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:
Experimental Procedures: Date Started: 13 July 1990; Date Completed: 30 August 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
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): KS-235 1 ,2-Diphenoxyethane
- Physical state: white powder
- Analytical purity: 100%
- Lot/batch No.: 900501
- Storage condition of test material: room temperature
- Other:
- Supplied by Kanzaki Paper Manufacturing Co. Ltd.
- Container: plastic screw-top bottle
- Date of arrival: 21 May 1990

Test animals

Species:
mouse
Strain:
other: Albino BKW strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Bantin & Kingman Ltd., Grimston, Aldborough, Hull, U.K
- Age at study initiation: approximately five to eight weeks old
- Weight at study initiation (at the start of the main study):
males = 22 - 30 g
females = 20 - 30 g
- Assigned to test groups randomly: yes - animals were selected at random and given a unique number within the study by ear punching and a number written on a colour coded cage card.
- Fasting period before study: 3 - 4 hour fast immediately before dosing and for approximately two hours after dosing
- Housing: animals were housed in groups of up to five by sex in solid-floor polypropylene cages with sawdust bedding.
- Diet and water (e.g. ad libitum): With the exception of a 3 - 4 hour fast immediately before dosing and for approximately two hours after dosing, free access to mains drinking water and food (Rat and Mouse Expanded Diet No. 1, Special Diet Services Limited, Witham, Essex, U.K.) was allowed throughout the study.
- Acclimation period: minimum acclimatisation period of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 50 - 58 (relative humidity)
- Air changes (per hr): approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): controlled by a time switch to give 12 hours light and 12 hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil B.P
- Supplied by: Analytical Supplies Limited
- Description: clear, straw-coloured oily liquid
- Container: plastic screw-top bottle
- Supplier’s identification: Oil Arachis B.P.
- Safepharm serial number: Co/319
- Storage conditions: room temperature
Details on exposure:
Route = oral
PREPARATION OF DOSING SOLUTIONS:

For the purpose of this study the test material was freshly prepared as required as a solution/suspension at the appropriate concentration in arachis oil B.P. The identification and stability of the test material and the preparations were not determined.

For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water. The identification and stability of the control material and the preparation were not determined.

The identification and stability of the vehicle control were not determined.
Duration of treatment / exposure:
Once
Frequency of treatment:
Single dose
Post exposure period:
- Range-finding Toxicity Study: Animals were observed 1 hour after dosing and subsequently once daily for 3 days.
- Micronucleus Study: All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable.
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
other: nominal in arachis oil
No. of animals per sex per dose:
5 per time of termination
Control animals:
yes, concurrent vehicle
Positive control(s):
- Supplied by: Sigma Chemical Company
- Description: white powder
- Container: brown glass screw-top bottle
- Supplier’s identification: Cyclophosphamide Monohydrate
- Safepharm serial number: Co/311
- Date of arrival: 23 March 1990
- Storage conditions: +4°C in the dark

Examinations

Details of tissue and slide preparation:
Slide Preparation: Immediately following sacrifice (i.e. 24, 48 or 72 hours following dosing), one femur was dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, and stained in May-Grünwald/Giemsa.
Evaluation criteria:
Evaluation of Slides: Stained bone marrow smears were examined at random using light microscopy at x 1000 magnification. The incidence of micronucleated cells per 1000 polychromatic erythrocytes PCE (blue stained immature cells) per animal was scored. Micronuclei are circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes NCE (pink stained mature cells) associated with 1000 polychromatic erythrocytes were counted; these cells were also scored for incidence of micronuclei.

The ratio of normochromatic to polychromatic erythrocytes was calculated together with appropriate group mean values for males and females separately and combined.

Interpretation of Results: A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the three test material groups and the number occurring in the corresponding vehicle control groups.

A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24, 48 or 72-hour kill times.

If the above criteria are not demonstrated, then the test material is considered to be non-mutagenic under the conditions of the test.
A positive response for bone marrow toxicity is demonstrated when the treatment group mean polychromatic to normochromatic ratio is half the vehicle control value or when a treatment related decrease is shown to be statistically significant.
Statistics:
If necessary, and where possible, all data were statistically analysed using the Kruskal-Wallis one-way analysis of variance by ranks (Kruskal W.H. and Wallis W.A. 1952 J. Am. Statist. Soc. 47 583).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

RANGE-FINDING TOXICITY STUDY:

- The mortality data are summarised in the Table on page 13 of the attached report.

- Adverse clinical signs were observed in three of the animals treated with KS-235 these included: hunched posture, lethargy, pilo-erection and ptosis. There was one premature death in the 5000 mg/kg treatment group. 5000 mg/kg was selected as the maximum recommended dose for use in the micronucleus study.

 

MICRONUCLEUS STUDY:         

- Mortality Data and Clinical Observations: No adverse clinical signs were observed in any of the animals treated with KS-235 at 5000 mg/kg and there were no premature deaths.

- Evaluation of Bone Marrow Slides:

- A summary of the results of the micronucleus study is given in Table 1 of attached report. Individual and group mean data are presented in Tables 2 to 8 of attached report.

- There were no significant increases in the frequency of micro- nucleated PCE’s in any of the KS-235 treatment groups when compared to their concurrent control groups.

- There were no significant increases in the frequency of micro- nucleated NCE’s in any of the KS-235 treatment groups when compared to their concurrent control groups.

- There was no significant change in the PCE/NCE ratio in any of the three KS-235 treatment groups when compared to their concurrent vehicle control groups.

- The test material, KS-235, was found not to produce micronuclei in polychromatic erythrocytes of mice under the conditions of the test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
KS-235 was considered to be non-mutagenic under the conditions of the test.
Executive summary:

1. A study was performed to assess the potential of KS-235 to produce damage to chromosomes or the mitotic apparatus of mice when administered by the oral route. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 474 “Genetic Toxicology: Micronucleus Test” and Method B12 of Commission Directive 84/449/EEC and the requirements of the Japanese MITI/MHW Chemical Substance Law.

2. Following a preliminary range-finding study to confirm the oral toxicity of the test material, the micronucleus study was conducted using KS-235 at the maximum recommended dose level (5000 mg/kg).

3. In the micronucleus study, groups of ten mice (five males and five females) were given a single oral dose of KS-235 at 5000 mg/kg. Animals were killed 24, 48 or 72 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

4. Further groups of mice were treated with arachis oil B.P. or cyclophosphamide, to serve as vehicle and positive controls respectively.

5. There was no evidence of an increase in the incidence of micronucleated polychromatic or normochromatic erythrocytes in animals treated with KS-235 when compared to the vehicle control groups.

6. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

7. The test material, KS-235, was considered to be non-mutagenic under the conditions of the test.