Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue substante DL-Borneol, the test item is considered to be not mutagenic with or without metabolic activation in any of the tested strains.

In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue substante DL-Borneol, the test item is determined to be not mutagenic.

In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue substante DL-Borneol, the test item is determined to be not mutagenic.

In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue substante D-Borneol, the test item is determined to be not mutagenic.

In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue isobornyl propionate, the test item is determined to be not mutagenic under the test conditions.

In vitro gene mutation study in mammalian cells. Key study: Read-across approach. Based on the read-across approach from the analogue fenchyl acetate, the test item was determined to be not mutagenic either in the presence or in the absence of metabolic activation system under the conditions of a Mouse Lymphoma Assay.

In vitro cytogenicity in mammalian cells/micronucleus study. Data waiving.According to column 2 of REACH Annex VIII, an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted since adequate data from an in vivo test are available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The standard plate incorporation as described by Ames et al. (1975) and modified by Batzinger et al. (1978) and Babish et al. (1983) was used in this research.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Assay 1: Strains TA 97, TA 98 and TA 100 with preincubation time of 20 min both in the presence and absence of 0.5 ml of S9 mix.
Assay 2: Strain TA 100 with S9 activation and incorporation of another sample preincubated for 60 min.

DURATION
- Preincubation period: 20 min

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
Evaluation according to "MR = mean revertants per plate / mean spontaneous revertants per plate" with the following criteria:
Positive: MR greater than 2
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
No evidence of mutagenicity was observed at test conditions for Borneol.
Executive summary:

A bacterial reverse mutation test (Ames test) was performed on test substance Borneol using Salmonella typhimurium strains TA 97, TA 98 and TA 100 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent at a concentration of 1 mg/ml. Positive and negative/solvent controls were included. No evidence of mutagenicity was observed at test conditions.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: No GLP. Equivalent or similar to OECD 471. Documentation insufficient for assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
up to 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incorporation method



Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Under the conditions of the study, DL-Borneol was found to be not mutagenic with or without metabolic activation in any of the tested strains.
Executive summary:

The mutagenic potential of DL-Borneol was assessed in an Ames study following experimental outlines similar to those in OECD Guideline 471 using the plate incorporation method. Salmonella

typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with DL-Borneol at concentrations up to 5000 μg/plate in the presence and absence of metabolic activation (S9 mix). Under the conditions of the study, DL-Borneol is considered not mutagenic in bacteria.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: No data on GLP. Test method not reliable for full assessment of genetic toxicity in bacteria.
Principles of method if other than guideline:
A mutation test in Escherichia coli WP 2 uvrA (trp-) was performed in Borneol and some other synthetic flavoring agents widely used in foodstuffs.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
not specified
Test concentrations with justification for top dose:
Test concentration range: 0.4 to 3.2 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data
Evaluation criteria:
Evaluation in mutation test according to "ratio = maximal revertants / spontaneous revertants" with the following criteria:
Positive: when ratio >=2
Negative: when ratio < 2
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified

The results were as follows:

Reagents

Dose range (mg/plate)

Ratio

Conclusion

Borneol

0.4-3.2

0.9

-

Conclusions:
No evidence of mutagenicity was observed at test conditions for Borneol.
Executive summary:

No mutagenic activity was observed for Borneol in the test mutation using E. Coli WP2 uvrA within a concentration range for the test substance of 0.4 -3.2 mg/plate.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May 2013 - 15 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver)
Test concentrations with justification for top dose:
3h, ± S9 mix: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 µg/mL
3h, + S9 mix: 110; 100; 95; 90; 85; 80; 70; 60; 40; 20; 10 and 5 µg/mL
24h, - S9 mix. 80; 75; 70; 65; 60; 55; 50; 45; 40; 30; 20; 10 and 5 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was non-soluble in Distilled water or Dimethyl sulfoxide at 500 mg/mL concentration. However, a proper formulation could be made at the same concentration using Acetone or Ethanol (96%) as vehicle. Due to the better biocompatibility to the test system, Ethanol (96%) was selected for vehicle of the study.
Untreated negative controls:
yes
Remarks:
(Untreated)
Negative solvent / vehicle controls:
yes
Remarks:
(Ethanol 96%, DMSO)
Remarks:
Test item vehicle: Ethanol (10 µL/mL), positive control vehicle: DMSO (10 µL/mL)
Positive controls:
yes
Remarks:
(Without metabolic activation)
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.15 µg/mL (3h treatment), 0.10 µg/mL (24h treatment), in DMSO
Positive controls:
yes
Remarks:
(With metabolic activation)
Positive control substance:
cyclophosphamide
Remarks:
4 µg/mL in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (RPMI-5 medium)

DURATION
- Exposure duration: 3h (with and without S9-mix), 24h (without S9-mix)
- Expression time (cells in growth medium, to allow expression of the TK- mutation ): 3 days
- Selection time (plating for -trifluorothymidine (TFT) resistance): Two weeks

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment.

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was measured by relative survival

OTHER EXAMINATIONS:
- Determination of Survival or Viability:
After the exposure period and the expression time, cells were also diluted to be plated for survival and viability respectively. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Parameter calculated:
- Relative survival
- Mutant Frequency
- Small and large colony mutant frequencies

Others:
The number of viable cells in the individual samples was counted manually using a haemocytometer.
Measurement of pH and osmolality was performed after the treatment period.
Evaluation criteria:
The test item was considered to be mutagenic in this assay if all the following criteria were met (based on M. Moore 2006):
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.
Statistics:
Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(Assay 1: ±S9mix, 3h: >= 100 µg/L; Assay 3: +S9mix, 3h: >= 95 µg/L; -S9mix, 24h: >= 55 µg/L)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no large changes in pH after treatment.
- Effects of osmolality: There were no large changes in osmolality after treatment.
- Water solubility: No insolubility was detected in the final treatment medium at the end of the treatment (although discoloured medium was detected at some concentrations)

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed to select dose levels for the main assays. During the preliminary test, a 3 hour treatment in the presence and absence of S9-mix and a 24 hour treatment in the absence of S9-mix were performed with a range of test item concentration to determine toxicity. The highest concentration tested in the preliminary experiment was 2500 μg/mL. Insolubility and cytotoxicity were observed in the preliminary experiment. Concentrations for the main experiments were selected to cover the range from cytotoxicity to little or no cytotoxicity according to the instructions of the relevant OECD guideline.

ADDITIONAL INFORMATION:
No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations.
No significant dose-response to the treatment was indicated by the linear trend analysis.

OBSERVATION:
During Assay 2, a contamination was detected in the samples, therefore it was considered invalid and terminated. An additional experiment (Assay 3) was performed using the same experimental conditions as in Assay 2 to provide valid and interpretable data.
Remarks on result:
other: all strains/cell types tested

Mutagenicity Results of Assay 1

S9 mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn)♦

Mutation frequency

+

3

140 µg/mL

ND

ND

ND

ND

ND

130 µg/mL

ND

ND

ND

ND

ND

120 µg/mL

ND

ND

ND

ND

ND

110 µg/mL

ND

ND

ND

ND

ND

100 µg/mL

NE

NE

NE

NE

NE

90 μg/mL

642/768

64/768

62/768

0.047

125.0

80 µg/mL

640/768

71/768

57/768

0.056

124.5

60 µg/mL

640/768

74/768

54/768

0.087

122.7

40 µg/mL

641/768

74/768

53/768

0.009

128.8

20 µg/mL

652/768

68/768

48/768

0.108

143.1

10 µg/mL

654/768

65/768

49/768

0.785

105.8

5 µg/mL

636/768

75/768

57/768

0.003

133.5

Vehicle control

640/768

71/768

57/768

--

131.8

Vehicle control for CP

645/768

64/768

59/768

--

118.3

Untreated control

642/768

64/768

62/768

--

135.2

Positive control
(CP: 4 μg/mL)

277/768

204/768

287/768

♦♦
3.55E-13

1076.7*

In linear trend analysis β2/var (β) = 0.002, not significant.

S9 mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn) ♦

Mutation frequency

-

3

140 µg/mL

ND

ND

ND

ND

ND

130 µg/mL

ND

ND

ND

ND

ND

120 µg/mL

ND

ND

ND

ND

ND

110 µg/mL

ND

ND

ND

ND

ND

100 µg/mL

ND

ND

ND

ND

ND

90 μg/mL

671/768

38/768

59/768

0.710

111.7

80 µg/mL

672/768

42/768

54/768

0.229

100.8

60 µg/mL

652/768

28/768

88/768

0.818

112.6

40 µg/mL

683/768

38/768

47/768

0.139

79.0

20 µg/mL

673/768

39/768

56/768

<0.001

87.4

10 µg/mL

679/768

47/768

42/768

0.094

80.6

5 µg/mL

672/768

36/768

60/768

0.254

101.5

Vehicle control

672/768

52/768

44/768

--

88.0

Vehicle control for NQO

670/768

39/768

59/768

--

100.8

Untreated control

653/768

40/768

75/768

--

119.8

Positive control
(NQO: 0.15 μg/mL)

407/768

199/768

162/768

♦♦
2.54E-12

749.6*

In linear trend analysis β2/var (β) =1.199, not significant.

Mutagenicity Results of Assay 3

S9-mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn)♦

Mutation frequency

+

3

110 µg/mL

ND

ND

ND

ND

ND

100 µg/mL

NE

NE

NE

NE

NE

95 µg/mL

NE

NE

NE

NE

NE

90 μg/mL

NE

NE

NE

NE

NE

85 µg/mL

646/768

67/768

55/768

0.014

120.7

80 µg/mL

655/768

52/768

61/768

<0.001

123.5

70 µg/mL

646/768

59/768

63/768

<0.001

124.2

60 µg/mL

646/768

60/768

62/768

0.276

109.1

40 µg/mL

661/768

50/768

57/768

0.464

103.9

20 µg/mL

661/768

51/768

56/768

0.531

103.2

10 µg/mL

667/768

51/768

50/768

1.148

94.2

62.5 µg/mL

671/768

51/768

46/768

1.564

88.9

Vehicle control

649/768

57/768

62/768

--

124.4

Vehicle control for CP

654/768

66/768

48/768

--

105.1

Untreated control

658/768

57/768

53/768

--

100.4

Positive control
(CP: 4 μg/mL)

183/768

240/768

345/768

♦♦
1.58E-15

1571.2*

In linear trend analysis β2/var (β) = 1.619, not significant.

S9-mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn) ♦

Mutation frequency

-

24

80 µg/mL

ND

ND

ND

ND

ND

75 µg/mL

ND

ND

ND

ND

ND

70 µg/mL

ND

ND

ND

ND

ND

65 µg/mL

ND

ND

ND

ND

ND

60 µg/mL

ND

ND

ND

ND

ND

55 µg/mL

NE

NE

NE

NE

NE

50 μg/mL

684/768

44/768

40/768

0.270

80.2

45 μg/mL

678/768

51/768

39/768

0.245

80.9

40 μg/mL

663/768

51/768

54/768

0.043

97.5

30 μg/mL

672/768

68/768

28/768

0.058

86.7

20 μg/mL

683/768

47/768

38/768

0.049

87.0

10 μg/mL

670/768

49/768

49/768

0.001

93.2

5 μg/mL

668/768

65/768

35/768

<0.001

91.9

Vehicle control

674/768

55/768

39/768

--

92.4

Vehicle control for NQO

678/768

65/768

25/768

--

87.0

Untreated control

681/768

53/768

34/768

--

82.1

Positive control
(NQO: 0.1 μg/mL)

384/768

269/768

115/768

♦♦
2.27E-13

830.8*

In linear trend analysis β2/var (β) =0.359, not significant.

* = Statistically significant.

♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) >5.48(at p<0.05).

♦♦ = Evaluated by T-test for independent samples. Significant at p<0.05.

Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control

var(Dn) = variance of Dn; β = slope of the curve; var(β) = variance of the slope

+ = in the presence of S9-mix

- = in the absence of S9-mix

Vehicle control = Ethanol

Vehicle control for CP = DMSO

CP = Cyclophosphamide

Vehicle control for NQO = DMSO

NQO = 4-Nitroquinoline-N-oxide

DMSO = Dimethyl sulfoxide

ND = no data (No cells were plated for colony growing due to the observed cytotoxicity during treatment or in the expression period.)

NE = not evaluated (Due to the high level of cytotoxicity)

Note: Mutation frequency refers to 106viable cells

The overall study was considered to be valid:

The spontaneous mutation frequency of the negative (vehicle) control was in the recommended range (50-170 mutants per 106 viable cells) in each experiment.

The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays.

The plating efficiencies for the negative (vehicle) control at the end of the expression period (PEviability) were within the acceptable range (65-120 %) in all assays.

The number of test concentrations evaluated for each treatment was at least seven in each case,.

The tested concentration range in the study was considered to be adequate as the highest examined concentration produced approximately 80-90% toxicity (i.e. approximately 10-20% relative survival or relative total growth) and lower test concentrations were evenly spaced by a factor of no more than two. For determination of the cytotoxicity, the relative total growth was taken into consideration, as the test item showed late phase cytotoxicity during the expression period.

Conclusions:
No mutagenic effect was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD 476) to test the potential to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). Ethanol was used as vehicle of the test item in this study. The test item was examined up to 2500 μg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL

Assay 1, 3-hour treatment without metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL

Assay 3, 3-hour treatment with metabolic activation: 110; 100; 95; 90; 85; 80; 70; 60; 40; 20; 10 and 5 μg/mL

Assay 3, 24-hour treatment without metabolic activation: 80; 75; 70; 65; 60; 55; 50; 45; 40; 30; 20; 10 and 5 μg/mL

No insolubility was detected in the final treatment medium at the end of the treatment. There were no large changes in pH or osmolality after treatment. Cytotoxicity was observed in both assays (Assay 1: ± S9mix, 3h: >= 100 µg/L; Assay 3: + S9mix, 3h: >= 95 µg/L; - S9mix, 24h: >= 55 µg/L). No biologically relevant or statistically significant increase in the mutation frequency was observed. No dose response to the treatment was indicated by the linear trend analysis. The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases and the study was considered to be valid. In conclusion, no mutagenic effect was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The standard plate procedure was followed (Ames, McCann and Yamasaki, 1975)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine-requiring gene in Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of liver rats
Test concentrations with justification for top dose:
5 doses (up to 3.6 mg/plate) in all five tester strains ±S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incorporation method:
S-9 liver fractions were prepared from Aroclor-pretreated rats (Aroclor 1254, 500 mg/kg ip) and adjusted to 25 mg protein/ml; 0.5 ml S-9 mix, equivalent to 50 µl S-9, was incorporated into the plates.
Vogel-Bonner medium (Vogel & Bonner, 1956) was used throughout.
Plates were incubated for 48 hr.
- Cell density at seeding (if applicable): Overnight bacterial cultures had cell titres of at least 10^9 cells/ml.

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
Criteria regarded as positive: a reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency.
Criteria regarded as marginally mutagenic under the experimental conditions: reproducible, dose-related and significant (p<=0.01) but less than two-fold elevations.


Statistics:
Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970).
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
No evidence of mutagenicity was observed at test conditions for isobornyl propionate.
Executive summary:

A bacterial reverse mutation test (Ames test) was performed on test substance isobornyl propionate using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA1537 and TA 1538 with and without S9 mix metabolic activation. Five doses up to 3.6 mg/plate of test item were used and 48 h incubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls with sodium azide and benzo(a)pyrene were included. No evidence of mutagenicity was observed at test conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance dextro alpha fenchyl acetate undergoes rapid hydrolysis to acetic acid and dextro alpha fenchyl alcohol which is the target substance.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(Read-across from an analogue)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
(read-across from an analogue determined to be not mutagenic in mammalian cell assay)
Conclusions:
Based on the read-across approach from the analogue fenchyl acetate, the test item was determined to be not mutagenic either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD 476) to test the potential of analogue substance fenchyl acetate to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). Ethanol was used as vehicle of the test item in this study. The test item was examined up to 2500 μg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL

Assay 1, 3-hour treatment without metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL

Assay 3, 3-hour treatment with metabolic activation: 110; 100; 95; 90; 85; 80; 70; 60; 40; 20; 10 and 5 μg/mL

Assay 3, 24-hour treatment without metabolic activation: 80; 75; 70; 65; 60; 55; 50; 45; 40; 30; 20; 10 and 5 μg/mL

No insolubility was detected in the final treatment medium at the end of the treatment. There were no large changes in pH or osmolality after treatment. Cytotoxicity was observed in both assays (Assay 1: ± S9mix, 3h: >= 100 µg/L; Assay 3: + S9mix, 3h: >= 95 µg/L; - S9mix, 24h: >= 55 µg/L). No biologically relevant or statistically significant increase in the mutation frequency was observed. No dose response to the treatment was indicated by the linear trend analysis. The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases and the study was considered to be valid. In conclusion, no mutagenic effect was observed either in the presence or in the absence of metabolic activation system. Based on these results, the read-across was applied and the test item was determined to be not mutagenic under the conditions of this Mouse Lymphoma Assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance isobornyl propionate undergoes rapid hydrolysis to propionic acid and isoborneol which shares the same functional groups with the substance D-alpha fenchol and also has comparable values for the relevant molecular properties.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from supporting substance. All data sources agree to consider the test substance as not mutagenic in bacteria and are sufficient to fulfil the information requirements.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in bacteria
Conclusions:
Based on the read-across approach from the analogue isobornyl propionate, the test item is determined to be not mutagenic under the test conditions.
Executive summary:

A bacterial reverse mutation test (Ames test) was performed on test substance isobornyl propionate using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA1537 and TA 1538 with and without S9 mix metabolic activation. Five doses up to 3.6 mg/plate of test item were used and 48 h incubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls with sodium azide and benzo(a)pyrene were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across was applied and the test item was determined to be not mutagenic under the test conditions.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance DL-Borneol which shares the same functional groups with the substance D-alpha fenchol also has comparable values for the relevant molecular properties.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from supporting substance. All data sources agree to consider the test substance as not mutagenic in bacteria and are sufficient to fulfil the information requirements.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in bacteria
Conclusions:
Based on the read-across approach from the analogue substante DL-Borneol, the test item is determined to be not mutagenic.
Executive summary:

A bacterial reverse mutation test (Ames test) was performed on analogue substance Borneol using Salmonella typhimurium strains TA 97, TA 98 and TA 100 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent at a concentration of 1 mg/ml. Positive and negative/solvent controls were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance DL-Borneol which shares the same functional groups with the substance D-alpha fenchol also has comparable values for the relevant molecular properties.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from supporting substance. All data sources agree to consider the test substance as not mutagenic in bacteria and are sufficient to fulfil the information requirements.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in bacteria
Conclusions:
Based on the read-across approach from the analogue substante DL-Borneol, the test item is considered to be not mutagenic with or without metabolic activation in any of the tested strains.
Executive summary:

The mutagenic potential of analogue substance DL-Borneol was assessed in an Ames study following experimental outlines similar to those in OECD Guideline 471 using the plate incorporation method. Salmonella

typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with DL-Borneol at concentrations up to 5000 μg/plate in the presence and absence of metabolic activation (S9 mix). Under the conditions of the study, DL-Borneol is considered not mutagenic in bacteria. Based on these results, the read-across was applied and the test item is considered to be not mutagenic in the Ames test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance DL-Borneol which shares the same functional groups with the substance D-alpha fenchol also has comparable values for the relevant molecular properties.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from supporting substance. All data sources agree to consider the test substance as not mutagenic in bacteria and are sufficient to fulfil the information requirements.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in bacteria
Conclusions:
Based on the read-across approach from the analogue substante DL-Borneol, the test item is determined to be not mutagenic.
Executive summary:

A mutation test was conducted on the analogue substance DL-Borneol with E. Coli WP2 uvrA (trp-) at a dose range of 0.4 -3.2 mg/plate. No evidence of mutagenicity was observed. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The mutation test was carried out by the preincubation procedure described by Ames et al. (1975). The test chemical was preincubated with S9 mix or phosphate buffer (pH =7.4) for 20 min.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0 (control), 0.001, 0.005, 0.01, 0.05 and 0.1 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation procedure
Strains TA 97 and TA 102 with preincubation time of 20 min both in the presence and absence of S9 mix.

DURATION
- Preincubation period: 20 min

NUMBER OF REPLICATIONS: 3
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid

Table 1. Results of mutation test

Chemical

Solvent

Dose (mg/plate)

Nº revertants/plate a)

TA97

TA102

-S9

+S9

-S9

+S9

D-borneol

DMSO

0.1

128

183

222

437

0.05

152

181

243

412

0.01

123

194

266

448

0.005

129

179

273

436

0.001

120

191

269

424

0

135

196

271

467

9-Aminoacridine

DMSO

50 (µg)

902±257 c)

 

 

 

Mitomycin C

DMSO

0.2 (µg)

 

 

1993±616

 

2-Aminoanthracene

DMSO

5 (µg)

 

1122±365

 

799±99

a) Mean of three plates.

c) Mean and standard deviation (n=17)

Conclusions:
No evidence of mutagenicity was observed at test conditions for D-borneol.
Executive summary:

A bacterial reverse mutation test (Ames test) was performed on test substance D-borneol using Salmonella typhimurium strains TA 97 and TA 102 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls were included. No evidence of mutagenicity was observed at test conditions.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance D-Borneol which shares the same functional groups with the substance D-alpha fenchol also has comparable values for the relevant molecular properties.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from supporting substance. All data sources agree to consider the test substance as not mutagenic in bacteria and are sufficient to fulfil the information requirements.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in bacteria
Conclusions:
Based on the read-across approach from the analogue substante D-borneol, no evidence of mutagenicity is observed on the test item.
Executive summary:

A bacterial reverse mutation test (Ames test) was performed on test substance D-borneol using Salmonella typhimurium strains TA 97 and TA 102 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to column 2 of REACH Annex VIII, an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted since adequate data from an in vivo test are available
Reason / purpose:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In-vivo micronucleus assay: Key study: Read-across approach. Test method OECD 474, GLP study: Based on read-across approach, the test item was determined to be not mutagenic in mice.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 January 1991 - 17 January 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: 28.7 g (males) and 22.8 g (females)
- Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sesame oil
- Concentration of test material in vehicle: 20 % (w/v)
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test sample dilutions were prepared fresh each day. 500 mg test item were weight in a 25 mL flask, mixed with sesame oil and topped up to the calibration mark.
Frequency of treatment:
Once
Post exposure period:
24, 48 and 72 hours (killing times)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
70 animals (35 male and 35 female):
Group 1: 0 mg/kg bw (5 males and 5 females) killing time: 24 h post administration
Group 2: 2000 mg/kg bw (5 males and 5 females) killing time: 24 h post administration
Group 4: 0 mg/kg bw (5 males and 5 females) killing time: 48 h post administration
Group 5: 2000 mg/kg bw (5 males and 5 females) killing time: 48 h post administration
Group 6: 0 mg/kg bw (5 males and 5 females) killing time: 72 h post administration
Group 7: 2000 mg/kg bw (5 males and 5 females) killing time: 72 h post administration
Control animals:
yes, concurrent vehicle
Positive control(s):
Endoxan(R). Group 3 (5 males and 5 females) killing time: 24 h post administration
- Route of administration: oral, gavage
- Doses / concentrations: 50 mg/kg bw (0.5% w/v in distilled water , 10 mL/kg bw)
Tissues and cell types examined:
Bone marrow erythrocytes cells (from both femora)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Preliminary studies were conducted to determine the highest administrable non lethal dose level. 3 mice per sex and per dose were exposed to 5000, 4000, 3000 and 2000 mg/kg bw test item.

TREATMENT AND SAMPLING:
After treatment, animals were killed by carbon dioxide asphyxiation 24, 48 and 72 hours after application.

For each animal, about 3 mL foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged (5 min, 1200 rpm) and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide and air-dried for 24 hours. The slides were then stained.

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was determined.
Statistics:
The number of polychromatic erythrocytes with micronuclei and the number of normocytes with micronuclei were evaluated statistically. The comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase). The results of the treatment groups were compared with the corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). All statistical results were based on a 95% level of significance.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
(see below)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000, 4000, 3000 and 2000 mg/kg
- Mortality:
At 5000 mg/kg bw: 1/3 males and 2/3 females died;
At 4000 mg/kg bw: 0/3 males and 1/3 females died;
At 3000 mg/kg bw: 0/3 males and 3/3 females died;
At 2000 mg/kg bw: No death were observed.
- Clinical signs of toxicity in test animals: Several clinical signs were observed at 3000-5000 mg/kg bw doses. At 2000 mg/kg bw, uncoordinated gait, increased spontaneous activity and stilted gait was observed.

RESULTS OF DEFINITIVE STUDY
- Mortality: All animals survived.
- Signs of toxicity: uncoordinated gait, increased spontaneous activity. These signs were fully reversible by 5-6 hours after application.
- Induction of micronuclei (for Micronucleus assay): The number of polychromatic (PCE) and normochromatic (NCE) erythrocytes containing micronuclei was not increased.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE/NCE in both male and female animals remained unaffected by the treatment, and was statistically not different from the control values.
- Positive control: Endoxan induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. the ratio of PCE/NCE was not changed to a significant extent.

Summary of findings in bone marrow erythrocytes:

Sex

Dose (mg/kg)

Sample time

No. animals

Erythrocytes

Erythrocytes with micronuclei

Poly mean

Normo

mean

 mean

No

%

 

Mut I

No

%

 

Mut I

Male

0

24 h

5

1000

1000

0.92

2

0.16

I

1.0

1

0.08

I

1.0

2000

5

1000

1000

1.07

2

0.18

-I

1.1

1

0.08

-I

1.0

Endoxan

5

1000

1000

0.82

23

2.32

*A

14.5

1

0.14

-I

1.7

Female

0

5

1000

1000

1.03

1

0.12

I

1.0

1

0.08

I

1.0

2000

5

1000

1000

0.86

1

0.08

-I

0.7

0

0.04

-I

0.5

Endoxan

5

1000

1000

0.79

21

2.10

*A

17.5

2

0.22

*A

2.8

Male

0

48 h

5

1000

1000

0.90

2

0.20

I

1.0

2

0.16

I

1.0

2000

5

1000

1000

0.78

1

0.08

-I

0.4

0

0.04

-I

0.2

Female

0

5

1000

1000

0.96

1

0.12

I

1.0

1

0.08

I

1.0

2000

5

1000

1000

0.86

1

0.14

-I

1.2

0

0.04

-I

0.5

Male

0

72 h

5

1000

1000

1.06

2

0.18

I

1.0

0

0.04

I

1.0

2000

5

1000

1000

1.08

1

0.10

-I

0.6

1

0.10

-I

2.5

Female

0

5

1000

1000

1.05

2

0.20

I

1.0

1

0.08

I

1.0

2000

5

1000

1000

1.20

1

0.14

-I

0.7

0

0.04

-I

0.5

Mut. I = Mutagenic index

- = No difference from control (P>0.05

I = within the normal range

* = Significantly different from control (P<0.05)

A = Outside the normal range

Conclusions:
Isobornyl acetate was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.
Executive summary:

An in-vivo micronucleus test was performed with isobornyl acetate according to OECD 474. Five mice per sex and per group (70 in total) were exposed to a single dose of test item at 2000 mg/kg bw, based on preliminary results. Cyclophosphamide was used as a positive control (5 mice per sex). Animals were killed 24, 48 and 72 hours after administration of the test compound. For each animal, bone marrow smears were flushed from both femora and the slides were prepared for erythrocyte micronuclei observation. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment and was statistically not different from the control values, indicating no mutagenicity. Based on these results, isobornyl acetate was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance isobornyl acetate undergoes rapid hydrolysis to acetic acid and isoborneol which shares the same functional groups with the substance D-alpha fenchol and also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in the mammalian erythrocyte micronucleus test
Conclusions:
Based on the read-across approach from the analogue Isobornyl acetate, the test item was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.
Executive summary:

A mammalian in-vivo erythrocyte micronucleus test was performed with isobornyl acetate according to OECD 474. Five mice per sex and per group (70 in total) were exposed to a single dose of test item at 2000 mg/kg bw, based on preliminary results. Cyclophosphamide was used as a positive control (5 mice per sex). Animals were killed 24, 48 and 72 hours after administration of the test compound. For each animal, bone marrow smears were flushed from both femora and the slides were prepared for erythrocyte micronuclei observation. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment and was statistically not different from the control values, indicating no mutagenicity. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.

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

Additional information

In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. The mutagenic potential of analogue substance DL-Borneol was assessed in an Ames study following experimental outlines similar to those in OECD Guideline 471 using the plate incorporation method. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with DL-Borneol at concentrations up to 5000 μg/plate in the presence and absence of metabolic activation (S9 mix). Under the conditions of the study, DL-Borneol is considered not mutagenic in bacteria. Based on these results, the read-across was applied and the test item is considered to be not mutagenic in the Ames test.

In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. A mutation test was conducted on the analogue substance DL-Borneol with E. Coli WP2 uvrA (trp-) at a dose range of 0.4 -3.2 mg/plate. No evidence of mutagenicity was observed. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.

In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. A bacterial reverse mutation test (Ames test) was performed on analogue substance Borneol using Salmonella typhimurium strains TA 97, TA 98 and TA 100 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent at a concentration of 1 mg/ml. Positive and negative/solvent controls were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.

In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. A bacterial reverse mutation test (Ames test) was performed on test substance D-borneol using Salmonella typhimurium strains TA 97 and TA 102 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.

In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. A bacterial reverse mutation test (Ames test) was performed on test substance isobornyl propionate using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA1537 and TA 1538 with and without S9 mix metabolic activation. Five doses up to 3.6 mg/plate of test item were used and 48 h incubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls with sodium azide and benzo(a)pyrene were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across was applied and the test item was determined to be not mutagenic under the test conditions.

In vitro gene mutation study in mammalian cells. Key study: Read-across approach. An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD 476) to test the potential of analogue substance fenchyl acetate to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). No mutagenic effect was observed either in the presence or in the absence of metabolic activation system. Based on these results, the read-across was applied and the test item was determined to be not mutagenic under the conditions of this Mouse Lymphoma Assay.

In vitro cytogenicity in mammalian cells/micronucleus study. Data waiving.According to column 2 of REACH Annex VIII, an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted since adequate data from an in vivo test are available.

In-vivo micronucleus assay: Key study: Read-across approach. A mammalian in-vivo erythrocyte micronucleus test was performed with isobornyl acetate according to OECD 474. Five mice per sex and per group (70 in total) were exposed to a single dose of test item at 2000 mg/kg bw, based on preliminary results. Cyclophosphamide was used as a positive control (5 mice per sex). Animals were killed 24, 48 and 72 hours after administration of the test compound. For each animal, bone marrow smears were flushed from both femora and the slides were prepared for erythrocyte micronuclei observation. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment and was statistically not different from the control values, indicating no mutagenicity. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.

Justification for classification or non-classification

Based on the available information, the substance is considered to be negative for genetic toxicity, and therefore the substance is not classified in accordance with CLP Regulation (EC) no 1272/2008.