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:
November-December 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 474 protocol study under GLP without significant deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
equivalent or similar to
Guideline:
other: Guidelines for Testing Drugs for Toxicity, Pharmaceutical Affairs Bureau, Notice No. 118 (1984), Japanese Ministry of Health and Welfare
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Information on the composition of the batch used was obtained from another report in which the same batch had been used (see
Durward, 1994, report on Ames test).
Contains more impurities than submission substance.

Sponsor's identification : Nervanaid GBS 5 powder
Batch number : GLS 30P
Date received : 23 June 1994
Description : white powder
Storage conditions : room temperature over silica gel

Composition according to Certificate of Analysis (taken from Durward, 1994, report on Ames test)
Glutamic acid - N,N -diacetic acid, tetrasodium salt 70.70%

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd. Margate, Kent, UK
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 22-29 g (males), 20-25 g (females)
- Assigned to test groups randomly: yes, no further info given
- Fasting period before study: not indicated
- Housing: groups of 5 by sex in solid-floor polypropylene cages with woodflake bedding
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 50-55
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 November 1994 To: 3 December 1994

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: sterile distilled water (Steripak Ltd.)
- Concentration of test material in vehicle: 10, 20 and 40 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Type and concentration of dispersant aid (if powder): not used
- Lot/batch no. (if required): 401019
Details on exposure:
Exposure = intraperitoneal

Freshly prepared solutions (dilstilled water) were used.
The volume administered was calculated according to its bw at the time of dosing.
Duration of treatment / exposure:
Single injection
Frequency of treatment:
Single injection
Post exposure period:
24, 48 or 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
mg/kg bw
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (supplied by Sigma Chemical Company)
- Justification for choice of positive control(s): indicated in OECD TG 474
- Route of administration: orally
- Doses / concentrations: 50 mg/kg bw (solution of 5 mg/mL in distilled water given at a dose volume of 10 mL/kg)
- Lot number: 43H0269

Examinations

Tissues and cell types examined:
femural bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on a range-finding toxicity study (one male/one female per group)
- oral levels: 2500 and 5000 mg/kg. No animals died.
- intraperitoneal levels: 50, 400, 800, 1200, 2500, 5000 mg/kg. Animals died within 5 min of being dosed at levels of 800 mg/kg and
above. At the level of 400 mg/kg, hunched posture and irritation at the injection site were noted. Therefore, the dose of 400 mg/kg
bw was considered as the MTD, and 200 and 100 mg/kg bw were chosen as the lower dose levels.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Control and high dose animals were killed 24, 48 or 72 hours after dosing, low and mid dose animals and positive control animals 24 hours after dosing.

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice 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-Grunwald/Giemsa.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind using light microscopy at x 1000 magnification. The incidence of
micronucleated cells per 1000 polychromatic erythrocytes (PCEblue stained immature cell s) per animal was scored. Micronuclei
are normally 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 polychromatic to normochromatic erythrocytes was calcul ated together with appropriate group mean val ues .

Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the 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 when compared to each of the concurrent
vehicl e control groups.
If these criteria are not demonstrated, then the test material is considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio is
shown to be statistically significant from the concurrent vehicle control group.
4
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS sub-committee on
guidelines for mutagenicity testing, Report, part 111 (1989). The data was analysed following a √(x+1) transformation 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/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250 and 500 mg/kg (oral), 50-5000 mg/kg (ip)
- Solubility: good
- Clinical signs of toxicity in test animals: Animals treated intraperitoneally died within 5 min of being dosed at levels of 800 mg/kg
and above. At the level of 400 mg/kg, hunched posture and irritation at the injection site were noted.
- Evidence of cytotoxicity in tissue analyzed: RF study only used to determine MTD, no tissues analysed.
- Rationale for exposure: two routes were chosen; the ip route was chosen as the oral route did not show mortality at the high
levels used.
- Harvest times: 24, 48 and 72 h after dosing


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): small but statistically significant increase in the prequency of PCE's in the 72-h
400 mg/kg test group when compared to the concurrent vehicle control group. The response would not have been significant if
the 72-hour vehicle control frequency had been equivalent to the 48 and 24-hour vehicle controls. The response seen in the 72-
hour 400 mg/kg dose group was modest and within the usual range of 0 to 4 micronuclei per 1000 PCEs for vehicle control animals,
it was therefore considered to be spurious and of no toxicological significance.

- Ratio of PCE/NCE (for Micronucleus assay): no significant changes

- Appropriateness of dose levels and route: Although there was no significant change in the PCE/NCE ratio in any of the test
groups when compared to their concurrent vehicle control groups, clinical signs (increased activity, decreased respiratory rate, laboured respiration, and ataxia) and premature deaths were observed in animals dosed with GBS-5 (400 mg/kg bw) and this
suggests that systemic absorption had occurred and that the bone marrow was exposed. The positive control group showed a
marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to theknown mutagenic activity of cyclophosphamide under the conditions of the test.

- Statistical evaluation: The test material, GBS-5, was found not t o produce a toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Any other information on results incl. tables

Results

Treatment

(mg/kg bw)

Sampling period

(h)

Number of PCE with micronuclei per 1000 PCE

PCE/NCE ratio

mean

SD

mean

SD

0

72

0.4

0.5

1.42

0.37

0

48

1.0

1.2

1.51

0.45

0

24

1.1

0.9

1.51

0.35

Pos. control

24

18.4 ***

7.5

1.45

0.39

400

72

1.4 *

0.9

1.53

0.29

400

48

0.7

0.8

1.32

0.37

400

24

0.2

0.4

1.41

0.35

200

24

0.4

0.7

1.57

0.50

100

24

1.0

1.3

1.17

0.22

* p0.05; *** p0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance was considered to be non-genotoxic under the conditions of the test.
Executive summary:

A study was performed to assess the potential of GBS-5 to produce damage to chromosomes or aneuploidy when administered via the

intraperitoneal route to mice. The method used has been designed to comply with the OECD Guidelines for Testing of Chemicals (1981) No. 474 "Genetic Toxicology: Micronucleus Test" and Method 812 of Commission Directive 84/449/EEC (which constitutes Annex V of Council Directive

67/548/EEC) and the Ministry of Health and Welfare, Japan, Guidelines for Testing of Drugs for Toxicity, Pharmaceutical Affairs Bureau, Notice No. 118 (1984).

Following a preliminary range-finding study to evaluate the toxicity of the test material and route of administration, the micronucleus study was conducted using GBS-5 via the intraperitoneal route in groups of ten mice (5 males and 5 females) at the maximum tolerated dose (MTD) of 400 mg/kg, with 200 and 100 mg/kg as the two lower dose levels. Oral exposure at levels of 2500 or 5000 mg/kg bw induced slight toxicity only, and therefore the i.p route was chosen. Animals were killed 24, 48 or 72 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single intraperitoneal dose of distilled water or oral dose of cyclophosphamide, to serve as vehicle and

positive controls respectively.

There was a small but statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with GBS-5

in the 72-hour harvest group when compared to the concurrent vehicle control group. The response seen would not have been significant if the

72-hour vehicle control frequency had been equivalent to the 24 and 48 -hour vehicle controls. The response seen in the 72 -hour 400 mg/kg dose

group was modest and within the usual range of 0 to 4 micronuclei per 1000 PCEs for vehicle control animals, it was therefore considered to be

spurious and of no toxicological significance. No significant change in the PCE/NCE ratio was observed after dosing with GBS-5, However, clinical

signs and premature deaths were observed in animals dosed with GBS-5 and this suggests that systemic absorption had occurred and that the bone

marrow was exposed.

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

The test material, GBS-5, was considered to be non-genotoxic under the conditions of the test.