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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
The genetic toxicity of 2-butyl chloride (sec-butylchloride, sBC) has been assessed in 1 in vitro study (bacterial reverse mutation assay) as well as in an in vivo studies (a micronucleus assay). Negative results were reported in all studies.
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:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1983 followed, reliability scoring based on 1997 guideline.
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: BOR:NMRI SPF (Han.)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Firma Winkelmann, Versuchtierzucht, D- 4791 Borchen, Germany
- Age at study initiation: Adult (about 3 months)
- Weight at study initiation: 32.9 to 38.6 g (males); 30.9 to 35.0 g (females)
- Assigned to test groups randomly: Yes, by lot
- Housing: Collective caging
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 ± 1.5
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Lot/batch no. (if required): Batch: 2466515
- Manufacturer: Roth GmbH, Karlsruhe
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: By suspending appropriate amounts in corn oil
Duration of treatment / exposure:
Single administration
Frequency of treatment:
Single administration
Post exposure period:
24, 48, or 72 hours
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5/sex in control groups and 15/sex in test-article groups (with 5/sex being sacrificed for smear evaluation at each time point)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/kg bw in 10 mL/kg corn oil
- positive control group 5 males and 5 females
- Batch No 13096473, supplied by Asta-Werke, D-4800 Bielefeld
Tissues and cell types examined:
Bone marrow from femurs.
Details of tissue and slide preparation:
The femurs were removed and the bone marrow was suspended in fetal calf serum. Samples were centrifuged at 1.600 x g, decanted and then one drop of each single suspension was smeared on a slide by means of a second slide.

These preparations were dried, fixed in absolute (99 %) methanol for 5 min. and then allowed to air dry. The slides were stained using a May-Grünwald and Giemsa solution.

From each animal 2 preparations were made. Prior to analysis all the slides were randomized and coded (blind evaluation).
Evaluation criteria:
The test substance is considered to be active if a statistically significant increase in the number of polychromatic erythrocytes with micronuclei in comparison to the control values occurs at any point in time.
Statistics:
One factorial analysis of variance.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 2500 and 5000 mg/kg bw
- Clinical signs of toxicity in test animals:
Animals treated with 5000 and 2500 mg/kg showed severe ataxia, a distinct writhing-reflex, a lateral body position, sedation and piloerection i.e. 5 min. p.a. and up to 4 hours p.a., whereby the lower dosed animals only showed slight ataxia, a slight sedation and a slight piloerection at this point of time. Animals treated with 1000 mg/kg were slightly sedated for about 1 hour and showed a very slight piloerection.

For animals treated with a single dose of 2000 mg/kg bw a very slightly reduced activity (sedation) and piloerection was noted 1 to 3 hours following administration, but the animals were normal in appearance and behaviour afterwards. No animal died throughout the study.

Conclusions:
Interpretation of results (migrated information): negative
2 -chlorobutane is considered negative for mutagenicity in this in-vivo test.
Executive summary:

The number of polychromatic erythrocytes with micronuclei was significantly incresed 24h post injection in the positive control animals. This confirms the sensitivity of the animal strain used. No significant differences between animals treated with 2 -chlorobutane and control animals were noted at 24, 48 and 72 hours following application. The number of polychromatic and normochromatic erythrocytes and the ratio polychromatic/normochromatiuc erythrocytes was not significantly different to the controls in animals treated with 2 -chlorobutane at any time. Thus, 2 -chlorobutane is considered negative for mutagenicity in this test.

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

Additional information

Additional information from genetic toxicity in vivo:

In the GLP compliant, key bacterial reverse mutation assay (equivalent to OECD guideline 471), 2 -chlorobutane was tested at doses of 0, 8, 40, 200, 1000, or 5000 µg/plate in Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 both in the absence and presence of exogenous metabolic activation [rat liver S9 (compound used to induce cytochrome P450 enzymes was not reported)] (IBR, 1989).  The experiment was conducted in quadruplicate and an independent repeat experiment was performed. Ethanol was used as the vehicle and positive controls were included in all incubations. No cytotoxicity was observed at any sBC concentration and no increases in the reverse mutation rate were noted in any strain either in the absence or presence of metabolic activation. Incubation with positive control substances in the absence or presence of metabolic activation resulted in anticipated increases in reverse mutation rates.

In a key, GLP compliant in vivo micronucleus assay (according to OECD guideline 474), sBC was administered via intraperitoneal (IP) injection to male and female NMRI mice at a single dose of 2,000 mg/kg body weight (IBR, 1989).  Corn oil was used as the vehicle and cyclophosphamide was administered via IP injection as the positive control compound. Animals were sacrificed 24, 48, or 72 hours after administration of the test article or controls (5 to 15 mice/sex/dose). No deaths were reported; however, sedation and piloerection were observed following test article administration. Statistically significant increases in the number of micronucleated polychromatic erythrocytes were not noted following sBC administration. The positive control compound caused anticipated increases in micronucleated polychromatic erythrocytes. 

 


Justification for selection of genetic toxicity endpoint
Valid in-vivo GLP study

Justification for classification or non-classification

The substance sec-butylchloride (sBC) produced negative results in both in vitro and in vivo genetic toxicity studies. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.5 nor according to DSD (Directive 67/548/EEC).