Registration Dossier

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:
2001-05-23 - 2001-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentapotassium bis(peroxymonosulphate) bis(sulphate)
EC Number:
274-778-7
EC Name:
Pentapotassium bis(peroxymonosulphate) bis(sulphate)
Cas Number:
70693-62-8
Molecular formula:
H3K5O18S4
IUPAC Name:
pentapotassium bis(peroxymonosulphate) bis(sulphate)
Details on test material:
- Name of test material (as cited in study report): Oxone® Monopersulfate Compound
- Description: White powder
- Analytical purity: 95.7 % (based on KMPS triple salt)
- Lot/batch No.: H 24607
- Stability under test conditions: Not stated
- Maximum tolerable dose: 1750 mg/kg bw (male mice);
>= 2000 mg/kg bw (female mice)

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, UK
- Age at study initiation: not stated
- Weight at study initiation: 28 – 30 g (males) 22 – 24 g (females)



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Purified water
Details on exposure:
Stability/homogeneity:
Concentration and stability of the dosing solutions was not assessed during the study. This is not considered to be required for this kind of tests for the following reason:
According to the description in the report on the in vivo MNT in mice, the dosing solutions were freshly prepared and immediately administered to the animals (“Solutions of the test substance were freshly prepared on the day of the test and were diluted to the concentrations…in purified water”. This dosing regime is usual practice and it is very unlikely that the test substance degraded in the short time period between preparation of dosing solutions and administration to the animals. Therefore, the lack of the determination of the stability and homogeneity of the test material is not considered to have compromised the study. In addition, KMPS triple salt is a readily water-soluble substance (25 – 30 % (w/v)) and, thus, homogeneity of the dose preparations was warranted.


Duration of treatment / exposure:
Animals were killed and examined after 24 and 48 hours, respectively
Frequency of treatment:
One application only
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Preliminary toxicity test: 75, 87.5, 100 mg/mL; Main micronucleus test: - males: 21.88, 43.75, 87.5 mg/mL - females:25, 50, 100 mg/mL, - Mitomycin C: 0.6 mg/mL
Basis:
nominal in water
Remarks:
Doses / Concentrations:
20 mL/kg bw
Basis:
other: total volume applied
Remarks:
Doses / Concentrations:
Preliminary toxicity test: 1500, 1750, 2000 mg/kg bw, Main micronucleus test: - males: 0, 437.5, 875, 1750 mg/kg bw - females:0, 500, 1000, 2000 mg/kg bw, Mitomycin C: 12 mg/kg bw
Basis:
other: dose applied
No. of animals per sex per dose:
Preliminary toxicity test: 2 per sex and group

Micronucleus test:
vehicle control: 10 per sex
KMPS triple salt (437.5, 875,500 and 1000 mg/kg bw): 5 per sex and group
KMPS triple salt (1750 and 2000 mg/kg bw): 12 per sex and group (2 additional animals to replace any that died)
Positive control: 5 per sex
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C: 12 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: Bone marrow

Type of cells: Erythrocytes

Further details on Examinations are given under "Any other information on materials and methods incl. tables".
Details of tissue and slide preparation:
not indicated
Evaluation criteria:
not indicated
Statistics:
Statistical analysis was performed. For incidences of micronucleated immature (polychromatic) erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, USA). Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected; for individual inter-group comparisons (i.e. the positive control group) this procedure simplifies to a straightforward permutation test. For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, i.e. exact versions of Wilcoxon’s sum of ranks test and Jonckheere’s test for trend.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
only in females
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CLINICAL SIGNS, MORTALITY

Preliminary toxicity test:
1500 mg/kg bw – males: fast respiration, hunched posture, underactivity, abnormal gait and piloerection
1500 mg/kg bw – females: fast respiration, underactivity and piloerection
2000 mg/kg bw – males: deep, fast slow or irregular respiration, unresponsive, pallor, hunched posture, underactivity, abnormal gait, partially closed eyelids or closed eyes, piloerection, prostrate, fasiculations and reduced body temperature.
One animal was dead app. 20 hours after dosing, the remaining animal was killed in extremis app. 44hours after dosing.
2000 mg/kg bw – females: fast respiration, hunched posture, underactivity, abnormal gait and piloerection
1750 mg/kg bw – males: irregular respiration, hunched posture, underactivity, abnormal gait, partially closed eyes and piloerection.
1750 mg/kg bw – females: fast and irregular respiration, hunched posture, underactivity, abnormal gait and piloerection

Main micronucleus test:
1750 mg/kg bw males: fast and irregular respiration, flat and hunched posture, underactivity, abnormal gait, partially closed eyes, piloerection/ungroomed and incidence of weight loss was recorded for eight of ten animals.
2000 mg/kg bw – females: fast respiration, flat and hunched posture, underactivity, abnormal gait, partially closed eyes and piloerection/ungroomed
No mortalities were observed at any dose level given.
No clinical signs were observed for the vehicle and positive control groups.


HAEMATOLOGY / TISSUE EXAMINATION

Not stated


GENOTOXICITY

The test substance did not cause any statistically significant increases in the number of micronucleated immature (polychromatic) erythrocytes at either sampling time in both male and female animals (P>0.01) when compared to vehicle control values.
It also did not cause any substantial increases in the incidence of micronucleated mature (normochromatic) erythrocytes at either sampling time in either sex when comparison to vehicle control values was made.
The test substance failed to cause any significant decreases in the proportion of immature erythrocytes at either sampling time in male animals (P>0.01).
There was a dose-related decrease in the proportion of immature erythrocytes at the 24 hour sampling time in female animals. The Jonckheere’s test for trend was significant at the 1 % level when all test substance treated groups were included in the analysis, compared to the vehicle control group values (P<0.001). When the high treatment group (2000 mg/kg bw) was excluded from the analysis, there was some evidence of a significant trend but this was not formally statistically significant (P>0.01).
At the 48 hour sampling interval, the difference in the proportion of immature erythrocytes between the 2000 mg/kg bw level and the control group of female animals was not significant.

Any other information on results incl. tables

Table 1:          KMPS triple salt – Mammalian Erythrocyte Mouse Micronucleus Test: Summary of results and statistical analysis

MALES

Sampling time

Treatment

Dose
(mg/kg bw)

%ie/(ie+me)+
(mean)

Incidence mie
(mean)

Incidence mme
(group mean)

 

24 hours

Purified water

-

43

0.2

0.7

 

-

KMPS triple salt

437.5

43

0.4

0.0

 

-

KMPS triple salt

875

42

0.4

1.3

 

-

KMPS triple salt

1750

38

0.2

0.0

 

-

Mitomycin C

12

44

31.2**

0.7

 

48 hours

Purified water

-

54

0.6

0.0

 

-

KMPS triple salt

1750

42

0.0

0.0

 

FEMALES

 

Sampling time

Treatment

Dose
(mg/kg bw)

%ie/(ie+me)+
(mean)

Incidence mie
(mean)

Incidence mme
(group mean)

 

24 hours

Purified water

-

49

0.0

0.8

 

-

KMPS triple salt

500

49

0.6

0.8

 

-

KMPS triple salt

1000

37

0.0

0.0

 

-

KMPS triple salt

2000

36***

0.4

0.6

 

-

Mitomycin C

12

45

23.8**

0.0

 

48 hours

Purified water

-

51

0.4

0.0

 

-

KMPS triple salt

2000

37

0.2

0.6

 

%ie/(ie+me)+          Proportion of immature (polychromatic) erythrocytes
mie
                     Number of micronucleated cells observed per 2000 immature erythrocytes examined
mme
                   Number of micronucleated cells calculated per 2000 mature (normochromatic) erythrocytes

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

***          P<0.001 (highly significant)
**
           P<0.01 (significant)

otherwise P>0.01 (not significant)

+ Occasional apparent errors of ± 1 % may occur due to rounding of values for presentation in the table

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability). KMPS triple salt did not show any evidence of causing chromosome damage in either male or female mice, but according to the interpretation in the study report, showed some evidence of bone marrow cell toxicity in female mice only, when administered orally by intragastric gavage in this in vivo test. The bioavailability was ensured by the clinical signs of toxicity observed in both sexes of mice.

The applicant further concludes that the depression in the PCE/NCE ratio noted in the in vivo MNT with KMPS triple salt at the high dose level of female mice at the 24 hour time point but not at the 48 hours sampling time point is due to the acute toxicity of KMPS triple salt. In view of the degradation of KMPS triple salt to hydrogen peroxide an influence of hydrogen peroxide on this particular effect cannot be excluded which has been demonstrated to depress the PCE/NCE ratio in an in vivo MNT in mice also but, most importantly, not to increase the incidence of micronucleated polychromatic erythrocytes.
Executive summary:

Materials and methods

Groups of CD 1 mice were orally administered a single dose of KMPS triple salt at concentrations of 1500, 1750, and 2000 mg/kg bw in a preliminary toxicity test. In the micronucleus test doses of 0, 437.5, 875,1750 mg/kg bw (males) and 0, 500, 1000 and 2000 mg/kg bw were administered. The animals were killed by cervical dislocation 24 and 48 hours, respectively, after the administration. Femoral marrow cells were flushed out and pooled in a total volume of 2 mL of pre-filtered foetal calf serum. The cells were sedimented by centrifugation and resuspended in a small volume of fresh serum. Three smears were made from each animal. Cells were fixed in methanol and stained with 10 % Giemsa. Following rinsing in purified water and differentiation in buffered purified water, the smears were rinsed in purified water and air-dried. The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. The proportion of immature erythrocytes for each animal was assessed of at least 1000 erythrocytes.

Results and discussion

Please refer to Table 1; which is presented under "Remarks on results including tables and figures".

No statistically significant increases in the frequency of micronucleated immature erythrocytes were observed in mice treated with KMPS triple salt and killed 24 or 48 hours later, compared to vehicle control values (P>0.01 in each case).

No statistically significant decrease in the proportion of immature erythrocytes was recorded at either sampling time in male mice. In female mice, a significant decrease was observed at the high treatment level (2000 mg/kg bw) at the 24 hour sampling time only while no such an effect was evident at the 48 hour sampling time point.

The statistically significant reduction in the PCE/NCE ratio observable at the high dose level of female mice at the 24 hour sampling time point is considered to be a direct consequence of the acute toxicity of KMPS triple salt rather than related to systemic toxicity. The oral doses given to male and female mice were in the range of the LD50 observed in rats (please refer to Document IIIA, Section 6, A6.1.1/01) and although no mortalities occurred in mice there were clinical signs of toxicity evident at the top dose levels. These signs were noted during the first 24 hours of treatment which is consistent with the observations made in the acute oral study in rats. This supports the conclusion above that the decreased PCE/NCE ratio at the high dose level of female mice at the 24 hours sampling time point is related to the acute toxicity of KMPS triple salt.

Furthermore, in view of the chemical nature of KMPS triple salt and considering the mode of action, it will rapidly degrade to hydrogen peroxide which has also been demonstrated to depress the PCE/NCE ratio in an  in vivo MNT in mice but, most importantly, no increases in the incidence of micronucleated polychromatic erythrocytes were noted. In an overview of the genotoxicity of hydrogen peroxide which has been compiled in the available EU Risk Assessment Report, hydrogen peroxide was demonstrated to be clearly negative in the  in vivo tests performed amongst of which was an  in vivo UDS. The overall conclusion was that the available studies are not in support of significant genotoxicity/mutagenicity of H2O2 under  in vivo conditions. According to the principles followed in the EU, hydrogen peroxide is not classified as a mutagen.