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Key value for chemical safety assessment

Genetic toxicity in vivo

Link to relevant study records
Reference
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:
4th June 2008 - 5th August 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed to GLP
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)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
Source: Charles River (UK)
Acclimatisation period: 7 days
Environmental conditions
Housed in groups of 7 in solid floor polypropylene cages with wood flake bedding.
Free access to mains drinking water and food.
Temperature: 19-25ºC
Humidity: 30-70%
Air changes: 15 per hour
Lighting: 12 hours light, 12 hours darkness.
Route of administration:
oral: gavage
Vehicle:
Arachis Oil
Details on exposure:
All animals were dosed once only at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
Duration of treatment / exposure:
Gavage
Frequency of treatment:
Each group was dosed once only.
Post exposure period:
All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediatley prior to termination.
Remarks:
Doses / Concentrations:
750 (MTD), 375, 187.5 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Seven
Control animals:
yes
Positive control(s):
Cyclophosphamide
Tissues and cell types examined:
Bone marrow smears from both femurs
Details of tissue and slide preparation:
Both femurs were 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, stained in May-Grünwald/Giemsa, allowed to air dry and cover slipped using mounting medium.
Statistics:
Statistical analysis was a t-test following a √(x+1) transformation of the data, significant values were confirmed by one-way analysis of variance as recommended by the UKEMS (Lovell et al, 1989)
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative
The test material was considered to be non-genotoxic under the conditions of the test
Executive summary:

A GLP study was performed in accordance with OECD Guideline 474 to assess the potential of Inhibitor AHM P500 to produce damage to the chromosomes or aneuploidy when administered to mice.

A range-finding test was performed to find suitable dose levels of the test material, route of administration and to investigate to see if there was a marked difference in toxic response between sexes. There was no marked difference in the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven mice (Males) at the maximum tolerated dose (MTD) 750 mg/kg and with 375 and 187.5 mg/kg as the two lower dose levels. Animals were killed 24 or 48 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 oral dose of arachis oil (two groups each of 7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle controls were killed 24 or 48 hours later, positive control animals were killed after 24 hours.

There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 375 mg/kg in both the 24 and 48-hour groups. Where applicable, these were; hunched posture, ptosis and ataxia. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48 hour test material dose groups when compared to their concurrent control groups. However, the observation of a modest reduction in PCE/NCE ratio values in the 48-hour material dose group, together with the observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue achieved.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control group showed a marked 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.

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Two in vivo studies were available, an Ames test (Thompson, 2006) and a mammalian cell gene mutation study in the L5178Y cell line (Flanders, 2008). A clear negative result was obtained in the Ames test, however the gene mutation study showed a dose related increase in the mutant frequency in the presence of the S9 fraction. The mutagenic response was partly due to an increase in the frequency of small colonies and was considered to demonstrate a modest clastogenic response. Considering the results from the two in vitro studies a possible weak mutagenic effect is indicated.

 

However an in vivo micronucleus study (Flanders, 2008) did not reproduce the mutagenic effect of the in vitro study. A conclusive negative result was obtained, therefore based on the available data; Inhibitor AHM P500 is not considered to be mutagenic.


Justification for selection of genetic toxicity endpoint
In vivo guideline study is considered to supersede results obatined in vitro.

Justification for classification or non-classification