4,5,6,7-tetrahydrothieno[3,2-c]pyridinium chloride

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:

from 13 july 2010 to 27 july 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A well documented GLP study according to OECD guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride

IUPAC Name:

4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride

Constituent 2

Reference substance name:

4,5,6,7-tetrahydrothieno[3,2-c]pyridinium chloride

EC Number:

249-220-0

EC Name:

4,5,6,7-tetrahydrothieno[3,2-c]pyridinium chloride

Cas Number:

28783-41-7

Molecular formula:

C7H9NS.ClH

IUPAC Name:

4,5,6,7-tetrahydrothieno[3,2-c]pyridin-5-ium chloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Test article identification: PCR0665
Test article batch number: CS02047
Purity (gas chromatography): 99.6%
Storage conditions: At room temperature
Stability/homogeneity: PCR0665 in water was homogeneous and stable at concentrations of 1 to 100 mg/mL for up to 15 days at ambient room temperature or in a freezer.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at initiation of dosing:
on arrival 5 to 6 weeks
on dosing (Day 1) 6 to 7 weeks
- Weight at initiation of dosing (main group animals at Day 1 including controls and replacement animals):
males from 209 to 240 grams
females from 163 to 195 grams
- Assignement to test groups: Animals were selected based on pretreatment evaluations and assigned to dosage groups based on weight ordered randomization using the Provantis software in Client / Server – Environments.
- Housing: During acclimatization, pretest and dosing periods, the animals were housed at the most 5 per cage in polysulfone solid bottom floor cages (type IV) on autoclaved NestPak TM Aspen 2HK, containing wood shaving with a small piece of wood, with a grid cover through which food and a water bottle were passed.
- Diet: Ssniff R/M-H (V1534) pellets ad libitum in feed racks
- Water: Tap water given ad libitum by polypropylene bottles with stainless-steel sipper tubes. Periodic analyses of the water are performed and the results of these routine analyzes are available on request (data retained in the archives). The bottles were changed at least once a week.
- Acclimation period: For at least 5 days

ENVIRONMENTAL CONDITIONS
- Room temperature: 20° to 24°C (short lasting deviations are acceptable)
- Relative humidity: 40% to 70% (short lasting deviations are acceptable, eg, during cleaning processes)
- Air flow: 15 to 20 changes/hour without recirculation in an air conditioned room
- Light cycle (time): 12-hour light/12-hour dark cycle (6:00 am to 6:00 pm)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: deionized water

Duration of treatment / exposure:

2 days

Frequency of treatment:

Once daily

Post exposure period:

24 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
Dose levels: 156.25, 312.5, and 625 mg/kg corresponding respectively to the following nominal concentrations: 15.625, 31.25, and 62.5 mg/mL. Dosing volume: 10 mL/kg
Basis:
nominal conc.

No. of animals per sex per dose:

Total number of animals on study: 116 (males 58/females 58).
Group 1 (negative control): 8 animals per sex
Group 2 (dose 156.25 mg/kg/d): 14 animals per sex
Group 3 (dose 312.5 mg/kg/d): 14 animals per sex
Group 4 (dose 625 mg/kg/d): 17 animals per sex (including 3 replacement animals per sex)
Group 5 (positive control, dose 20 mg/kg/d): 5 animals per sex

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control identification: Cyclophosphamide (CP)
- Preparation of formulations: The reference compound was prepared freshly in an aqueous solution under the responsibility of the study technician.
- Dosing duration: 1 day
- Dosing route: oral
- Dosing method: gavage (esophageal intubation)
- Dose level: 20 mg/kg
- Concentration: 2 mg/mL
- Dosing volume: 10 mL/kg
- Frequency of administration: once (24 hours before sampling of bone marrow)

Examinations

Details of tissue and slide preparation:

Slides for microscopic examination were prepared and scored manually.
Slides preparation:
The femurs were removed from the main group animals by sectioning the ligaments and rotating the joint. The muscles were carefully detached from the femurs and the epiphyses were sectioned. Five mL of fetal calf serum (FCS) were injected into the bone marrow cavity of one femur in order to collect the bone marrow suspension for determination of genotoxicity and cytotoxicity. For each animal, the tube containing the bone marrow suspension was centrifuged for 5 minutes at 250 g. After centrifugation, the supernatant was removed and the pellet was resuspended in an appropriate volume of fresh fetal calf serum, depending on cell density. One drop of the suspension was smeared onto a clean slide, identified by project code and animal number and air-dried for approximately 12 hours.
Subsequently, the slides were stained as follows with a May-Grünwald/Giemsa staining procedure:
Time (min) Steps
10 Methanol
5 May-Grünwald: phosphate buffer (pH 6.8) 1:1
5 May-Grünwald solution
20 Giemsa: phosphate buffer (pH 6.8) 1:6
5 Phosphate buffer (pH 6.8)
10 Deionized water
then Drying and coating with Entellan®
The other femur was used to prepare bone marrow smears for the analysis of the total bone marrow cell population. Two smears per animal were prepared, air-dried, fixed for 30 seconds with methanol. These bone marrow smears were not processed further as evaluation of the total bone marrow cell populations was not considered to be necessary based on hematology results.

Scoring system:
All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator. Coding of the slides was performed using MNU software.
Cytotoxicity scoring:
Giemsa staining differentiates PCE from mature NCE. One slide per animal was analyzed to evaluate bone marrow cytotoxicity. A total number of 1000 erythrocytes were counted to determine the ratio PCE / (PCE + NCE). Micronucleus scoring in polychromatic erythrocytes:
The following criteria were used for scoring micronuclei in polychromatic erythrocytes:
- round or slightly almond-shaped;
- generally single;
- color similar to that of nuclear chromatin;
- located in the same plane as the erythrocyte.
2000 PCE were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei.

Evaluation criteria:

ASSAY ACCEPTANCE CRITERIA
The assay is considered valid only when:
- the incidence of spontaneous MPCE in individual animals in the negative control group remains consistent with the range of our individual historical negative control data (99 percentile)
- cyclophosphamide induces a statistically significant increase in the incidence of MPCE;
- the mean percentage of PCE at the highest dose level group is not less than 20% of the mean percentage at the negative control group (ie, 10% for 50% in the control)
TEST EVALUATION CRITERIA
A test article is considered to induce a positive response in the bone marrow micronucleus test if all of the following criteria are fulfilled:
- the test article induces a statistically significant increase in the incidence of the MPCE in the treated group(s) as compared to the negative control group;
- the increase is dose-related or observed at the highest dose level;
- the incidence of MPCE observed in individual animals treated with the test article clearly exceeds the upper value of the range of individual historical negative control data (99 percentile);
- in addition, the biological significance is taken into consideration for positivity evaluation.
When the above criteria are not fulfilled, the test article is considered negative.

Statistics:

STATISTICAL METHODS:

CYTOTOXICITY ANALYSIS: For the PCE / (PCE + NCE) ratio, the 95% Dunnett adjusted confidence intervals (CI) of the ratio of each treated group to the negative control group were calculated for each sex using the variance estimation of a one-way ANOVA (dose) on the log of the values, as described below.
In order to express the results as a ratio and to stabilize variances, a log transformation was performed. Then, an analysis of variance was calculated. Therefore, 95% confidence intervals of differences between treated groups and the negative control group were calculated and values back transformed in order to express the results as confidence intervals of the ratios treated to control.
As performing multiple comparisons increases the number of false positive results, a Dunnett’s
adjustment for multiplicity was applied on both p values and confidence intervals.

GENOTOXICITY ANALYSIS: Micronuclei are either present or absent in a PCE. Their relative rarity in control animals suggests that their incidence follows a Poisson distribution.
The MPCE are therefore analyzed using a Poisson model with a log link function using GENMOD procedure in SAS. The effect of a test item is included as a factor with one level corresponding to each concentration in the model.
The effect of the test article is evaluated using two statistical approaches:
• a linear trend test, build as a linear combination of the logs of the proportions of micronuclei, for dose-response relationship investigation.
• pairwise comparisons to the control, using the one-sided Likelihood Ratio test, to detect the doses significantly different from the control. As performing multiple comparisons increases the number of false positive results, a Bonferroni-Holm adjustment for multiplicity is applied.
All the tests are one-sided, a decreasing trend being not of interest. So in case of decrease and calculated p value <0.5, the final p value is estimated as 1-[p value].

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

See "Any other information on results incl. tables".

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Mortality and Clinical signs: The clinical signs observed in male and female rats after administration of PCR0665 included decreased activity, discharge from nose and salivation, (partly) closed eyelids, hunched or recumbent posture, trembling, convulsions, incoordinated gait, and secretion of the Harderian fluid. Findings at 625 mg/kg/d were severe and associated with mortality in 5/8 females and 2/8 male rats of the main group and 3/9 females in the toxicokinetics group. Also, one male rat out of five died at 312.5 mg/kg/d. The definite cause of death remained undetermined. All surviving animals did not show clinical signs at Day 3 before the necropsy.
- Body weight: Mean body weight was significantly decreased from Day 1 to Day 3 in all male and female groups treated with PCR0665.
- Hematology: Red blood cell mass values (RBC, hematocrit, and hemoglobin) were increased dose-related by 11% to 18% in males at all dose levels. Neutrophils were decreased by about 40% and lymphocytes were increased by about 40% at all dose levels in males and females without a consistent dose relationship. The findings in hematology were considered to be of minor toxicological relevance and were not considered to impair the bone marrow evaluation.
- Bone marrow examination: Male animals treated with 625, 312.5, and 156.25 mg/kg/d were evaluated for the induction of micronucleated polychromatic erythrocytes, although mortality was observed in the two highest dose groups and the maximum tolerated dose was slightly exceeded in these respective dose groups. However, the validity of the data was not impaired, as no relevant change in the ratio of polychromatic to all erythrocytes was observed. Based on the high incidence of mortality in the main group (5/8 females), and the resulting insufficient number of remaining animals, no evaluation of the female animals treated with 625 mg/kg/d was conducted. Therefore, only two dose levels (312.5, and 156.25 mg/kg/d) were evaluated for females.
A statistically significant decrease of the ratio of polychromatic erythrocytes to all erythrocytes in all treatment groups analyzed was regarded as being not biologically relevant, as the observed values were within the range of the historical negative control data.
For the evaluated incidence of micronucleated polychromatic erythrocytes, none of the groups treated with PCR0665 was significantly different from the negative control group. There was therefore no evidence for induction of clastogenicity or aneugenicity after treatment with PCR0665.

Any other information on results incl. tables

The ratio of polychromatic erythrocytes PCE/(PCE + NCE) was reduced with statistical significance in all analyzed groups treated with PCR0665. As the changes were not dose-related and within the range of the historical negative control data they were considered to be biologically irrelevant and did not have an impact on the validity of the study.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the experimental conditions of the study, PCR0665 administered by the oral route once daily for two consecutive days was found negative in the rat bone marrow micronucleus test up to a dose level of 625 mg/kg/d in males and 312.5 mg/kg/d in females.