Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
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 Cyclamen Aldehyde Extra (Target substance) It is proposed to read across to Florhydral (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 QSAR toolbox . From the profiling in this table , 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:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: colourless liquid
Details on test material:
- Name of test material (as cited in study report): Ro 82-4130/000
- Molecular formula (if other than submission substance): C13H18O
- Molecular weight (if other than submission substance): 190
- Smiles notation (if other than submission substance): CC(C)C1=CC(=CC=C1)C(C)CC=O
- InChl (if other than submission substance):
InChI: InChI=1S/C13H18O/c1-10(2)12-5-4-6-13(9-12)11(3)7-8-14/h4-6,8-11H,7H2,1-3H3
InChI key: InChIKey=OHRBQTOZYGEWCJ-UHFFFAOYSA-N
- Substance type: Fragrance ingredient
- Physical state: colourless liquid
- Analytical purity: 97.9%
- Purity test date: 12/03/1991
- Lot/batch No.: 185809, Givaudan Vernier
- Structure: image attached below

Test animals

Species:
mouse
Strain:
other: Füllinsdorf Moro Albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Füllinsdorf Moro Albino mice of a closed randomized outbred strain. The mice were bred under SPF conditions and kept under conventional conditions during the experiment.
- Source: Biological Research Laboratories Ltd., CH-4414 Füllinsdorf, Switzerland.
- Age at study initiation: no data
- Weight at study initiation: Females: 34.3g, Males: 40.2g (mean)
- Assigned to test groups randomly: randomly upon reciept.
- Fasting period before study: no data
- Housing: Macrolon cages, in fully-aircontrolled animal room under periodic bacteriological control.
- Diet (e.g. ad libitum): Kilba 343 cubes (GLP 85/91), ad libitum
- Water (e.g. ad libitum):tap water in drinking bottles, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ~22°C ±2C
- Humidity (%): ~55% ± 10%
- Air changes (per hr): 20-25 cycles per hour
- Photoperiod (hrs dark / hrs light): Fluorescent tubes, 12h dark / 12h light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
no data ("standard suspension vehicle" is stated in the report)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
doses delivered by gavage in a volume of 10 ml/kg SSV

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

Duration of treatment / exposure:
single dose
Frequency of treatment:
singel dose
Post exposure period:
24 hours, 48 hours, and 72 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Treated:
0 mg/kg - 18M/18F
1000 mg/kg - 6M/6F
2000 mg/kg - 18M/18F

Evaluated:
0 mg/kg - 15M/15F
1000 mg/kg - 5M/5F
2000 mg/kg - 15M/15F
Control animals:
yes, concurrent no treatment
Positive control(s):
Procarbazine-HCL
- Justification for choice of positive control(s): no data
- Route of administration: oral
- Doses / concentrations: 50 mg/kg
- Vehicle: Phosphate buffered saline (PBS)

Treated:
50 mg/kg - 6M/6F
Evaluated:
50 mg/kg - 5M/5F

Examinations

Tissues and cell types examined:
Bone marrow smears from three different sampling times (24, 48, 72 hours) were evaluated after administration of the higher dose (2000mg/kg) and one sampling time (24 h) was evaluated for the low dose (1000mg/kg).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
24h: 0mg/kg & 1000mg/kg & 2000mg/kg
48h: 0mg/kg & 2000mg/kg
72h: 0mg/kg & 2000mg/kg

DETAILS OF SLIDE PREPARATION: Both femurs were dissected from each animal. The marrow was flushed out by a serum jet from a syringe needle injected into the marrow canal after dissection of the femoral condyli. The cells were suspended in serum and centrifuged for 5 minutes at 1250 rpm before making bone marrow smears onto slides. At least two slides per animal were prepared. The smears were air dried, fixed and stained with may- Grünvald-Giemsa (Heddle et al., 1984; Schmid, 1975)

METHOD OF ANALYSIS: The slide were coded and 1000 PCE per animal were checked for the existence of MN. The ration of PCE/NCE was determined on 1000 counts of erythrocytes. The MN-NCE were recorded while counting the 1000 PCE per animal and the frequency of MN-NCE should be excluded from the evaluation because the kinetics of the maturing process of erythrocytes makes it likely that the MN appearing in those animals are not substance related.

Evaluation criteria:
The slide were coded and 1000 PCE per animal were checked for the existence of MN. The ration of PCE/NCE was determined on 1000 counts of erythrocytes. The MN-NCE were recorded while counting the 1000 PCE per animal and the frequency of MN-NCE should be excluded from the evaluation because the kinetics of the maturing process of erythrocytes makes it likely that the MN appearing in those animals are not substance related.
Statistics:
The observed yields of MN-PCE were evaluated by means of the Mann-Whitney-U-Test. The algorithm is described in detail by Lüdin (1985). For a meaningful statistical analysis is it recommended that at least 1000 PCE should be scored per animal.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
no observed clastogenic or statisically significant effects
Toxicity:
yes
Remarks:
3/18 mice did not survive the 2000mg/kg dose
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Since no LD50 data were reported fo mice (LD50 for rats >2000mg/kg [Bremner, 1988]) a preliminary experiment was performed using 3 animals per treatment group. Doses rangig from 2000-6000 mg/kg were administered. All animals died after administration of 5000 and 6000 mg/kg while 1 of 3 animals died after administration of 3000 or 4000 mg/kg. At 2000 mg/kg all animals survived the treatment, therefore thos dose was selected as the top dose to be tested. In the main experiment 3/18 mice did not survive the administration of 2000 mg/kg, it is therefore documenterd that no higher doses could have been evaluated.
- Dose range: 2000, 3000, 4000, 5000, 6000 mg/kg


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): polychromatic erythrocytes containing micronuclei
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay):
- Appropriateness of dose levels and route:
- Statistical evaluation:

The effects of orally administered Ro 82-4130/000 on the incidence of polychromatic erythrocytes containing micronuclei (MN-PCE) in the mouse bone marrow at three sampling times (24,48 and 72 hours) are presented in the tables below.
The compound showed lethal effects at a dose of 2000 mg/kg: 3 of 36 animals died after administration of the test substance. However no indication of bone marrow toxicity in terms of the reduction of the PCE/NCE ration was evidenced.
24 hours after application, bone marrow smears were prepared from the mice treated with 1000 and 2000 mg/kg. The results shown in the 24 hour table below did not raise the frequency of MN-PCE.
Additionally, mice treated with 2000 mg/kg were sacrificed 48 and 72 hours after application. The data of the 48 h sampling time are presented in the 48 hour table and those of the 72 h sampling time in 72 hour table below. These also show no increase in the rate of MN-PCE.
The validity of the presented data was confirmed by the fact that:
- The incidence of normochromatic erythrocytes containing micronuclei (MN-NCE) was in the normal range throughout the control and treated animals.
- The spontaneous frequency of MN-PCE was in the standard range of historical control data possessed by the testing laboratory.
The sensitivity of the in vivo test system was demonstrated by the response of the positive control animals which received a single dose of the 50 mg/kg Procarbazine-HCl. The percentage of MN-PCE was significantly increased 24 hours after application if compared to the concurrent negative control animals (see the positive control table below)
Consequently the tested doses of 1000 and2000 mg/kg did not raise the frequency of MN-PCE. It is therefore concluded that no chromosome-breaking or spindle-disturbances producing activity was demonstrated at any of the sampling times.

Any other information on results incl. tables

Micronucleus Test with Füllinsdorf Albino Mice (SPF) Treated with RO 82-4130/000 (Florhydral).

Oral: Gavage - Sampling time: 24 hours

Single Dose (mg/kg) Animal NCE with MN Ratio PCE/NCE Median PCE with MN Median + Significance Levels
No. Sex No. % No. %
0 111 m 2 0.22 1.08 1.38 0 0 0.3
112 m 4 0.47 1.17 4 0.4
113 m 2 0.22 1.11 3 0.3
114 m 1 0.14 1.43 3 0.3
115 m 3 0.4 1.33 4 0.4
117 f 2 0.32 1.58 3 0.3
118 f 2 0.32 1.58 2 0.2
119 f 2 0.45 2.25 5 0.5
1110 f 2 0.41 2.06 7 0.7
1111 f 0 0 1.33 1 0.1
1000 211 m 3 0.72 2.4 1.54 3 0.3 0.3 (n.s.)
212 m 3 0.46 1.53 3 0.3
213 m 5 0.47 0.95 10 1
214 m 3 0.38 1.26 8 0.8
215 m 0 0 1.73 3 0.3
217 f 0 0 1.25 1 0.1
218 f 0 0 1.8 6 0.6
219 f 2 0.24 1.2 3 0.3
2110 f 1 0.17 1.66 4 0.4
2111 f 0 0 1.56 2 0.2
2000 311 m 2 0.25 1.26 1.31 4 0.4 0.4 (n.s.)
312 m 8 0.82 1.02 1 0.1
314 m 10 0.6 0.6 6 0.6
315 m 2 0.3 1.48 6 0.6
316 m 1 0.14 1.4 4 0.4
318 f 0 0 1.32 5 0.5
319 f 2 0.25 1.23 1 0.1
3110 f 2 0.26 1.31 3 0.3
3111 f 1 0.17 1.67 8 0.8
3112 f 1 0.18 1.84 4 0.4

No. of PCE scored per animal: 1000

n.s. = no significance

Micronucleus Test with Füllinsdorf Albino Mice (SPF) Treated with RO 82 -4130/000 (Florhydral).

Oral: Gavage - Sampling time: 48 hours

Single Dose (mg/kg) Animal NCE with MN Ratio PCE/NCE Median PCE with MN Median + Significance Levels
No. Sex No. % No. %
0 121 m 2 0.32 1.61 1.31 4 0.4 0.3
122 m 4 0.52 1.3 2 0.2
123 m 2 0.23 1.14 4 0.4
124 m 2 0.26 1.31 3 0.3
125 m 1 0.11 1.09 1 0.1
127 f 3 0.4 1.32 5 0.5
128 f 1 0.09 0.87 3 0.3
129 f 4 0.69 1.72 2 0.2
1210 f 0 0 1.67 3 0.3
1211 f 0 0 1.53 3 0.3
2000 321 m 6 0.56 0.93 1.19 14 1.4 0.25 (n.s.)
323 m 3 0.24 0.79 7 0.7
324 m 0 0 1.4 2 0.2
325 m 0 0 1.18 0 0
326 m 0 0 1.4 2 0.2
327 f 1 0.15 1.48 3 0.3
328 f 4 0.26 0.66 2 0.2
329 f 4 0.53 1.33 3 0.3
3210 f 2 0.24 1.19 2 0.2
3211 f 1 0.12 1.19 5 0.5

No. of PCE scored per animal: 1000

n.s. = no significance

Micronucleus Test with Füllinsdorf Albino Mice (SPF) Treated with RO 82 -4130/000 (Florhydral).

Oral: Gavage - Sampling time: 72 hours

Single Dose (mg/kg) Animal NCE with MN Ratio PCE/NCE Median PCE with MN Median + Significance Levels
No. Sex No. % No. %
0 131 m 1 0.12 1.16 1.16 1 0.1 0.35
132 m 2 0.42 2.12 4 0.4
133 m 3 0.34 1.14 5 0.5
134 m 4 0.5 1.25 3 0.3
135 m 3 0.28 0.93 3 0.3
137 f 1 0.14 1.38 7 0.7
138 f 1 0.17 1.72 2 0.2
139 f 4 0.47 1.17 4 0.4
1310 f 2 0.21 1.06 1 0.1
1311 f 2 0.23 1.16 4 0.4
2000 331 m 3 0.25 0.84 1.29 7 0.7 0.25 (n.s.)
332 m 0 0 1.72 3 0.3
333 m 0 0 1.36 1 0.1
334 m 5 0.34 0.69 2 0.2
335 m 4 0.39 0.97 1 0.1
337 f 0 0 2.21 8 0.8
338 f 2 0.29 1.44 1 0.1
339 f 1 0.12 1.21 8 0.8
3310 f 1 0.1 0.96 2 0.2
3311 f 0 0 2.32 4 0.4

No. of PCE scored per animal: 1000

n.s. = no significance

Micronucleus Test with Füllinsdorf Albino Mice (SPF) Treated with RO 04 -6467/001(Procarbazine HCl).

Oral: Gavage - Sampling time: 24 hours

Single Dose (mg/kg) Animal NCE with MN Ratio PCE/NCE Median PCE with MN Median + Significance Levels
No. Sex No. % No. %
0 111 m 2 0.22 1.08 1.38 0 0 0.3
112 m 4 0.47 1.17 4 0.4
113 m 2 0.22 1.11 3 0.3
114 m 1 0.14 1.43 3 0.3
115 m 3 0.4 1.33 4 0.4
117 f 2 0.32 1.58 3 0.3
118 f 2 0.32 1.58 2 0.2
119 f 2 0.45 2.25 5 0.5
1110 f 2 0.41 2.06 7 0.7
1111 f 0 0 1.33 1 0.1
50 411 m 2 0.35 1.73 1.41 67 6.7 6.35 xx(+)
412 m 3 0.27 0.91 39 3.9
413 m 3 0.32 1.06 29 2.9
414 m 3 0.46 1.5 51 5.1
415 m 1 0.17 1.69 45 4.5
417 f 4 0.38 0.94 34 3.4
418 f 4 0.64 1.59 32 3.2
419 f 4 0.52 1.3 16 1.6
4110 f 3 0.52 1.72 45 4.5
4111 f 2 0.19 0.95 24 2.4

No. of PCE scored per animal: 1000

n.s. = no significance

x = for P =< 0.05

xx for P=<0.01

(+) = increasing trend

(-) = decreasing trend

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions described in this report Ro 82-4130/000 (Florhydral) does not induce clastogenic or stathmokintec effects in vivo.
Executive summary:

The fragrance ingredient Ro 82-4130/000 Flohydral was evaluated for its ability to induce chromosome breakage or spindle disturbances in vivo using the micronucleus assay in mouse bone marrow.

Doses of 1000 mg/kg and 2000 mg/kg bodyweight were evaluated. The test compound was administered by oral gavage. Some animals did not survive the administration of the top dose. However no antiproliferative effect on bone marrow cells in terms of a reduction of the PCE/NCE ratio was observed.

Bone marrow smears from three different sampling times (24, 48, 72 hours) were evaluated after administration of the higher dose and one sampling time (24 hours) was evaluated for the low dose.

Ro 82-4130/000 did not raise the frequency of MN-PCE and therefore no chromosome-breaking of spindle-disturbances producing activity was demonstrated at any of the sampling times.

The sensitivity of the test system was demonstrated by using procarbazine hydrochloride as a positive control substance. It increased the number of micronucleated polychromatic erythrocytes significantly.

It can be concluded that under the experimental conditions described in the report Ro 82-4130/000 (Florhydral) does not show genotoxic activity in mouse bone marrow cells.