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Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is in accordance to the test guidelines in place at the time when the study was performed and GLP and therefore rated as reliable without restrictions.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 26 May 1983
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): AR005
- Substance type and Physical state: pale cream free-flowing solid
- Analytical purity: 99.5%
- Storage condition of test material: room temperature

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River (UK) Ltd, Manston, Kent
- Age at study initiation: appr. 5-7 wks
- Weight at study initiation: 23-29 g (males), 20-24 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: -
- Housing: in groups of up to 5 in solid floor polypropylene cages with woodflake bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 20-21°C
- Humidity (%): 50-51%
- Air changes (per hr): appr. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
- Vehicle used: arachis oil
- Justification for choice of solvent/vehicle: formulation of the test item as a homogenous suspension, stability of the test item in the vehicle
- Concentration of test material in vehicle: 125, 250, 500 mg/ml (range finder); 125, 62.5, 31.25 mg/ml (main study)
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Details on exposure:
The test item was freshly prepared as required as a suspension at the appropriate concentration on arachis oil.
Duration of treatment / exposure:
Frequency of treatment:
once by oral gavage
Post exposure period:
Vehicle and test item group animals were killed 24, 48 and 72 hrs following dosing. Positive control animals were killed 24 hrs following dosing.
Doses / concentrations
Doses / Concentrations:
1250, 2500, 5000 mg/kg bw (range finder); 312.5, 625, 1250 mg/kg bw (main study)
nominal conc.
No. of animals per sex per dose:
5 male and 5 female mice per dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Justification for choice of positive control(s): known to induce micronuclei under the conditions of the test
- Route of administration: oral gavage
- Dose: 50 mg/kg bw


Tissues and cell types examined:
tissue: bone marrow, cell types: normochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
A range-finding toxicity study was performed to determine a suitable dose level for the micronucleus study . The dose levels selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 2000 mg/kg (EC guidelines) or 5000 mg/kg (US EPA guidelines) .
Groups of mice were dosed as follows :
All animals were dosed once only by oral gavage at the appropriate dose level, using a metal needle attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing. Animals were observed 1 hour after dosing and subsequently once daily for 3 days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Groups, each of ten mice (five males and five females), were dosed orally once only with test material at 312 .5, 625 or 1250 mg/kg .
One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment, two other groups dosed at 1250 mg/kg were sacrificed at 48 hours and 72 hours . In addition four further groups of ten mice (five males and five females) were included in the study ; three groups were given a single oral dose of the vehicle (arachis oil) and the fourth group was dosed orally with cyclophosphamide, a positive control material known to induce micronuclei under the conditions of the test. The vehicle control groups were killed 24, 48 and 72 hours following dosing and positive control group animals were killed 24 hours following dosing .
All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily .

Immediately following sacrifice (ie . 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-Gruenwald/Giemsa, dried and coverslipped using mounting medium.

Stained bone marrow smears were coded and examined 'blind' using light microscopy at x1000 magnification . The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) 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 was counted ; these cells were also scored for incidence of micronuclei .
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values for males and females separately and combined .
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 a test material dose group at either the 24, 48, or 72-hour kill times when compared to its
corresponding control group .
If this criteria is not met, 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 significantly different from the concurrent vehicle control group .
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines
for Mutagenicity Testing Report, Part III (1989) . The data was analysed following a V(x + 1) transformation using Student's ttest
(two tailed) and any significant results were confirmed using the one way analysis of variance .

Results and discussion

Test results
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
The mortality data are summarised as follows :
1250 mg/kg: 2 males and 2 females treated; deaths: 0/4
2500 mg/kg: 2 males and 2 females treated; deaths: 2/4 (2 females on day 1 and 2 post treatment)
5000 mg/kg: 2 males and 2 females treated; deaths: 3/4 (2 females on day 1 and 2 post reatment and 1 male on day 1 post treatment)

Premature deaths were seen in animals dosed with the test material at and above 2500 mg/kg. Clinical signs were observed in animals dosed with test material at and above 1250 mg/kg, and were as follows : lethargy, ptosis, decreased respiratory rate, laboured respiration, splayed gait, occasional body tremors, clonic and tonic convulsions, ataxia, hunched posture and pilo-erection .
There were no premature deaths at 1250 mg/kg, clinical signs were observed, however and this was the dose level selected as the maximum tolerated dose for the main study, with 625 and 312 .5 mg/kg as the two lower dose levels.

- Mortality Data and Clinical Observations:
Three premature deaths were seen in animals dosed with test material at 1250 mg/kg . Clinical signs observed in animals dosed with the test material were as follows : decreased respiratory rate, lethargy, laboured respiration, ataxia, ptosis, hunched posture, pilo-erection and splayed gait .
The observations of clinical signs were dose-related such that most of the clinical signs were observed in all of the 625 and 1250 mg/kg animals one hour after dosing, whilst at 312 .5 mg/kg only three clinical signs were observed in 6 of 10 animals . It was only in the 1250 mg/kg dose groups that the clinical signs were observed at all time-points although not in all animals at 24, 48 and 72 hours .

- Induction of micronuclei:
There was no statistically significant increase in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.

- Ratio of PCE/NCE:
There was no statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups, but premature deaths and the presence of clinical signs indicated that systemic absorption had occurred .

- Positive Control
The positive control group showed a significant increase in the incidence of micronucleated polychromatic erythrocytes, hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test .

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
The test material did not produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice and was considered to be non-genotoxic under the conditions of the test.
Executive summary:

In a micronucleus test (Safepharm Laboratories) groups of 10 albino CD-1 mice (5 males and 5 females) were given a single oral dose of TAED at 312.5, 625 and 1250 mg/kg BW. Negative and positive control groups receiving the vehicle, arachis oil, or cyclophosphamide were also included. Control and high dose animals were killed 24, 48 and 72 hours after dosing; animals of intermediate and low dosage groups as well as the positive control group were killed 24 hours after dosing. Bone marrow was extracted from the femurs and smear preparations were made and stained. Three premature deaths were observed after dosing with 1250 mg/kg BW. There were no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes in animals dosed with TAED when compared to the concurrent vehicle control groups. No significant change in the ratio of polychromatic/normochromatic erythrocytes was observed, but the presence of clinical observations and premature deaths at the high dose level indicated that systemic absorption had occurred. In conclusion, TAED was considered to be non-genotoxic under the conditions of the test. The test was performed according to OECD test guideline 474 and GLP and is reliable without restrictions.