Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl propionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Isobornyl propionate is negative in the Ames test in an OECD TG 471, where the E.coli strain is missing, additional Ames test information from Cyclacet is presented to complete this endpoint.

Isobornyl propionate is negative in the Mouse Lymphoma assay based on read across from Cyclacet, which was tested in an OECD TG 476.

Isobornyl propionate is negative in the chromosome aberration assay based on read across from Cyclobutanate, which was tested in the OECD TG 473.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Incomplete strain selection/ test concentrations not cleary specified and top-dose is below the recommended maximum concentration without a clear justification

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

5 concentrations up to 3.6 mg/ plate

Vehicle / solvent:

Water or, if poorly soluble in water, DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The standard plate procedure was followed (Ames, McCann & Yamasaki, 1975)

DURATION
Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
At least two

Evaluation criteria:

Results that met the following criteria were regarded as positive:
- a reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency.
- Agents producing reproducible, dose-related and significant (P ≤ 0.01) but less than two-fold elevations were classified as marginally mutagenic under the experimental conditions

Statistics:

Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970).

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA98 and 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 applicable

Positive controls validity:

valid

Conclusions:

Under the conditions of the test, the test substance is not mutagenic in the Ames test in absence and presence of metabolic activation.

Executive summary:

The mutagenic activity of the test substance was evaluated in a study equivalent to OECD 471. The substance was added by principles of the standard plate procedure at five dose levels up to 3.6 mg/plate in absence and presence of Aroclor induced rat liver S9. The five tester strains included Salmonella typhimurium TA 1535, TA 100, TA 1537, TA1538 and TA98, and water or DMSO was used as vehicle. No data on cytotoxicity was provided. Sodium azide and benzo[a]pyrene were taken along as positive controls at dose levels of 0.5 µg/plate and 5 µg/plate, respectively. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 2

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a guideline study

Justification for type of information:

The information is based on read across from Cyclacet. The read across rationale is presented in the Genetic toxicity Endpoint summary. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Result obtained from read-across substance Cyclacet (EC 911-369-0)

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of 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: read-across from a guideline study

Justification for type of information:

The chromosomal aberration toxicity is based on read across from Cyclobutanate. The read across rationale is presented in the Genetic toxicity Endpoint summary. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Result obtained from read-across substance Cyclabutanate (CAS 113889-23-9)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Isobornyl propionate was negative when tested in an in vivo micronucleus study in NMRI mice using i.p dosing (similar to OECD TG 474, but not fully according to current guidelines).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

In a micronucleus test groups of male and female NMRI mice, 4 per dose (10-14 weeks old) were treated once intraperitoneally at dose levels of 841, 1893 and 2944 mg/kg in olive oil. Control animals were dosed with olive oil alone. Mice from each dose level were sacrificed at 30 hours, the bone marrow extracted and smear preparations made and stained according to the method of Schmid (1976). Polychromatic erythrocytes were then scored for the presence of micronuclei.

GLP compliance:

no

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: lvanovas GmbH, Kisslegg
- Age at study initiation: 10-14 weeks
- Diet: standard chow (Altromin) ad libitum
- Water: ad libitum

Route of administration:

intraperitoneal

Vehicle:

Vehicle used: Olive oil

Duration of treatment / exposure:

Single intraperitoneal dose

Frequency of treatment:

Once

Post exposure period:

The mice were killed and bone-marrow smears were prepared 30 hr after treatment.

Dose / conc.:

841 mg/kg bw (total dose)

Dose / conc.:

1 893 mg/kg bw (total dose)

Dose / conc.:

2 944 mg/kg bw (total dose)

No. of animals per sex per dose:

4 animals in total per dose group, ratio of male:female not specified

Control animals:

yes, concurrent vehicle

Positive control(s):

no data

Tissues and cell types examined:

Bone marrow was extracted and smear preparations were made and stained. Polychromatic erythrocytes were scored.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
The smears were stained according to the method of Schmid (1976).

METHOD OF ANALYSIS:
The mean number of micronucleated polychromatic erythrocytes was expressed per 1000 cells

Evaluation criteria:

A substance is considered positive if a significant increase in the frequency of polychromatic erythrocytes from the concurrent control value is observed.

Statistics:

Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

2/4 animals died in the high dose group

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

- Mean number of micronucleated polychromatic erythrocytes (per 1000 cells) was 2.3, 1.0, 1.0 and 2.0 for the dose groups receiving vehicle (control), 841 mg/kg bw, 1893 mg/kg bw and 2944 mg/kg bw, respectively.
- In the high dose group 2/ 4 animals died, no mortality was observed in the other groups.

Conclusions:

Under the conditions of the test, the substance is not clastogenic in NMRI mice in vivo.

Executive summary:

In a micronucleus test groups of male and female NMRI mice, 4 mice per dose (10-14 weeks old) were treated once intraperitoneally at dose levels of 841, 1893 and 2944 mg/kg in olive oil tested similar to OECDTG 474). Control animals were dosed with olive oil alone. Mice from each dose level were sacrificed at 30 hours, the bone marrow extracted and smear preparations made and stained. Polychromatic erythrocytes were then scored for the presence of micronuclei. In the top dose group 2/4 animals died. No significant increase in the incidence of micronucleated polychromatic erythrocytes was observed with any of the tested doses.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For the test substance an Ames test and a (limited) in vivo micronucleus assay are available. Therefore additional information is derived from the structural analogues Cyclacet (gene mutations in mammalian cells) and Cyclabutanate (chromosome aberration)

 

Ames test with the test substance itself (similar to OECD TG 471, with limitations)

The mutagenic activity of the test substance was evaluated in a study somewhat similar to OECD TG 471 (Wild 1983) but without E.coli. The substance was added by principles of the standard plate procedure at five dose levels up to 3.6 mg/plate in absence and presence of Aroclor induced rat liver S9. The five tester strains included Salmonella typhimurium TA 1535, TA 100, TA 1537, TA1538 and TA98, and water or DMSO was used as vehicle. No data on cytotoxicity was provided. Sodium azide and benzo[a] pyrene were taken along as positive controls at dose levels of 0.5 µg/plate and 5 µg/plate, respectively. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Ames test with Cyclacet

The test substance, Cyclacet, was evaluated in a bacterial reverse mutation assay employing Salmonella typhimurium strains TA97a, TA98, TA100, and TA1535 along with Escherichia coli strain WP2 uvrA (328) both in the presence and absence of an exogenous metabolic activation system. The test substance was evaluated using an initial plate incorporation assay and a confirmatory preincubation procedure.

The solvent, diluent and negative control used in this assay was dimethyl sulfoxide (DMSO). Test substance concentrations of 5, 10, 50, 100, 500, 1000, 2500, and 5000 ug per plate were assessed with respect to negative (solvent) controls in the plate incorporation assay. Due to test article associated toxicity, test substance concentrations were lowered for the confirmatory assay. Concentrations of 5, 10, 50, 100, and 250 ug per plate were assessed with respect to negative (solvent) controls in the confirmatory preincubation assay. In the plate incorporation assay, test substance associated toxicity, as evidenced by a concentration related reduction in the mean number of revertants per plate and/or the reduction of the microcolony background lawns, was observed at 500 ug per plate and above in S. typhimurium tester strains TA97a, TA98, TA100, and TA1535 with and without exogenous metabolic activation along with E. coli strain WP2 uvrA (328) without exogenous metabolic activation. Toxicity was observed at 1000 ug per plate and above in E. coli strain WP2 uvrA (328) with exogenous metabolic activation. Test substance associated precipitate was not observed at any concentration level.

In the preincubation assay, toxicity was observed at 100 ug per plate and above in S. typhimurium test strain TA98 without exogenous metabolic activation and at 250 ug per plate in S. typhimurium strains TA97a, TA100, and TA1535 along with E. coli strain WP2 uvrA (328) without exogenous metabolic activation. No toxicity was observed at the tested concentrations for any strain with exogenous metabolic activation.

The mean number of revertants observed in the negative controls (DMSO) for each of the test strains was within acceptable historical negative control ranges. All test strains demonstrated appropriate phenotypic characteristics.

Under the conditions of this study, no evidence of mutagenic activity was detected for the test substance, Cyclacet, in S. typhimurium test strains TA97a, TA98, TAI00, and TA1535 along with E. coli strain WP2 uvrA (328). The findings of this study show the test substance to be negative for the induction of mutagenicity in the bacterial reverse mutation test.

Mouse Lymphoma Assay with Cyclacet (OECD TG 476)

The substance was tested for its potential to induce mutations at the thymidine kinase locus of L5178Y TK+ mouse lymphoma cells in vitro according to OECD TG 476 and GLP principles (SITEK 2009). The concentrations tested with and without S-9 activation in the Range Finding Test were 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500, 1000, and 5000 µg/mL. Relative Suspension Growth (RSG) was used to measure toxicity. The RSG for treated cultures without activation indicated that the substance was toxic at 50 µg/mL and above. Cultures treated with 50 µg/mL had 16% RSG. The cultures treated with higher concentrations had 0% RSG. The RSG for cultures treated in presence of S-9 activation indicated that the substance was completely toxic, i.e., 0% RSG, at 500 µg/mL and above. The culture treated with 100 µg/mL had 74% RSG.

The Definitive Mutation Assay was performed using a 4-hour treatment period at test article concentrations ranging from 19 to 170 µg/mL without activation and from 109 to 300 µg/mL with S-9 activation. Cultures were selected for cloning for mutant selection based on their RSG. All of the cloned cultures, both with and without activation, had Mutant Frequencies (MF) that were similar to the average MF of their concurrent solvent control cultures. The Relative Total Growth (RTG) for the cloned cultures ranged from 17% to 99% for cultures treated without activation and from 42% to 90% for cultures treated in conjunction with exogenous activation.

Since it is ideal to have some cloned cultures that have between 10% and 30% RTG for evaluating a test articles mutagenic potential, a repeat assay with a 4-hour exposure period with activation was conducted. Cultures were treated with concentrations ranging from 200 to 500 ug/mL with 20 ug/mL increments between doses. The results for the repeat of the with S-9 activation part of the Definitive Mutation Assay also showed that the treated cultures all had MFs that were similar to the average MF of the solvent control cultures. The RTG for these cultures ranged from 0% to 104%. Under the test conditions, the results of the Definitive Mutation Assay are considered negative. The Confirmatory Mutation Assay was conducted without activation with a 24-hour exposure period. Cultures were treated with concentrations of 1.0, 5.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, and 140 µg/mL. The cultures treated with 30 to 140 µg/mL were cloned for mutant selection. All of the cultures had MFs that were similar to the average MF of the solvent controls. The RTG for the cloned cultures ranged from 3% to 59%. Under the test conditions, the results of the Definitive and Confirmatory Mutation Assays are considered negative (i.e. the test substance is not mutagenic).

 

Chromosomal aberration test with Cyclobutanate (OECD TG 473)

This study was conducted according to a method which was designed to assess the potential chromosomal mutagenicity of the substance on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line according to the requirements of OECD Guideline 473 and the updated Annex V EU B10 Method (Safepharm 2002). Duplicate cultures of CHL cells were treated at several dose levels, together with vehicle (DMSO) and positive controls. Five exposure groups were used: Experiment 1 included a 6(18)-hour exposure, both with and without the addition of an induced rat liver homogenate metabolizing system; Experiment 2 included a 24-hour continuous exposure without metabolic activation, a 48-hour continuous exposure without metabolic activation and a repeat of the 6(18)-hours exposure with metabolic activation. The dose levels evaluated in the main experiments were selected from a range of dose levels based on the results of a preliminary toxicity test and were in the range of 8.70 to 34.5 µg/mL for the 6(18)-hour exposure, without S9, 137.7 to 550.8 µg/mL for the with-S9 exposure, in both Experiment 1 and 2, and 4.35 to 34.5 µg/mL for the 24 and 48-hour treatments. The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control chemicals induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolizing system. The substance did not induce any toxicologically significant increases in the frequency of cells with aberrations in any of the exposure groups. The substance was shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all exposure groups. Based on the results, the substance was considered non-clastogenic to CHL cells in vitro.

 

In vivo Micronucleus test with the substance itself

In a micronucleus test groups of male and female NMRI mice, 4 per dose (10-14 weeks old) were treated once intraperitoneally at dose levels of 841, 1893 and 2944 mg/kg in olive oil similar to OECD TG 474 (Wild 1983). Control animals were dosed with olive oil alone. Mice from each dose level were sacrificed at 30 hours, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were then scored for the presence of micronuclei. In the top dose group 2/4 animals died. No significant increase in the incidence of micronucleated polychromatic erythrocytes was observed with any of the tested doses.

Genetic toxicity of Isobornyl propionate (CAS# 2756-56-1) using read-across from Isobornyl acetate (CAS# 125-12-2), Cyclacet (CAS54830-99-8)and Cyclobutanate (CAS#113889-23-9)

Introduction and hypothesis for the analogue approach

Isobornyl propionate (CAS# 2756-56-1) consists of an exo-1,7,7-Trimethylbicyclo[2.2.1]heptane (Isoborneol ) structure with a propionic ester attached to this ring. For this substance insufficient data on genotoxicity is available. In accordance with Article 13 of REACH, lacking information can be generated by other means, i.e. applying alternative methods such as QSARs, grouping and read-across. The analogue approach is selected since for the structurally related analogues Isobornyl acetate, Cyclacet and Cyclobutanate reliable data is available on the genotoxicity endpoints.

Hypothesis: Isobornyl propionate will present the same results in the Ames, in the MLA and cytogenicity as Cyclacet, Cyclacet and Cyclobutanate, respectively.

Available information:For Isobornyl propionate an Ames test is available. The study, similar to OECD 471 (Rel.2, lacking TA102 or a DNA repair-proficient strain of E. coli) was negative. For Isobornyl acetate also a limited Ames test is available and therefore not used. For Cyclacet and Cyclobutanate negative Ames results are available both according to OECD TG 471, Rel. 1. Information on gene mutations in mammalian cells is available for Cyclacet, which was tested negative in an MLA (OECD 476, Rel. 1). Isobornyl propionate was negative in an in vivo micronucleus assay (similar to OECD TG 474, Rel. 2). This result is supported with a negative in vitro chromosome aberration assay (OECD 473) carried out with Cyclobutanate.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical(s) are shown in the data matrix, including relevant physico-chemical properties.

Purity / Impurities

Isobornyl propionate is a mono-constituent >=88%, with impurities similar to the parent substance and < 10%.

Analogue approach justification

According to REACH Annex XI 1.5 read-across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read-across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation, which is presented below.

For Isobornyl propionate the one methyl shorter Isobornyl acetate was selected as an analogue being the closest analogue for which genotoxicity information is available. E.g. for Isobornyl butyrate no such information was found on the ECHA site or in the RIFM database. Cyclacet and Cyclobutanate are used as analogues because these analogues have other genotoxicity information which is not available for Isobornyl acetate.

Structural similarities and differences:Isobornyl propionate is similar to Cyclacet and Cyclobutanate: These substances have a very similar hydrocarbon backbone and the similar functional ester group. The double bond in Cyclacet and Cyclobutanate backbone is not near the ester bond and will not present additional electrophilicity and thus reactivity. The propionate versus the acetate or the butanoate ester is also not affecting the electrophilicity because of the weak electron feeding effect of CH3 groups.

Absorption:Isobornyl –propionate and acetate as well as the two Cycla-esters are liquids, have similar molecular weight and other physico-chemical properties i.e. log Kow, which indicate (some) absorption via all routes.

Reactivity: Isobornyl-propionate and acetate are similarly reactive because these are almost the same substances with one methyl difference in the alkyl-ester chain, which has a slight impact on the log Kow but not on reactivity. The propionate versus the acetate or butanoate group will not influence the reactivity. The OECD Toolbox 2.2 shows a similar non-reactive profile (data not shown).

Uncertainty of the prediction: There are no uncertainties other than those already addressed above.

Conclusion on hazard and risk assessment

For Isobornyl propionate limited genotoxicity information is available. Information from two analogues can be used for read across. When using read across the result should be applicable for classification and labelling and risk assessment as well as presented with reliable and adequate documentation. This documentation is presented in the current document. The Ames information from Cyclacet and Cyclobutanate can be used for read across and these two substances are negative in the Ames (OECDTG 471). The MLA information (OECD TG 476) from Cyclacet and the in vitro chromosomal aberration test from Cyclobutanate )OECD TG 473) are both negative and based on the structural similarity these can be used for read across to Isobornyl propionate.

Final conclusion:Isobornyl is negative for genemutations in bacterial and mammalian cells and for cytogenicity in vitro and in vivo.

Data matrix to support the read across to Isobornyl propionate from, Cyclacet and Cyclobutanate.

Common names	Isobornyl propionate	Isobornyl acetate	Cyclacet	Cyclobutanate
	Target	Supporting source	Source	Source
Chemical structures
CAS no.	2756-56-1	125-12-2	54830-99-8 (generic)	17511-61-4
EINECS	220-410-5	204-727-6	911-369-0	241-515-2
REACH registered	2018	Registered	Registered	Registered
Empirical formula	C13H22O2	C12H20O2	C12H16O2	C14H20O2
Molecular weight	210.32	196.29	192.25	220.31
Phys-chem properties
Physical state	Liquid	Liquid	Liquid	Liquid
Log Kow (measured)	5.0 (IFF measured)	4.3 (Simonich, EpiSuite)	3.9 (IFF measured)	4.48 (IFF measured)
Human health-Genotoxicity
Gene mutation in bacterial cells: Ames	Negative (OECD 471, but incomplete)	Negative (OECD TG 471, but incomplete)	Negative (OECD TG 471)	Negative (OECD TG 471)
Gene mutation in mammalian cells: MLA	Read-across	-	Negative (OECD 476)	-
In vitro cytogenicity	Read-across	-	-	Negative (CA, OECD 473)
In vivo micronucleus	Negative OECD TG 474	-	-	-

CA=Chromosomal aberrations

Justification for classification or non-classification

Based on the available data, classification and labelling of the substance for genetic toxicity is not warranted according to EU CLP (EC No. 1272/2008 and its amendments).