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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Sufficient documentation; comparable to guideline study with acceptable restrictions
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2003
Reference Type:
study report
Title:
Unnamed
Year:
1995
Reference Type:
other: abstract
Title:
TR-505 Toxicology and Carcinogenesis Studies of Citral (Microencapsulated) (CAS No. 5392-40-5) in F344/N Rats and B6C3F1 Mice (Feed Studies) - Draft Abstract
Author:
National Toxicology Program NTP
Year:
2003
Bibliographic source:
http://ntp-server.niehs .nih .gov/htdocs/LT-studies/tr505 .html

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
(No positive control included; other exposure subchronic feeding)
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Citral
- Physical state: colourless liquid
- Analytical purity: ca. 97.6%
- Impurities (identity and concentrations): seven substances corresponding to 2.4 % in total
- Isomers composition: ratio geranial:neral = 2:1
- Lot/batch No.: 06930PG
- Expiration date of the lot/batch: no data
- Stability under test conditions: no degradation of the neat chemical after storage for 2 weeks at temperatures up to 60°C when protected from light; no loss of citral from the microcapsules during 14 weeks

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services, Germantown, N.Y.
- Age at study initiation: 6 weeks
- Weight at study initiation: males ca. 20 g, females ca. 16.5 g
- Fasting period before study: no
- Housing: males inidividually, females 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 to 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C (calculated from 72° +- 3° F)
- Humidity (%): 50% +- 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: June 7 or 8, 1995 To: September 7 or 8, 1995

Administration / exposure

Route of administration:
oral: feed
Vehicle:
VEHICLE
- Justification for use and choice of vehicle: microcapsules prepared from food-grade sugar and starch were loaded with citral to prevent loss of test substance
- Concentration in vehicle: 31.3 % citral (analytical method GC)
- Lot/batch no.: 20295
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 to 4 weeks
- Mixing appropriate amounts with nonirradiated NTP-2000 feed: final concentrations of 3,900, 7,800, 15,600, or 31,300 ppm citral were achieved by adding loaded and/or placebo microcapsules at a total concentration of 10% microcapsules
- Storage temperature of food: room temperature
Duration of treatment / exposure:
13 w
Frequency of treatment:
continuously
Post exposure period:
No
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
3900, 7800, 15600, or 31300 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
745, 1840, 3915, and 8110 mg/kg bw/d
Basis:
other: actual ingested by male mice
Remarks:
Doses / Concentrations:
790, 1820, 3870, and 7550 mg/kg bw/d
Basis:
other: actual ingested by female mice
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
none

Examinations

Tissues and cell types examined:
peripheral blood
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
based on pre-study of Dieter et al., 1993 (see Section 7.5.1)

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
after 13 weeks of continuous feeding

DETAILS OF SLIDE PREPARATION:
Smears were immediately prepared and fixed in absolute methanol. The fixed slides were stained with acridine orange.

METHOD OF ANALYSIS:
1000 mature erythrocytes (normochromatic erythrocytes or NCEs) are scored per animal for presence of micronuclei. In addition, the percentage of PCEs among the total erythrocyte population in the periphral blood was scored for each exposure group as a measure of chemical-induced toxicity to the bone marrow. All data were analyzed separately for male and female mice.
Evaluation criteria:
Test positive if
the trend test P value is < or = 0.025
or if the P value for any single dosed group is < or = 0.025 divided by the number of dosed groups; for this study the relevant P value is < or = 0.006
Final call of positive for micronucleus induction based on reproducibly positive trials
Statistics:
Test for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test
Pairwise comparisons between each dosed group and the control group

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
Toxicity: For details see Section 7.5.1, 14 week repeated dose toxicity study

Any other information on results incl. tables

Table 1: Frequency of micronuclei in peripheral blood erythrocytes of mice following administration of citral in Feed for 13 Weeks

Sex

Dose group

(mg/kg bw/d)

Number of test animals evaluated

Micronucleated NCEs/1,000 NCEs

P value

PCEs

(%)

Mean

SE

Male

Untreated control

10

1.30

0.32

2.2

Vehicle control

10

1.90

0.22

2.1

745

10

1.50

0.32

0.5818

2.2

1,840

10

1.70

0.28

0.4199

1.9

3,915

10

2.50

0.25

0.0458

1.8

8,110

6

1.83

0.28

0.3482

1.8

Trend: P=0.145

Female

Untreated control

10

1.00

0.35

2.3

Vehicle control

10

1.50

0.32

2.6

790

10

1.00

0.24

0.7251

2.4

1,820

10

1.10

0.36

0.6382

2.4

3,870

10

0.90

0.22

0.8022

2.3

7,550

10

1.60

0.29

0.2196

2.2

       Trend: P=0.217      

NCE normochromatic erythrocyte; PCE polychromatic erythrocyte

Tables with a compilation of data from the individual test animals are available from Appendix 2

Applicant's summary and conclusion