Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Adequacy of study:
weight of evidence
Study period:
28AUG2015 - 21OCT2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD/EC guidelines and GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(adopted 26 September 2014)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(31 May 2008)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 5-(Sodiosulfo)isophthalic Acid
- Substance type: Organic
- Physical state: White powder
- Storage condition of test material: At room temperature

Test animals

Species:
mouse
Strain:
other: NMRI BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 31-36g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: Group housed (maximum 5 animals per sex per cage) in Macrolon cages
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: tapwater, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8 – 22.7
- Humidity (%): 41 - 80
- Air changes (per hr): Approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intravenous
Vehicle:
- Solvent used: physiological saline (0.9% NaCl)
- Rationale: In the solubility test physiological saline (Eurovet Animal Health, Bladel, the Netherlands) appeared to be a suitable solvent
- Method: In the main assay, 5-(Sodiosulfo)isophthalic Acid was dissolved in physiological saline by vortexing and sonication (maximum temperature 30.0 °C).
5-(Sodiosulfo)isophthalic Acid concentrations were dosed within 4 hours after preparation.
Details on exposure:
- The dosing volume was 10 mL/kg body weight.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
Two treatments were performed, administered with a 24-hour interval.
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
62.5, 125, 250 mg/ kg bw
Basis:
other: injected
No. of animals per sex per dose:
5 (males only)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- Route of administration: one intravenous injection at t=0 (control animals sacrificed at t=48 hours)
- Concentration: 40 mg/ kg bw

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Doses were selected based on the results of the dose range finding test. As no differences were seen between males and females, the main test was performed with one sex.

DETAILS OF SLIDE PREPARATION:
Cell suspensions of bone marrow cells were placed on clean slides previously immersed in a 1:1 mixture of 96% (v/v) ethanol. The drop was spread and preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Slides were stained using the "Wright-stain-procedure" (based on Giemsa) and fixed in a 1:10 mixture of xylene.

METHOD OF ANALYSIS: Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

OTHER: To prevent bias, all slides were randomly coded before examination.
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data will be analysed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05). ToxRat Professional version 3.1 will be used for statistical analysis of the data.
If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical effect on the study, the test will be rejected and repeated.
Statistics:
Equivocal results will be clarified by further testing using modification of experimental conditions.
A test item is considered positive in the micronucleus test if:
a) At least one of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) All results are within the 95% control limits of the negative historical control data range. In case the micronucleus data is not normally distributed the data will be transformed by using the formula y = 1/y. Thereafter the Dunnett’s test will be performed. In case the Dunnett’s test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
toxic effect on erythropoiesis at 250 mg/kg bw.
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: The male and female animal dosed with 2000 mg/kg 5-(Sodiosulfo)isophthalic Acid died directly after dosing. Subsequently one male animal was dosed with 500 mg/kg bw 5-(Sodiosulfo)isophthalic Acid. This animal died also directly. Based on these results it was concluded that both 2000 and 500 mg/kg would be too high as maximum dose in the main study. Therefore a lower dose was selected. One male and one female animal were dosed with 50 mg/kg 5-(Sodiosulfo)isophthalic Acid for two days (24h interval). These two animals showed no treatment related clinical signs of toxicity. It was concluded that 50 mg/kg would be too low as maximum dose in the main study. Therefore the animals were treated with a higher dose of 250 mg/kg 5-(Sodiosulfo)isophthalic Acid for two days (24h interval). The three male and female animals showed treatment related clinical signs of toxicity directly after dosing (slow breathing and effect on heartbeat). Based on the results of the dose range finding study a dose level of 250 mg/kg body weight was selected as the highest dose level tested in the main study. Since there were no substantial differences in toxicity between sexes only males were used in the main study.

RESULTS OF DEFINITIVE STUDY
- No mortality occurred.
- No treatment related clinical signs of toxicity were observed, with exception of three animals in the 250 mg/kg bw group which had problems with breathing
and showed low muscle tension for approximately 10 seconds after the second dose.
- Induction of micronuclei (for Micronucleus assay): No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of 5-(Sodiosulfo)isophthalic Acid treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database. Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups, which were treated with 62.5 and 125 mg/kg bw 5-(Sodiosulfo)isophthalic Acid showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test item on the erythropoiesis at these concentrations. At 250 mg/kg bw 5-(Sodiosulfo)isophthalic Acid a slight decrease in the ratio of polychromatic to normochromatic erythrocytes was observed indicating a toxic effect on erythropoiesis at 250 mg/kg bw. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Any other information on results incl. tables

Chemical analysis of dose preparations:

The concentrations of the test substance high, mid and low dose groups were in agreement with target concentrations (i.e. mean accuracies of respectively 90, 91 and 93%). No test substance was detected in the vehicle formulation. The formulations of the high and low dose group were homogeneous (i.e. coefficient of variation of ≤2.0%). Analysis of the high and low dose group after storage at room temperature under normal laboratory light for 4 hours yielded a relative difference of -0.11 and -0.32%, respectively. Based on this it is concluded that formulations were stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

Historical control data (negative and positive control data for micronucleus studies (males)):

 

Negative

Positive

Mean number of micronucleated

polychromatic erythrocytes

1.41

28.6

SD

1.39

11.9

n

150

149

Upper control limit

(95% control limits)

4.16

55.85

Lower control limit

(95% control limits)

-1.34

1.33

 

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between January 2012 and June 2015.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Based on the outcome of a bone marrow micronucleus assay performed according to OECD/ EC guidelines and GLP principles, it is concluded that 5-(Sodiosulfo)isophthalic Acid is not clastogenic or aneugenic in vivo.
Executive summary:

A bone marrow micronucleus assay was performed according to OECD/ EC guidelines and GLP principles. 5-(Sodiosulfo)isophthalic Acid was injected at 250, 125 and 62.5 mg/ kg bw at t=0 and t=42 hours in male mice. Bone marrow was sampled 48 hours after the first dosing. No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with 5-(Sodiosulfo)isophthalic Acid compared to the vehicle treated animals. At 250 mg/kg bw 5-(Sodiosulfo)isophthalic Acid, a slight decrease in the ratio of polychromatic to normochromatic erythrocytes was observed indicating a toxic effect on erythropoiesis at 250 mg/kg. Positive and negative controls were valid, formulation analysis showed accurate exposure. Based on these results, it is concluded that 5-(Sodiosulfo)isophthalic Acid is not clastogenic or aneugenic in vivo.