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:
30 March 2010 to 27 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted in accordance with a relevant guideline with no or minor deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Diammonium bis[carbonato-O]dihydroxyzirconate
EC Number:
269-682-7
EC Name:
Diammonium bis[carbonato-O]dihydroxyzirconate
Cas Number:
68309-95-5
Molecular formula:
C2H2O8Zr.2H4N
IUPAC Name:
diammonium bis[carbonato-O]dihydroxyzirconate
Details on test material:
Description : clear colourless liquid
Batch number : 09/092
Date received : 26 February 2010
Expiry date : 26 August 2010
Storage conditions :room temperature in the dark
CoA included in the report

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals or test system and environmental conditions:
Sufficient albino Hsd: ICR (CD-1®) strain mice were obtained from Harlan UK. At the start of the main test the mice weighed 22 to 28 g and were approximately five to eight weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.

The animals were housed in groups of up to seven, by sex, in solid-floor polypropylene cages with wood-flake bedding. Free access to mains drinking water and food (Harlan Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
The vehicle was supplied by Gibco, as follows:
Supplier's identification: Phosphate buffered saline
Supplier's lot number: 722040A
Description: Clear colourless liquid
Date received: 03 March 2010
Expiry date: January 2012
Storage conditions: Room temperature
Details on exposure:
Groups, each of seven male mice, were dosed once only via the intraperitoneal route with the test material at 1000, 500 or 250 mg/kg.
Duration of treatment / exposure:
Groups, each of seven male mice, were dosed once only via the intraperitoneal route.
Frequency of treatment:
Animals were dosed only once.
Post exposure period:
One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test material at 1000 mg/kg was killed after 48 hours. In addition, three further groups of male mice were included in the study; two groups (each of seven mice) were dosed via the intraperitoneal route with the vehicle alone (phosphate buffered saline) and a third group (five mice) was dosed orally with cyclophosphamide. Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test. The vehicle controls were killed 24 or 48 hours following dosing and positive control group animals were killed 24 hours following dosing.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
other: intraperitoneal injection
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
other: intraperitoneal injection
Remarks:
Doses / Concentrations:
250 mg/kg
Basis:
other: intraperitoneal injection
Remarks:
Doses / Concentrations:
Vehicle control
Basis:
other: intraperitoneal injection
Remarks:
Doses / Concentrations:
Positive control (5 mg/kg)
Basis:
actual ingested
oral dose
No. of animals per sex per dose:
7 (test and control animals)
5 animals only were dosed with positive control.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control material was supplied by Acros Organics, as follows:
Supplier's identification: Cyclophosphamide
Supplier's lot number: A0164185
In-house serial number: R-4447
Date received: 27 October 2008
Expiry date: 27 October 2010
Storage conditions: Approximately 4ºC in the dark

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 (Aguettant batch no. 300522801).

The concentration, homogeneity and stability of the positive control material and its preparation were not determined by analysis.

Examinations

Tissues and cell types examined:
Bone marrow
Polychromatic and normochromatic erythrocytes
Details of tissue and slide preparation:
Slide Preparation

Immediately following termination (i.e. 24 or 48 hours following dosing), 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 a cover slip applied using mounting medium.

Slide Evaluation

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 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 and standard deviations.

Evaluation criteria:
Interpretation of Results

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 group.

A positive mutagenic response would be demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group. If these criteria were not fulfilled, then the test material was considered to be non genotoxic under the conditions of the test.
A positive response for bone marrow toxicity would be demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
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 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
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Range-finding Toxicity Test

As available data in rats indicated no toxicity by the oral route at the limit dose of 2000 mg/kg, it was considered necessary to only investigate the intraperitoneal route of exposure. A single male and female mouse were treated at 1000 mg/kg by i.p. injection. No difference in sensitivity was observed and therefore two additional male mice were treated at the same dose level and by the same route of exposure. Clear evidence of toxicity was observed in animals dosed with test material via the intraperitoneal route with clinical signs observed as follows: Hunched posture, splayed gait, ptosis, and ataxia. These observations were taken as adequate evidence of systemic absorption. Because of no difference in sensitivity was observed, the main study was performed using male mice only. As adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration; therefore, this was selected for use in the main test. The maximum tolerated dose (MTD) of the test material, 1000 mg/kg, was selected for use in the main test, with 500 and 250 mg/kg as the lower dose levels.

Micronucleus Test

Mortality Data and Clinical Observations

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 250 mg/kg in both the 24 and 48 hour groups where appropriate, these were as follows: Hunched posture, ptosis and ataxia.

Evaluation of Bone Marrow Slides

Modest statistically significant decreases in the PCE/NCE ratio were observed in all three 24 hour test material dose groups and, whilst not statistically significant, a modest decrease was also observed in the 48 hour test 1000 mg/kg dose group when compared to the concurrent vehicle control group. Therefore, the decreases in PCE/NCE ratio, together with the observation of clinical signs at and above 250 mg/kg, were taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There were no statistically significant increases in the frequency of micronucleated PCE in any of the test material dose groups when compared to their 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 found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Applicant's summary and conclusion

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 compliant study has been conducted in accordance with OECD Guideline 474. The test material administered by intraperitoneal injections at dosages of 1000, 500 and 250 mg/kg caused clinical signs of toxicity and reductions in the PCE/NCE ratio, thus confirming exposure of the target organ. The test substance was considered to be non-genotoxic under the conditions of the test.