Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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
Study period:
02-12-99 to 06-07-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because of their structural similarities and is supported by their similar expected metabolism. This prediction is supported by the toxicological data on the substances themselves and predicted toxicokinetics of the substances. The target substance (Ligustral/Cyclal C/Trigustral) is a multi-constituent substance with 2 main constituents which are diastereomers (3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-, (1R,2R)-rel- and 3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-, (1R,2S)-rel-; Table 1). The source substance (HMPCC) is also a multi-constituent substance with 2 main constituents with a major (1,4-disubstituted-cyclohexenyl) and minor (1,3-disubstituted cyclohexenyl substance; Table 2) isomer. The target substance (Ligustral/Cyclal C/Trigustral) and source substance (HMPCC) have structurally similar backbones which are an unsaturated six member cyclohexyl ring with an attached ethylaldehyde group and the alkyl chain linked to the double bond. The structural differences consist of an alkyl chain which is methyl in Ligustral/Cyclal C/Trigustral and in HMPCC a longer 4-hydroxy-4-methylpentyl chain and lack of a methyl group beside the ethylaldehyde. There is also a methyl group at the meta position in Ligustral/Cyclal C/Trigustral. These differences are chemically simple structures and their impact on the read across is discussed further below. Similar expected metabolism is likely between the target and source substances. No reliable data from an in vivo micronucleus assay of Ligustral/Cyclal C/Trigustral is available. Therefore, read-across from the existing mammalian erythrocyte micronucleus study of the source substance is considered as an appropriate adaptation to the standard information requirements of Annex VIII, 8.4 of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Clear, colorless liquid
Details on test material:
- Name of test material (as cited in study report): 3 and 4-( 4-Hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde
- Physical state: Clear, colorless liquid
- Storage condition of test material: Room temperature; protected from exposure to light
- Other: Lyral_Multi-Constituent SIP from data holder is attached

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD.
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation:
Pilot Toxicity Study: Males, 27.9- 32.6 g, Females, 25.6- 27.8 g
Toxicity Study: Males, 26.3- 29.1g, Females, 24.7-27.7 g
Micronucleus Assay: Males,28.1 - 33.3 g, Females, 26.0 - 30.4 g

- Assigned to test groups randomly: yes
- Housing: be housed in an AAALAC-accredited facility. Heat-treated hardwood chips will be used for bedding.
- Diet (e.g. ad libitum): Certified rodent chow (Harlan TEKLAD certified rodent 7012C) ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: no less than 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72±3°F
- Humidity (%): 50±20%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil (CAS 8001-30-1; Sigma)
- Justification for choice of solvent/vehicle: Corn oil was determined to be the solvent of choice based on information provided by the Sponsor and compatibility of the vehicle with the test system animals.
- Concentration of test material in vehicle: The test article was soluble in corn oil at 100 mg/mL, the maximum concentration tested in the study

Details on exposure:
The test substance-vehicle mixture, the vehicle alone, or a positive control was administered by intraperitoneal (IP) injection at a constant volume of 20 mL/kg bodyweight. IP injection was selected to maximize delivery of the test article to the target system. Animals received the test article as a single administration. Dosing concentrations were delivered to the test system as light yellow solutions.
Duration of treatment / exposure:
Intraperitoneal injection
Frequency of treatment:
Single dose administration
Post exposure period:
Pilot Study and Toxicity Study: Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of chemical effect.
Micronucleus Assay: Bone Marrow Collection After Dose Administration 24 and 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Pilot Assay
Basis:
nominal conc.
Male: 1, 10, 100, or 1000 mg/kg bw. Female: 2000 mg/kg bw
Remarks:
Doses / Concentrations:
Toxicity Assay
Basis:
nominal conc.
200, 400, 600 and 800 mg/kg bw
Remarks:
Doses / Concentrations:
Micronucleus Assay
Basis:
nominal conc.
Corn oil: 0 mg/kg bw; Test substance: 225, 450, or 900 mg/kg bw; positive control : CP, 50 mg/kg bw
No. of animals per sex per dose:
Pilot Assay:
- 1, 10, 100, or 1000 mg/kg bw: 2 male animals per dose
- 2000 mg/kg/bw: 5 animals per sex

Toxicity Assay:
-200,400,600,800 mg/kg bw: 5 animals per sex per dose


Micronucleus Assay:
- corn oil group: 10 animals per sex
- 225, 450 mg/kg bw and positive control (CP, 50 mg/kg bw):: 5 animals per sex per dose
- 900 mg/kg bw: 15 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): On the basis of Annex I (Mouse Micronucleus Test Historical Control Data (1995-1997)), cyclophosphamide (CP) as positive control was range of 40-60 mg/kg, dosed by IV, IP or PO. In view of the historical control data, CP was chosen as the positive control with a dose of 50 mg/kg.
- Route of administration: intraperitoneal injection
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow was from the femur. Polychromatic erythrocytes were scored for presence of micronuclei. Number of micronucleated normochromatic erythrocytes was counted. Proportion of polychromatic erythrocytes to total erythrocytes was recorded.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice times, to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supematant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Evaluation criteria:
Positive response: a dose responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p≦0.05, Kastenbaum-Bowman Tables) at any sampling time.

Negative response: no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended.

Criteria for a Valid Test:
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p≦0.05, Kastenbaum-Bowman Tables).
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and sampling time.

Results and discussion

Test resultsopen allclose all
Sex:
male/female
Genotoxicity:
not determined
Remarks:
Pilot Toxicity Study
Toxicity:
yes
Remarks:
Mortality: Male: 1/2; 1000 mg/kg bw; Males and females: 5/5; 2000 mg/kg bw
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Sex:
male/female
Genotoxicity:
not determined
Remarks:
Toxicity Study
Toxicity:
yes
Remarks:
lethargy and piloerection in males and females at 400, 600 and 800 mg/kg
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Sex:
male/female
Genotoxicity:
negative
Remarks:
Micronucleus Assay
Toxicity:
yes
Remarks:
1/15 female mice was dead at 900 mg/kg. lethargy and piloerection in males and females at 225, 450 and 900 mg/kg and irregular breathing in males and females at 900 mg/kg.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Study report attachments:

Tables 1-2: Pilot Toxicity Study

Tables 3-4 Toxicity Study

Tables 5-8 Results of Micronucleus Assay

Annex I Mouse Micronucleus Test Historical Control Data (1995-1997)

Read-Across Justification - Full report is attached.

Analogue approach justification

Physicochemical properties

Physicochemical data shows that the physicochemical properties of the target and source substances are similar as outlined in the data matrix (Table 5). The structural differences in the side chains do not significantly influence the physicochemical properties of both substances, i.e. vapour pressure, partition coefficient and water solubility. There is more than one data point for the source substance (HMPCC) for vapour pressure due to different mixture ratios. The molecular weight of the target substance (Ligustral/Cyclal C/Trigustral) is 138 g/mol and the molecular weight of the source substance (HMPCC) is 210.32 g/mol. The partition coefficient (Log Kow) for both substances is in a similar range (2-3). The water solubility for both of the target and source substances is moderately soluble with a range of 100-1000 mg/L, (910 mg/L at 20 °C for the target substance and 184.6 mg/L at 25 °C for the source substance). Neither of the substances is volatile with a vapour pressure of 36 Pa at 20°C for the target substance and approximately 0.001 mm Hg (0.19 Pa) at 20 °C for the source substance.

Toxicokinetics

No experimental data on absorption, distribution, metabolism or excretion is available for the source or target substances. The toxicokinetic assessment is based on physicochemical properties of the substances.

Physicochemical data

The source and target chemical have similar toxicokinetic behaviour based on their similar physicochemical properties (Table 5): the molecular weights (>100 g/mol, <500 g/mol), moderate solubility in water (100 to 1000 mg/L) and moderate log Kow values (Ligustral/Cyclal C/Trigustral: Log Kow 2.7; HMPCC: Log Kow 2.1) indicate absorption via the oral and dermal routes. The physical forms (liquid) and low vapour pressures of both substances indicate low volatility, so respiratory exposure is expected to be low. Both of the target and source substances are not expected to undergo hydrolysis due to a lack of hydrolytic functional groups and so are likely to be present in the body in non-ionised forms. A wide distribution of both substances is favourable due to their relatively small molecular weight, moderate lipophilicity and non-ionised forms in the body. The physicochemical properties of both substances (low molecular weight, lipophilic, non-ionised) suggests it can cross the placenta. There is no direct evidence to indicate the major route of excretion of the substances however both are expected to be excreted in the urine due to their low molecular weight (<300) and water solubility will be increased during metabolic transformation. Postnatal exposure via the milk during lactation is a minor route of excretion for HMPCC and may also be for Ligustral/Cyclal C/Trigustral based on the physicochemical properties.

Predicted data for metabolism

Based on the SMARTCyp - Cytochrome P450 - Mediated Metabolism simulator (Toxtree v2.5.0), the primary and secondary sites of metabolism of the both the target and source chemicals are estimated to be oxidation of the aldehyde to a carboxylic acid group (Rank 1) and aliphatic hydroxylation (Rank 2) respectively. The tertiary and subsequent sites indicate epoxidation as a possibility for both the target and source chemicals (Annex II – Table 4).

Other data in the literature

There are no available experimental data for toxicokinetics for either the target or source substance. In the U.S. Environmental Protection Agency Hazard Characterization Document, March 2010 (U.S. EPA, 2010), it was anticipated that HMPCC will be rapidly absorbed via the oral route of exposure and primarily metabolized to the corresponding carboxylic acid and, to a lesser extent, the corresponding alcohol; both metabolites are excreted primarily in the urine (based on pharmacokinetic and metabolic studies in rabbit, rats, dogs, and humans with 7­hydroxycitronellal and perilla aldehyde derivatives).

Effect of structural differences between target and source chemical

The structural differences consist of an alkyl chain which is methyl in Ligustral/Cyclal C/Trigustral and in HMPCC a longer 4-hydroxy-4-methylpentyl chain and lack of a methyl group beside the ethylaldehyde. The methyl group in Ligustral/Cyclal C/Trigustral may be hydroxylated which will enhance polarity and excretion in the urine. The tertiary alcohol substituent may be considered in light of data available on metabolic fate of aliphatic and alicyclic tertiary alcohols e.g. linalool, alpha-terpineol in humans and animals. The alcohol group may be conjugated directly to glucuronic acid which facilitates excretion in the urine. Most tertiary alcohols have low toxicity after oral exposure and occur naturally in a wide variety of foods and show relative safety for long-term exposure in humans and animals (Joint FAO/WHO JECFA, 1999).

On the basis of all the available data, Ligustral/Cyclal C/Trigustral and HMPCC are expected to have similar metabolism routes.

Comparison of data from human health endpoints

Toxicity data of the target and source substances

As presented in the data matrix (Table 5), the acute oral and dermal toxicity data show similar acute toxicity for the source and the target chemicals. Moderate skin irritation and positive skin sensitization were noted for both. Both chemicals have negative in vitro bacterial gene mutation (Ames test) results. The US EPA accepted read across from Ligustral/Cyclal C/Trigustral to HMPCC for the in vitro bacterial gene mutation test (Ames test) for a HPV Chemical Hazard Characterization carried out for HPMCC in 2010 (US EPA, March 2010).

In the ICR mouse bone marrow micronucleus key study (OECD 474/GLP), groups of mice were treated intraperitoneally with HMPCC at doses of 0, 225, 400, 900 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hrs post-treatment. The vehicle was corn oil (intraperitoneal). Mortality was observed in 1/15 female mice at 900 mg/kg bw during the study. Lethargy and piloerection in male and female mice were observed at 225, 450 and 900 mg/kg and irregular breathing in males and females were observed at 900 mg/kg. 3 and 4-(4-Hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde was tested at an adequate dose based on the pilot study and toxicity assay. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

In the in vitro mammalian cell cytogenetics assay (Chromosome aberration) assay, there was a concentration related positive increase in structural chromosome aberrations, but not numerical aberrations, induced over background in the 4 hrs (with activation) treatment group. However in the in vivo micronucleus study, HMPCC did not induce a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow. In the OECD Toolbox 3.1, mechanistic analysis from two of the primary profilers (DNA and protein) was supported by the presence of a structural alert for an aldehyde within one endpoint specific profiler (e.g. Schiff base formation with aldehydes) for both the target and source substance. The ideal scenario is for all profilers to reveal complimentary results and the lack of consistency (i.e. only 3/5 indicating an alert) may reflect the differences noted in the in vitro and in vivo studies.

Based on the experimental evidence from this study, it is concluded that none of the metabolites of the source substance are genotoxic in vivo and based on the similar metabolic profile discussed in Section 3.2, it is expected that none of the metabolites of the target substance are genotoxic in vivo either. Taking into account all the evidence from the experimental studies and expected similar metabolism, Ligustral/Cyclal C/Trigustral is predicted to have a similar results in the in vivo micronucleus study to HMPCC, so using a read-across approach, a negative response is predicted for Ligustral/Cyclal C/Trigustral.

Classification and labeling

Based on available data in the IUCLID dossier, Ligustral/Cyclal C/Trigustral is classified according to CLP (1272/2008) as Skin Sensitization Category 1 and Skin Irritation Category 2 for human health hazards; it does not need to be classified for germ cell mutagenicity (see Section 3.3 Conclusion for further information on classification of Ligustral/Cyclal C/Trigustral for germ cell mutagenicity). In ECHA’s C&L inventory database, the current self-classification and labelling for HMPCC based on the CLP Regulation criteria indicate the following classification is Skin Sensitisation 1 (EC: 257-187-9; 25-04-13) and Skin Sensitization Category 1 and Eye Irritation Category 2 (EC No. 250-863-4; 25-04-13). It is not classified for germ cell mutagenicity (25-04-13).

Conclusion

The structural similarities between the source and the target substances and estimated similar toxicokinetic routes presented above support the read-across hypothesis. Adequate, reliable and available scientific information indicates that the source and target substances will have comparable results in the in vivo micronucleus assay.

In the in vivo mammalian erythrocyte micronucleus assay (OECD 474, GLP), HMPCC was concluded to be negative. Based on the information presented in this justification and the results in the dossier, we do not expect Ligustral/Cyclal C/Trigustral to present an increased hazard compared to HMPCC for the in vivo mammalian erythrocyte micronucleus assay endpoint. Ligustral/Cyclal C/Trigustral is predicted to be negative in the in vivo micronucleus study.

Uncertainty in the prediction: The main source of uncertainty is that the read across prediction indicates that the target substance is negative in the in vivo micronucleus study, when it may be positive. However, this read across justification is presented as an appropriate adaptation to the standard information requirements of Annex VIII, 8.4 of the REACH Regulation for the target substance and the results from this endpoint are considered with the results from the Annex VII, 8.4.1 and Annex IX, 8.4.2 endpoints to obtain an overall conclusion. The results from the Annex VII, 8.4.1 endpoint for Ligustral/Cyclal C/Trigustral demonstrated that the substance was not mutagenic in vitro. The results from the Annex VII, 8.4.2 endpoint for HMPCC (read-across) demonstrated that the substance was mutagenic in the presence of metabolic activation in vitro. The results from this Annex IX, 8.4 endpoint for HMPCC (read-across) demonstrated that the substance was not mutagenic in vivo. According to Table R.7.7.5 in ECHA Guidance 7a (2012): “If the Ames test results are negative, the in vitro cytogenecity test results are positive and the in vivo cytogenecity test results are negative, a substance can be considered as not genotoxic and no further tests are required". A qualitative read-across result is considered as an appropriate evidence for waiving Annex IX, 8.4.3 in vitro gene mutation study in mammalian cells, therefore, the in vitro mammalian cell gene mutation test (Annex VIII, 8.4.3) was not required. According to the CLP Regulation, based on the data available, Ligustral/Cyclal C/Trigustral is not classified for germ cell mutagenicity. The overall conclusion from the existing studies and classification is that Ligustral/Cyclal C/Trigustral is not genotoxic so the uncertainty in the prediction does not affect the overall conclusion.

Therefore, based on the considerations above, it can be concluded that the results of the erythrocyte micronucleus study conducted in mice with the source substance is likely to predict the properties of the target substance and are considered as adequate to fulfill the information requirement of Annex IX, 8.4.

Data matrix

A summary of key data for the target substance and source substance is presented below:

Corresponding standard information required

Target substance

Source substance

Synonyms

Ligustral; Cyclal C; Trigustral; 2,4-dimethylcyclohex-3-ene-1-carbaldehyde

Lyral®; Reaction mass of 4-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde and 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde

CAS No.

68039-49-6

31906-04-4 /51414-25-6

Information on the physicochemical properties

Molecular weight

138.21

210.32

Physical state

liquid

liquid

Vapour pressure

36 Pa (0.271 mmHg) at 20°C

 

Ref. 5

0.0012 mm Hg at 25°C(US EPA, 2010)1

< 0.001 mm Hg at 20 °C(SCCS, 2011)2

2.74×10-5mmHg(Chemical Zoo (2007) Chemical Spiders DB )

Partition coefficient (Log Kow)

2.7

 

Ref. 6

2.1(US EPA, 2010)1

 

Water solubility

0.91g/l at 20 °C

 

Ref. 7

184.6 mg/l at 25 °C(SCCS, 2011)2

Toxicological information

Toxicokinetics

Assessment based on phys-chem properties

Absorption rate: acute: 50%; dermal: 50%; inhalation:100%

Widely distribution, low bioaccumulation;

Excretion via urine

Ref. 8

Assessment based on phys-chem properties:

Absorption rate: acute: 50%; dermal: 50%; inhalation:100%

Widely distribution, low bioaccumulation;

Excretion via urine

Acute oral toxicity

rat

oral

LD50:3900mg/kg bw

2 (reliable with restrictions)

key study

 

Ref. 9

Rat

Oral: gavage

LD50 > 5000 mg/kg bw(US EPA, 2010)1; (SCCS, 2011)2

Acute dermal toxicity

rabbit

LD50: > 5000 mg/kg bw

2 (reliable with restrictions)

key study

 

Ref. 10

Rabbit

LD>5000 mg/kg bw(US EPA, 2010)1; (SCCS, 2011)2

Skin irritation/corrosiveness

 

 

 

 

Rabbit

OECD Guideline 404(skin irritation)

2 (reliable with restrictions)

weight of evidence

skin irritation

 

Ref. 11

 

some irritant potentialat higher exposures,no irritant effect is to be expected under conditions of actual use (SCCS, 2011)2

 

 

guinea pig (albino Dunkin Hartley guinea pig)

Coverage: occlusive (clipped)

Vehicle: arachis oil BP, ethanol/diethylphthalate 1:1

OECD Guidline 406 (skin sensitisation)

2 (reliable with restrictions)

weight of evidence

skin irritant

 

Ref. 11

rabbit

2 (reliable with restrictions)

weight of evidence

skin irritant

 

Ref. 11

Eye irritation

rabbit (New Zealand White albino rabbit)

Vehicle: unchanged (no vehicle)

FDA of the United States (fed, reg, 28 (119), 5582, 1963)/ Draize and Kelley (Drug Cosmet, Industr, 71(1952) 36)

2 (reliable with restrictions)

key study

Not irritation (Naarden test report, 1981)

 

Ref. 12

Eyeirritation (rabbit, GLP) (HMPCC full dossier)

 

Sensitization

guinea pig (albino Dunkin Hartley guinea pig) female

Guinea pig maximisation test

 

OECD Guideline 406 (Skin Sensitisation)

Sensitizing to skin (Symrisetest report, 1998)

 

1 (reliable without restriction)

key study

 

Ref. 13

Positive in LLNAat 25%concentration (US EPA, 2010)1

moderate skin sensitizer(SCCS, 2011)2

Genotoxicity – Ames test

bacterial reverse mutation assay (e.g. Ames test) (gene mutation)

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Negative

1 (reliable without restriction)

key study

 

Ref. 14

Negative (OECD 471)(US EPA, 2010)1

Genotoxicity in vitroChromosomal aberration

Read-across from HMPCC

Positive with metabolic activation (OECD 473) (RIFMtest report, 2007)

 

Ref. 14

in vitro mammalian chromosome aberration test (chromosome aberration)

mammalian cell line, other: CHO - K1 cells (met. act.: with and without)

OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test)

Positive with metabolic activation (OECD 473) (RIFMtest report, 2007)

2 (reliable with restrictions)

key study

 

Ref. 14

Mammalian Erythrocyte Micronucleus Test

Read-across from HMPCC

Negative (OECD 474) (RIFM test report, 2000)

 

Ref. 15

micronucleus assay (chromosome aberration)

mouse (ICR) male/female

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Negative (OECD 474)

2 (reliable with restrictions)

key study

 

Ref. 15

Repeated dose toxicity

Read-across from HMPCC

NOAEL=150 mg/kg bw/day

 

Ref.16

rat (Sprague-Dawley Crl:CD® (SD) IGS BR strain rat) male/female

subacute (oral: gavage)

0, 15, 150 and 1000 mg/kg bw/day (nominal conc)

Vehicle: Arachis oil BP

Exposure: twenty-eight consecutive days (daily)

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

NOAEL=150 mg/kg bw/day

2 (reliable with restrictions)

key study

 

Ref.16

 

Read-across from L-perillyl alcohol NOAEL=120 mg/kg bw/day

 

Ref.16

rat (Fischer 344) male/female

subchronic (oral: gavage)

40, 120 and 400 mg/kg bw/day (nominal concentration)

Vehicle: soybean oil

Exposure: 90 consecutive days (daily)

NOAEL=120 mg/kg bw/day in 90-day study

2 (reliable with restrictions)

key study

 

Ref.16

Reproductive and developmental toxicity (mg/kg bw/day)

Developmental: Read-across from HMPCC

NOAEL=25 mg/kg bw/day

 

Ref.17

rat (Sprague-Dawley Crl:CD( (SD) IGS BR strain)

oral: gavage

OECD Guideline 415 (One Generation Reproduction Toxicity Study)

Developmental: NOAEL=25 mg/kg bw/day

2 (reliable with restrictions)

key study

 

Ref.17

Environmental information

Hydrolysis

No hydrolysis

 

Ref. 18

Stable under environmental conditions(US EPA, 2010)1

Biodegradation

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

not readily biodegradable

 

1 (reliable without restriction)

key study

 

Ref. 19

notreadily biodegradable(US EPA, 2010)1

bioaccumulation

a low potential for bioaccumulation

 

Ref. 20

a low potential for bioaccumulation(US EPA, 2010)1

Adsorption coefficient (Koc)

OECD Guideline 121 (Estimation of the Adsorption Coefficient (Koc) on Soil and on Sewage Sludge using High Performance Liquid Chromatography (HPLC))

EU Method C.19 (Estimation of the Adsorption Coefficient (KOC) on Soil and Sewage Sludge Using High Performance Liquid Chromatography (HPLC))

 

log Koc = 2.2 (Givaudan, 2010)

 

1 (reliable without restriction)

key study

 

Ref. 21

log Koc =1.3(estimated data, US EPA, 2010) (US EPA, 2010)1

Note:

1U.S. EPA, 2010. U.S. Environmental Protection Agency Hazard Characterization Document, 2010. SCREENING-LEVEL HAZARD CHARACTERIZATION SPONSORED CHEMICAL 3 and 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde (CASRN 130066-44-3). It is a mixture of two isomers, CASRN 31906-04-4 and CASRN 51414-25-6, in an approximate 70:30 ratio.

 

2 SCCS, 2012. Scientific Committee on Consumer Safety (SCCS) OPINION ON HYDROXYISOHEXYL 3-CYCLOHEXENE CARBOXALDEHYDE (HICC). The isomer ratio A:B is approximately 2:1(A:4-(4-Hydroxy-4-methylpentyl)cyclohex-3-ene carboxaldehyde, andB: 3-(4-Hydroxy-4-methylpentyl)cyclohex-3-ene carboxaldehyde)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the assay described in this report, the test substance 3 and 4-(4-Hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the microneculeus test using male and female ICR mice. These results are suitable for REACH purposes and using a read-across approach, and a negative response is predicted for Ligustral/Cyclal C/Trigustral.
Executive summary:

In a ICR mouse bone marrow micronucleus assay (AA10BX.123.BTL), groups of mice were treated intraperitoneally with 3 and 4-(4-Hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde at doses of 0, 225, 400, 900 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hrs post-treatment. The vehicle was corn oil (intraperitoneal).

Mortality was observed in 1/15 female mice at 900 mg/kg bw during the study. Lethargy and piloerection in male and female mice were observed at 225, 450 and 900 mg/kg and irregular breathing in males and females were observed at 900 mg/kg. 3 and 4-(4-Hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde was tested at an adequate dose based on the pilot study and toxicity assay. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.