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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1985-09-13 to 1985-12-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted May 26, 1983
Deviations:
no
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Chemical name: C12-C16-alkyldimethylbenzylammoniumchlorid
CAS: 68424-85-1
Mean MW: 357 g/mol
Specific details on test material used for the study:
Test material: Hyamine 3500 (80%), Batch No. L5383
Received on September 13, 1985
Chemical designation: Alkyl dimethyl benzyl ammonium chlorid
pH (1% in water): 6.3
Solubility tested 0.5, 2.5, and 5% in water: clear
Ash (NaCl): <0.1%
Free amine: 0.2%
Aminehydrochloride: 0.1%

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
NMRI SPR mice of the strain Bom: NMRI, GI. Gomholtgard Ltd, DK-8680 Ry.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeders from GI. Bomholtgard Ltd., but mice used were born in testing facilities (Scantox Laboratories)
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: 25-30 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: type III Macrolone cages, 5 per cage, separated by sex. Bedding used was special softwood sawdust "Spanvall Special White" from Spanvall Ltd., DK-4535 Vallekilde.
- Diet (e.g. ad libitum): ad libitum complete rodent diet "Altromin 1314" from Chr. Petersen Ltd, DK-4100 Tingsted
- Water (e.g. ad libitum): free access to drinking water with hydrochloric acid added to pH 2.5

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2 °C
- Humidity (%): relative humidity 55 +/- 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: solutions of the test material, 400 mg/10 mL distilled water and of cyclophosphamide, 30 mg/10 mL distilled water was prepared.

Mice were administered test dose one time and sacrificed by cervical dislocation at 24 hours, 48 hours, and 72 hours after administration. The volume administered to the mice was constantly 1 mL/100 g body weight.
Duration of treatment / exposure:
Three groups (5 female, 5 male): 24, 48, 72 hours
Frequency of treatment:
single treatment
Post exposure period:
24, 48, 72 hours
Doses / concentrations
Dose / conc.:
400 mg/kg bw/day
No. of animals per sex per dose:
5 female, 5 male
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control group was administered 30 mg cyclophosphamide/kg (30 mg/10 mL distilled water), administered by oral gavage via metal tube, volume 1 mL/100 g body weight.

Examinations

Tissues and cell types examined:
Bone marrow from femur used for smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A few mice were treated orally by various concentrations of the test article diluted with distilled water. Thereby the maximum tolerated dose was estimated at 400 mg/kg. At this dosage bone marrow smears showed a reduced number of polychromatic erythrocytes as compared with normochromatic erythrocytes. At a dose exceeding 400 mg/kg the mortality was too high.

DETAILS OF SLIDE PREPARATION: Immediately after an animal was sacrificed, its femurs were dissected free, and by a 1 mL syringe with needle the bone marrow was flushed out into 5 mL of fetal calf serum. After thorough shaking, the mixture was centrifuged for 10 minutes at about 1000 rpm. Smears were made after removal of the supernatant. Specimens were fixed in methanol and stained with May-Gruenwald/Giemsa.

METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind.
- Number of normochromatic erythrocytes (NCE) per 1000 erythrocytes
- Number of polychromatic erythrocytes (PCE) per 1000 erythrocytes
- Number of micronuclei (MN) in 1000 normochromatic erythrocytes
- Number of micronuclei (MN) in 1000 polychromatic erythrocytes
Statistics:
The results were assessed by a one-way variance analysis. For PCE (%) of the test was performed on the values observed, and for MN (per thousand) the test was done on computed rank values transformed to normal scores according to Blom's method. (G. Blom Statistical Estimates and Transformed Beta Variables. New York: John Wiley and Sons, Inc., 1958).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Remarks:
one mouse in the 72-hour exposure group was found dead on day 3
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
the number of micronuclei was increased in the positive control group
Remarks on result:
not determinable
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 400, 500, 750, 1000, 1250, 2500, 5000 mg/kg body weight
- Solubility: not specified
- Clinical signs of toxicity in test animals: Mortality was too high in those treated with greater than 400 mg/kg doses.
- Evidence of cytotoxicity in tissue analyzed: At 400 mg/kg, the bone marrow smear showed a reduced number of PCE compared with NCE.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The number of micronuclei in the PCE was almost equal in the negative control group and the test groups. The number of micronuclei was definitely increased in the positive control group. The low micronucleus count found in NCE means that the micronuclei recorded in the PCE were not false micronuclei. The reduced count of PCE (%) in the test groups goes to show that the dosage 400 mg/kg was sufficiently high to affect erythropoiesis.
- Ratio of PCE/NCE (for Micronucleus assay): PCE count was reduced in the test groups and in the positive control group.
- Statistical evaluation: Statistically significant difference between the percentage of PCE in the negative control group and the test groups but no significant difference between the number of micronuclei in the negative control group and in the test group.
- Mortality: One mouse in test group 72-hours was found dead on day 3 after treatment

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions in this mouse erythrocyte micronucleus test, Hyamine 3500 (80%) is non-mutagnenic.
Executive summary:

In a NMRI mouse bone marrow micronucleus assay conducted according to OECD guideline 474, 5 animals per sex per dose were single treated orally by gavage with the test item Hyamine 3500 (80%) diluted in distilled water at a dose of 400 mg/kg bw. Bone marrow cells were harvested 24, 48 and 72 hours post-treatment.

One mouse in test group 72-hours was found dead on day 3 after treatment. A low micronucleus count was found in normochromatic erythrocytes. Thus, micronuclei recorded in the polychromatic erythrocytes were not false micronuclei and the reduced count of polychromatic erythrocytes in the test groups means that the dosage of 400 mg/kg was sufficiently high to affect erythropoiesis. The positive control induced the appropriate response. The test substance Hyamine 3500 was designated as non-mutagenic in the micronucleus test.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data.