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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:
supporting study
Study period:
December 21st, 1981 to January 20th, 1982
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Similar to guideline, GLP. The positive control in females did not induce a significant increase in micronuclei.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
1. The proportion of micronucleated PCEs was determined for 500 PCEs / animal. OECD 474 (1983) or OECD 474 (1997) indicate 1000 or 2000 PCEs /animal, respectively. 2. The positive control in females did not induce a significant increase in micronuclei.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclohexyldimethylamine
EC Number:
202-715-5
EC Name:
Cyclohexyldimethylamine
Cas Number:
98-94-2
Molecular formula:
C8H17N
IUPAC Name:
N,N-dimethylcyclohexanamine
Constituent 2
Reference substance name:
DMCHA
IUPAC Name:
DMCHA
Constituent 3
Reference substance name:
N,N-dimethylcyclohexylamine
IUPAC Name:
N,N-dimethylcyclohexylamine
Details on test material:
CAS 98-94-2, N,N-dimethylcyclohexylamine, purity not specified
- Substance type: organic
- Physical state: liquid
- Lot/batch No.: 20-133-CJ
- Other: Clear, straw coloured liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: Adult
- Weight at study initiation: First study: 248.7 g (males) and 209.2 (females). Second study: 191.2 g (males) and 186.2 g (females)
- Assigned to test groups randomly: yes
- Housing: Animals were housed up to 4 rats per cage.
- Diet (e.g. ad libitum): Purina Laboratory Chow ad libitum
- Water (e.g. ad libitum): ad libitum

IN-LIFE DATES: From: December 21st, 1981 To: January 20th, 1982

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Initial doses were based upon LD50 information and were 80% of the LD50 for high dose and one-half of the high dose for the low dose. High mortality at the high dose prompted a second study, using a third dose of one-fourth the high dose. The three doses employed were thus: 80% of the LDS0
or 450 mg/kg/day, 225 mg/kg/day and 112 mg/kg/day.

A subchronic dosing regimen was used. It consisted of two administrations approximately 24 hours apart. Harvest was at 48 and 72 hours after the first administration of the test article, and at 48 hours after the first administration of the control articles. Equal numbers of males and females were used at each dose/kill -time combination. The route of administration was oral gavage.
Duration of treatment / exposure:
Two administrations at an interval of approximately 24 hours
Frequency of treatment:
Two treatments at a 24-hour interval
Post exposure period:
Not documented
Doses / concentrationsopen allclose all
Dose / conc.:
225 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
Dose / conc.:
112 mg/kg bw/day (nominal)
No. of animals per sex per dose:
4 per sex (2 per sex per sampling time).
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine at 0.5 mg/kg dissolved in saline
Triethylenemelamine
- Route of administration: Intraperitoneal injection
- Doses / concentrations: at 0.5 mg/kg dissolved in saline

Examinations

Tissues and cell types examined:
% of polychromatic erythrocytes (PCE) in 500 erythrocytes. % micronucleated PCEs in 500 PCEs.
Number of cells scored: 500 PCEs were scored per animal.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The test compound was initially tested at 433 mg/ml in corn oil for the low dose. Due to the high morality of the high dose animals (450 mg/kg) wherein 15 out of 16 animals died, the study was repeated with a lower dose and concurrent 48-hour negative control.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): three doses were administered: high dose 450 mg/kg bw, 225 mg/kg bw and 112 mg/kg bw. Sampling time were carried out at 48 and 72 hours after the first treatment and at 48 hours after the first administration of the control articles.

DETAILS OF SLIDE PREPARATION: Following centrifugation to pellet the tissue, the supernatant was drawn off, cells resuspended in a drop of serum, and suspension spread on slides and air-dried. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa,
and rinsed in deionized water (Schmid, 1975).

METHOD OF ANALYSIS: Based on total numbers of PCE in the optic fields. The frequency of PCEs versus mature erythrocytes (RBCs) was
determined by scoring the number of RBCs observed in the optic fields while scoring the f i r s t 200 PCEs for micronuclei.
Evaluation criteria:
A test article is positive if two treatment groups are positive (p < 0.05). If only one treatment group is positive, the result is designated as unconfirmed positive.
Statistics:
Student t test and ANOVA using mean square for error (MSE)

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL: 
- Mortality and time to death:  6 at medium dose, 15 at high dose and one positive control animal were found dead during the study. Time to death not recorded.
- Clinical signs:  all medium and high dose animals showed convulsions after dosing on the first day. After seizure, animals were lethargic. 

EFFECT ON PCE/NCE RATIO: 
%PCE at 112 and 225 mg/kg was 82 and 117, respectively (males, 48 h); 61 and 113% (males 72h).
%PCE at 112, 225 and 450 mg/kg was 62, 126 and 88, respectively (females, 48h), and 110 and 55% respectively (females, 72 h, all females of 450 mg/kg died)

GENOTOXIC EFFECTS: 
Mean number of micronucleated PCE: at 48 h: at 112, 225 and 450 mg/kg: 0.65, 0.4 and 0.4 respectively (male and female)
at 72h:  at 112 and 225 mg/kg: 0.28 and 0.52 respectively (male and female).

MUTANT/ABERRATION/mPCE/ POLYPLOIDY FREQUENCY:  MPCE: no treatment related effects

Any other information on results incl. tables

All medium and high dose animals convulsed after dosing on first day; after seizure, animals were lethargic.

Applicant's summary and conclusion

Conclusions:
Although there was no increase in micronuclei in the females treated with the positive controls, DMCHA does not induce micronuclei in rat polychromatic erythrocytes and is therefore not clastogenic.
Executive summary:

A study (Cimino, 1982) was performed to assess the ability of DMCHA to induce micronuclei in polychromatic erythrocyte from rat bone marrow.

DMCHA was administered via intraperitoneal injection to groups of 4 animals/sex/dose level. Negative and positive control cultures were also prepared. Triethylenemelanine was used as the positive control at a dose level of 0.5 mg/kg bw. A preliminary study was carried out and due to the high mortality at 450 mg/kg bw, the study was repeated with a lower dose and concurrent 48 hour negative control. A total of three doses were administered 450 mg/kg bw/day, 225 mg/kg bw/day and 112 mg/kg bw/day. Two administrations were performed at a 24 hour intervals. Harvest of the cells was performed at 48 and 72 hours after the first administration and at 48 hours after the first administration of the control articles.

The animals were sacrificed and the bone marrow was extracted for preparation. The tissue was centrifuged and the supernatant was collected, resuspended in serum and spread on slides. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa and rinse in deionised water prior to analysis for scoring.

Five hundred PCEs per animal were scored.The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The frequency of PCEs versus mature erythrocytes (RBCs) was determined by scoring the number of RBCs observed in the optic fields while scoring the first 200 PCEs for micronuclei. The percentage micronuclei in marrow from the negative controls were 0.45% and 1.00% for males, and 0.85% and 0.65% for females, in the first and second studies respectively. The positive control compound, triethylenemelamine (TEM),induced an increase in frequency of micronuclei in males but not in females. The mean number of micronucleated PCE at 48 h were 0.65 at 112, 0.4 at 225 and 0.4 at 450 mg/kg (male and female). At 72 hours, the mean numbers of micronucleated PCE were  0.28 at 112 and and 0.52 at 225 mg/kg:  (male and female). 

 

Although there was no increase in micronuclei in the females treated with the positive controls, DMCHA does not induce micronuclei in rat polychromatic erythrocyte and is therefore not clastogenic. The negative response in the in vivo assay confirmed the negative in vitro responses in a battery of mutagenicity assays.

Based on these results, the test substance does not require classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.