked difference in toxicity between male and female mice. It was therefore considered acceptable to test male mice only in the micronucleus test.

TREATMENT AND SAMPLING TIMES:
7 male mice/dose/sampling time were administered the test substance orally by gavage at 93.8, 187.5 and 375 mg/kg, expressed as main ingredient. The positive control cyclophosphamide was administered to 5 mice at 50 mg/kg bw.
Sampling time was 24 hours for all groups, plus an additional sampling  period at 48 hours in the high dose and vehicle control groups.

DETAILS OF SLIDE PREPARATION: immediately following sacrifice, both femurs were dissected from each animal, aspirated with fetal calf serum and bone marrow smears prepared following centrifugation and resuspension. The smears were air-dried, fixed in absolute methanol and stained.
Evaluation criteria:
Incidence of micronucleated cells and polychromatic/normochromatic erythrocytes ratio were evaluated per 2000 cells for each animal.
A positive mutagenic response was demonstrated when a statistically significant dose-response increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48 hour kill times.
Statistics:
The data were analysed using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY for toxicity:
- Dose range: 250, 375 and 500 mg/kg bw, expressed as main ingredient.
- Clinical signs of toxicity in test animals: hunched posture, lethargy, pilo-erection, increased lacrimation, hypothermia, decreased respiratory rate, laboured respiration, ataxia, ptosis, splayed gait, occasional body tremors and dehydration. 375 mg/kg bw was established as the maximal tolerated dose. 
There was no marked difference in toxicity between male and female mice.  It was therefore considered acceptable to test male mice only in the micronucleus test.
See table 1 below

RESULTS OF DEFINITIVE STUDY

- clinical signs: There were two premature deaths in the 48 hour, 375 mg/kg bw test group.
Clinical signs observed at 24 and 48 hours in animals dosed with 375 mg/kg bw included hunched posture, lethargy, pilo-erection, decreased respiratory rate, laboured respiration, ataxia, pallor of extremities, ptosis, splayed or tiptoe gait, dehydration.
It was concluded that the test substance was absorbed.

- Induction of micronuclei: There was a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes at the maximal tolerated dose of 375 mg/kg bw sampled at 24 hours compared to vehicle control group.
Testing at 48 hours showed no response.
Examination of a further 2000 PCEs at 24 hours showed a non-significant slight increase only over vehicle controls. The increase at 24 hours in the first evaluation was within historical control values.

- Ratio of PCE/NCE: There were no statistically significant decreases in PCE/NCE ratio in test groups compared to concurrent vehicle control groups. A slight decrease in PCE/NCE ratio was observed in animals treated at 375 mg/kg bw after 48 hours, compared to current control group. This was considered indicative of slight cytotoxicity, showing that exposure of the target tissue had been achieved.

Any other information on results incl. tables

Table 8.5. 1 Range-finding toxicity test

Dose level (mg/kg bw main ingredient)

Sex

Number of animals

Deaths on day

Total deaths

0

1

2

250

Male

2

0

0

0

0/4

Female

2

0

0

0

375

Male

2

0

0

0

0/4

Female

2

0

0

0

500

Male

2

0

2

-

4/4

Female

2

0

2

-

Table 8.5.2 micronucleus study - summary of group mean data

Treatment Group

Number of PCE with Micronuclei per 2000 PCE

PCE/NCE Ratio

Group Mean

SD

Group Mean

SD

Vehicle control (48-hour sampling time)

1.1

0.9

1.51

0.82

Vehicle control (24-hour sampling time)

0.9 (0.9)a

1.2 (1.9)a

1.54

0.57

Positive control (24-hour sampling time)

47.6***

19.8

2.07

1.71

THPS (375 mg/kg 48-hour sampling time)

1.6

1.8

1.02

0.54

THPS (375 mg/kg 24-hour sampling time)

2.9**(2.3)a

1.9 (1.5)a

1.64

0.84

THPS (187.5 mg/kg 24-hour sampling time)

2.0

3.1

1.38

0.62

THPS (93.8 mg/kg 24-hour sampling time)

1.0

1.4

1.50

0.52

a : supplementary data from extra 2000 PCEs

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

SD: standard deviation

***: p < 0.001

**: p < 0.01

Applicant's summary and conclusion

Conclusions:
Under the test conditions of this study, THPS was considered to be non-genotoxic.
Executive summary:

A study was performed to assess the potential of THPS to produce damage to chromosomes or aneuploidy when administered to mice. The method used was OECD 474 and in compliance with GLP.

A range-finding toxicity study was carried out in male and female mice at 250, 375 and 500 mg/kg bw, expressed as main ingredient.

7 male mice/dose/sampling time were administered the test substance orally by gavage at 93.8, 187.5 and 375 mg/kg bw, expressed as main ingredient. The positive control cyclophosphamide was administered to 5 mice at 50 mg/kg bw.

Sampling time was 24 hours for all groups, plus an additional sampling period at 48 hours in the high dose and vehicle control groups.

In the initial toxicity test, 375 mg/kg bw was established as the maximal tolerated dose. There was no marked difference in toxicity between male and female mice. It was therefore considered acceptable to test male mice only in the micronucleus test.

There were two premature deaths in the 48-hour 375 mg/kg bw test group. These deaths were not considered to have affected the integrity of the study, since 5 animals remained for evaluation as required under the OECD guideline.

Clinical signs observed at 24 and 48 hours in animals dosed with 375 mg/kg bw indicated that the test substance was systemically absorbed. A slight decrease in PCE/NCE ratio observed in animals treated at 375 mg/kg bw after 48 hours compared to the vehicle control group was considered indicative of slight cytotoxicity, showing that exposure of the target tissue had been achieved.

There was a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes at the maximal tolerated dose of

375 mg/kg bwsampled at 24 hours compared to vehicle control group. Testing at 48 hours showed no response. Examination of a further 2000 PCEs sampled at 24 hours showed a slight, non-significant, increase over vehicle controls.

The increase at 24 hours in the first evaluation was within historical control values and consequently is not considered of toxicological significance. THPS is therefore considered to be non-genotoxic in this assay.

 

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