Cineole

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Between 22 September and 26 October 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

To address toxicological endpoints as part of the REACH registration of Cineole (Target Substance) it is proposed to read-across to Clarycet (Source Substance). The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised in using the categories and databases present in the OECD (Q)SAR Toolbox. From the profile, it can be seen that the two substances share structural similarities and also "mechanistic action" similarities which are both general and endpoint specific. Therefore read-across is justified.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydro-4-methyl-2-propyl-2H-pyran-4-yl acetate

IUPAC Name:

Tetrahydro-4-methyl-2-propyl-2H-pyran-4-yl acetate

Constituent 2

Reference substance name:

Clarycet

IUPAC Name:

Clarycet

Constituent 3

Reference substance name:

131766-73-9

EC Number:

603-508-6

Cas Number:

131766-73-9

IUPAC Name:

131766-73-9

Details on test material:

- Name of test material (as cited in study report): Clarycet
- Structural formula attached as image file (if other than submission substance): see Fig.
- Molecular weight (if other than submission substance): 200.28
- Smiles notation (if other than submission substance): C1(C)(OC(C)=O)CC(CCC)OCC1
- Physical state: Liquid
- Analytical purity: > 94 %; 45 - 60 % trans, 40 - 55 % cis.
- Lot/batch No.: 6370
- Expiration date of the lot/batch: December 1993
- Storage condition of test material: Room temperature in darkness

Test animals

Species:

rat

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate, Kent, England.
- Age at study initiation: 25 days old on dispatch
- Weight at study initiation: 22 - 24 g
- Assigned to test groups randomly: Yes
- Fasting period before study: Overnight
- Housing: In plastic disposable cages
- Diet (e.g. ad libitum): Pelleted Biosure LAD 1 rodent diet, ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

1 % methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions/emulsions of the test substance were prepared in 1 % methylcellulose on the morning of the test. Stability and homogeneity of the test compound and of the test compound in the vehicle were not determined. Chemical analysis of dosing formulations for acheived concentration was not performed.
All groups were dosed with the standard volume of 20 mL/kg bodyweight.

Duration of treatment / exposure:

Single administration

Frequency of treatment:

Single administration

Post exposure period:

None specified

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1080, 1800, 3000 and 5000 mg/kg
Basis:
actual ingested
Phase 1 preliminary toxicity study

Doses / concentrations 2

Remarks:

Doses / Concentrations:
750, 1050, 1470 and 2058 mg/kg
Basis:
actual ingested
Phase 2 preliminary toxicity study

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1600 mg/kg
Basis:
actual ingested
micronucleus test

No. of animals per sex per dose:

2/sex/dose in the preliminary toxicity study
15/sex/dose in the micronucleus study

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C was used as the positive control compound. It was prepared as a solution in 0.9 % saline at a concentration of 0.6 mg/mL just prior to administration in the preliminary toxicity study. In the micronucleus study the positive control was administered to 5 animals/sex/dose at a dosage of 12 mg/kg.

Examinations

Tissues and cell types examined:

The animals were examined regularly for mortalities or clinical signs of reaction.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Both femurs were dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. A direct bone marrow smear was made onto a slide containing a drop of calf serum. One smear was made from each femur. The prepared smears were fixed in methanol (>10 minutes). After air-drying the smears were stained for 10 minutes in 10 % Giemsa (prepared by 1:9 dilution of Gurr's improved R66 Giemsa with distilled water). Following rinsing in distilled water and differentiation in buffered distilled water (pH 6.8), the smears were air dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS:
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal.
The ratio of polychromatic erythrocytes for each animal was assessed by examiantion of at least 1000 erythrocytes. A record of the number of micronucleated normochromatic erythrocytes observed during assessment of this ratio was also kept.

Evaluation criteria:

A positive response is normally indicated by a substantial, statistically significant increase in the incidence of micronucleated polychromatic erythrocytes compared to the incidence for the concurrent vehicle control group for at least one of the sampling times. In borderline cases, e.g. where individual or group mean values for not fall outside historical control ranges, further slide reading or testing may be necessary.
Boen marrow toxicity (or depression) is normally indicated by a substantial, statistically significant decrease in the ratio of polychromatic to normochromatic erythrocytes. This decrease would normally be evident at both the 48 and 72 hour sampling points, a decrease at the 24 h time point is not necessarily expected because of the relatively long transition time of erythroid cells. A very large decrease in this ratio would be indicative of a cytotoxic effect.

Statistics:

Non-parametric statistical methods, based on rank are chosen for analysis of results because:
a) They are suited to analysis of data consisting of discrete/integer values such as the incidence of micronucleated polychromatic erythrocytes.
b) The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal disctribution, the results of non-parametric analysis and classical analysis of variance are very similar).
c) "Outliers" are frequently found in the polychromatic erythrocyte to normochromatic erythrocyte ratios for both control and treated animals; non-parametric analysis does not give such values an undue weighting.
For a comparison of an individual treated group with a concurrent control group, Wilcoxon's sum of ranks is used.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Two male and five female animals died after treatment with the test substance in the micronucleus test.

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: Two male and five female animals died after treatment with the test substance in the micronucleus test. At post mortem examination, none of the animals showed signs of misdosing. These animals were replaced by animals from the concurrently treated satellite group. No adverse clinical signs were obtained for the vehicle control or positive control treated animals over the duration of the test.


RESULTS OF DEFINITIVE STUDY
- Statistical evaluation:
The test substance did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes at any of the three sampling times (P > 0.01 using Wilcoxon's sum of ranks test).
Mitomycin C caused large, highly significant increases (P<0.001) in the frequency of micronucleated polychromatic erythrocytes.
The test substance did not cuase any substantial increases in the incidence of micronucleated normochromatic erythrocytes at any of the three sampling times.
A small but statistically singificant decrease in the ratio of polychromatic to normochromatic erythrocytes was obtained at the 72 hour sampling time after treatment of animals with the test substance (P<0.001 using Wilcoxon' sum of ranks test). This decrease may be indicative of slight bone marrow depression induced by this relatively high acute dose of the test substance.

Mitomycin C did not cause any statistically significant decreases in the ratio (P>0.01). It should be noted that even very cytotoxic compounds such as Mitomycin C do not always produce a substantial decrease in this ratio as early as the 24 h sampling time because of the lag caused by erythrocyte maturation.

Any other information on results incl. tables

Summary of results - group totals/means for the entire experiment and results of statistical analysis

Sampling time	Treatment	Dose (mg/kg)	Ratio p/n§	Incidence mnp	Incidence mnn (total)
24 h	Vehicle control		0.972	0.7	0.0
	Test substance	1600	1.077ns	1.0ns	0.4
	Mitomycin C	12	0.894ns	39.9**	1.3
48 h	Vehicle control	-	1.171	0.3	0.2
	Test substance	1600	0.945ns	0.9ns	04.4
72 h	Vehicle control	-	1.157	0.7	0.2
	Test substance	1600	0.754**	1.3	1.5

p/n = Ratio of polychromatic to normchromatic erythrocytes

mnp = Number of micronucleated cells onserved per 1000 polychromatic erythrocytes

mnn = Number of micronucleated cells observed per 1000 normochromatic erythrocytes

 

§ Very small apparent errors of +/- 0.001 may occur in the mean p/n value due to rounding absolute p/n ratios for presentation in the tables.

Results of statistical analysis using Wilcoxon's sum of ranks test (one-side probabilities):

ns P> 0.01

* P<0.01

** P<0.001

To address toxicological endpoints as part of the REACH registration of Cineole (Target Substance) it is proposed to read-across to Clarycet (Source Substance).

The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible.

The Target Substance and Source Substance have been characterised in using the categories and databases present in the OECD (Q)SAR Toolbox. From the profile, it can be seen that the two substances share structural similarities and also "mechanistic action" similarities which are both general and endpoint specific.

Therefore read-across is justified.

See Section 13 document, Read Across Justification_Clarycet

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The results of an in vivo genetic toxicity study on the structural analogue, Clarycet, are provided as part of a weight of evidence. Since the test substance did not cause any substantial increase in the incidence of micronucleated polychromatic erythrocytes, it is concluded that the test substance did not show any evidence of causing chromosome damage when administered orally in an in vivo test.

Executive summary:

This study was designed to assess the potential induction of micronuclei in bone marrow cells of mice.

Mice were treated with a single acute oral adminsitration of the test agent by intragastric gavage at a dosage of 1600 mg/kg. A preliminary toxicity test had previously shown this level to be approximately the maximum tolerated dosage.

Negative and positive control groups were dosed in an identical manner, orally by intragastric gavage. The negative control group received the vehicle, aqueous 1 % methylcellulose. The positive control group was treated with mitomycin C at 12 mg/kg bodyweight.

Bone marrow smears were obtained from five male and five female animals in the negative control and test compound groups at each of three sampling times; these being 24, 48 or 72 hours after dosing. Bone marrow smears were obtained from the positive control group 24 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

At all sampling times, mice treated with the test substance did not show any signifcant increase in the frequency of micronucleated polychromatic erythrocytes.

A slight but statistically significant decrease in the ratio of polychromatic to normochromatic erythrocytes was obtained at the 72 h sampling time after treatment of the animals with the test substance. This decrease may be indicative of slight bone marrow depression induced by the test substance.

The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated polychromatic erythrocytes.

It is concluded that the test substance has not shown any evidence of causing chromosome damage in this in vivo test.