Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
no positive controls were tested
GLP compliance:
yes
Remarks:
in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: NTP colony maintained at Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 22.5-23 g (male) and 19.3-19-7 g (female)
- Housing: individually. Cages: Stainless steel, wire bottom (Lab Products, Inc., Seaford, DE); rotated weekly; cageboard: Untreated paper cage pan liner (Shepherd Specialty Papers, Kalamazoo, MI), changed daily
- Diet (e.g. ad libitum): NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods)
- Water (e.g. ad libitum): Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
- aclimatation period: Animals were quarantined for 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Room fluorescent light: 12 hours/day
- Chamber air changes: 15 ± 2/hour

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
- Vehicle(s)/solvent(s) used: no data
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was held in an 8-gallon stainless-steel chemical reservoir. Test item was pumped into a heated glass column filled with glass beads that increased the surface area for vaporization. Heated nitrogen entered the column from below and assisted in vaporizing the chemical while conveying it into a short distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Metering valves at the manifold controlled flow to each chamber through individual Teflon® delivery lines that carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To initiate exposure, the chamber exposure valves were rotated to allow the test item vapor to flow to each exposure chamber inlet duct where it was further diluted with filtered, conditioned air to achieve the desired exposure concentration.
- Temperature, humidity, pressure in air chamber: 72 ± 3ºF; 50% ± 15%.
- Air change rate: 15 air changes per hour
- Method of particle size determination: A condensation particle detector (Model 3022A, TSI, Inc., St. Paul, MN) was used with and without animals in the exposure chambers. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.

TEST ATMOSPHERE
- Brief description of analytical method used: on-line gas chromatograph. Samples were analyzed using GC/FID to measure the stability and purity of test item in the generation and delivery system. To assess whether impurities or degradation products coeluted with test item or the solvent, a second GC/FID analysis of the samples was performed using a polar column capable of resolving compounds with similar boiling points and polarities.
- Samples taken from breathing zone: yes.
Duration of treatment / exposure:
14 weeks; 6 hours plus T90 (10 minutes) per day
Frequency of treatment:
five times per week, weekdays only
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
25 ppm
Remarks:
(0.14 mg/L)
Dose / conc.:
50 ppm
Remarks:
(0.28 mg/L)
Dose / conc.:
100 ppm
Remarks:
(0.56 mg/L)
Dose / conc.:
200 ppm
Remarks:
(1.13 mg/L)
Dose / conc.:
400 ppm
Remarks:
(2.26 mg/L)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Positive control(s):
none

Examinations

Tissues and cell types examined:
Peripheral blood samples
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
2-week preliminary study were conducted to determine the highest administrable non lethal dose level. 5 mice per sex and per dose were exposed to 0, 100, 200, 400, 800, and 1,600 ppm test item.

TREATMENT AND SAMPLING TIMES:
At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol.

DETAILS OF SLIDE PREPARATION:
Slides were air-dried, fixed and stained with a fluorescent DNA-specific stain (acridine orange).

METHOD OF ANALYSIS:
Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of five animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs) among a population of 1000 erythrocytes was scored for each exposure group as a measure of bone marrow toxicity.
Evaluation criteria:
In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction was preferably based on reproducibly positive trials. Ultimately, the final call was determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.
Statistics:
The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tail Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): See table 1
- Ratio of PCE/NCE (for Micronucleus assay): See table 1

Any other information on results incl. tables

Table 1: Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Treatment with alpha Pinene by Inhalation for 3 Months a

 

Concentration

(ppm)

Number of Mice with Erythrocytes Scored

Micronucleated NCEs/1,000 NCEs b

P Value c

PCEs b

(%)

Male

Aird

0

5

1.6 ± 0.33

2.50 ± 0.39

Alpha pinene

25

5

1.8 ± 0.30

0.3657

2.34 ± 0.19

50

5

1.9 ± 0.53

0.3059

2.20 ± 0.26

100

5

2.1 ± 0.43

0.2053

2.88 ± 0.31

200

5

1.9 ± 0.29

0.3059

2.74 ± 0.19

400

5

1.4 ± 0.40

0.6426

3.10 ± 0.20

P=0.742e

Female

Air

0

5

1.4 ± 0.19

2.40 ± 0.19

Alpha pinene

25

5

2.1 ± 0.43

0.1182

2.16 ± 0.26

50

5

1.8 ± 0.25

0.2396

2.16 ± 0.20

100

5

1.7 ± 0.44

0.2949

2.74 ± 0.36

200

5

1.7 ± 0.30

0.2949

2.06 ± 0.29

400

5

1.1 ± 0.19

0.7259

2.16 ± 0.06

P=0.899

a Study was performed at ILS, Inc. The detailed protocol is presented by MacGregor et al. (1990). NCE=normochromatic erythrocyte; PCE=polychromatic erythrocyte

b Mean ± standard error

c Pairwise comparison with the chamber control group, significant at P≤0.005

d Chamber control

e Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test; significant at P≤0.025

Applicant's summary and conclusion

Conclusions:
Alpha-Pinene was not mutagenic in the mouse peripheral blood micronucleus test
Executive summary:

In a peripheral blood micronucleus test conducted similarly to OECD Guideline 474, alpha pinene was administered through inhalation to groups of B6C3F1 mice (5/sex/dose) at dose levels of 0, 25, 50, 100, 200 or 400 ppm; 5 days/week for 14 weeks. At the end of the study, peripheral blood samples were obtained from mice. Smear slides were air-dried, fixed, stained with fluorescent DNA-specific stain (acridine orange) and scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) per animal. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined.

No increase in the frequency of micronucleated erythrocytes and no significant changes in the percentages of polychromatic erythrocytes were observed in peripheral blood samples in male or female B6C3F1 mice administered alpha-pinene.

Therefore, alpha-pinene was not mutagenic in the mouse peripheral blood micronucleus test.