Registration Dossier

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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:
22-08-99 to 23-05-00
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 close 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) isomer (Table 2). 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 on in vitro cytogenicity study in mammalian cells of Ligustral/Cyclal C/Trigustral is available. Therefore, read-across from the existing in vitro cytogenicity study in mammalian cells on the source substance is considered as an appropriate adaptation to the standard information requirements of Annex VIII, 8.4.2 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 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

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
- Molecular weight (if other than submission substance): 210.3
- 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.

Method

Species / strain
Species / strain / cell type:
mammalian cell line, other: CHO - K1 cells
Details on mammalian cell type (if applicable):
- Properly maintained: yes, McCoy's 5A medium with standard supplements
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, working cell stocks were not used beyond passage 20.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9: 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 µL S9 per mL medium
Test concentrations with justification for top dose:
Preliminary Toxicity Assay:
- with and without S9, 4 hrs and 20 hrs: 0.21, 0.63, 2.1, 6.3, 21, 63, 210, 630 and 2100 µg/mL

Chromosome Aberration Assay:
- without S9, 4 hours: 100, 200, 400, 600, 700, 800, 900 and 1000 µg/mL
- with S9, 4 hours: 100, 200, 400, 600, 700, 800, 900 and 1000 µg/mL
- without S9, 20 hours: 100, 200, 400, 600, 700, 800, 900 and 1000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was determined to be the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at a concentration of 500 mg/mL, the maximum concentration tested.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Mitomycin C (MMC; CAS No.: 50-07-7) from Sigma Chemical Company used as the positive control in the non-activated test system at 0.1 and 0.2 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Cyclophosphamide (CP;CAS No.: 6055-19-2) from Sigma Chemical Company used as the positive control in the S9-activated test system at 10 and 20 µg/mL.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with/without S9, 20 hours without S9
- Fixation time (start of exposure up to fixation or harvest of cells): all cells were harvested at 20 hours after treatment initiation

SPINDLE INHIBITOR (cytogenetic assays): Colcemid® 0.1 µg/mL
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS:
Preliminary test: 1
Chromosome test: 2

NUMBER OF CELLS EVALUATED:
Mitotic Index - the percentage of cells in mitosis per 500 cells scored was determined for each treatment group
Scoring of Chromosome Damage - a minimum of 200 metaphase spreads ( 100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Polyploid cells was evaluated from each treatment flask per 100 metaphase cells scored.
- Determination of endoreplication: endoreduplicated cells was evaluated from each treatment flask per 100 metaphase cells scored.

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:
Preliminary test-1
Chromosome aberration assay-2

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Evaluation criteria:
The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell was calculated and reported for each group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.

All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p≤0.05). Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative. Negative results with metabolic activation may need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not necessary, justification will be provided.

Criteria for a Valid Test
The frequency of cells with structural chromosome aberrations in the solvent control must be within the range of the historical solvent control. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p≤0.05, Fisher's exact test) relative to the solvent control.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
630 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2100 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2100 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
numerical and structural chromosome aberrations
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The toxicity was 60% at 600 µg/mL, the highest test concentration evaluated for chromosome aberrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
It was concluded to be positive for the induction of structural chromosome aberrations, and negative for the induction of numerical chromosome aberrations.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The toxicity was 52% at 900 µg/mL, the highest test concentration evaluated for chromosome aberrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
numerical and structural chromosome aberrations
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The toxicity was 60% at 400 µg/mL, the highest test concentration evaluated for chromosome aberrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest concentration of test article in treatment medium was approximately 7.
- Effects of osmolality: There was no significant change in osmolality. The osmolality in treatment medium of the highest concentration tested, 1000 µg/mL, was 401 mmol/kg. The osmolality ofthe solvent (DMSO) in treatment medium was 392 mmol/kg.
- Precipitation: The presence of test article precipitate was assessed using the unaided eye. The test article was soluble in treatment medium at all concentrations tested.

RANGE-FINDING/SCREENING STUDIES:
Based the preliminary toxicity assay, the results of the toxicity study, the dose levels selected for testing in the chromosome aberration assay were 100, 200, 400, 600, 700, 800, 900, and 1000 µg/mL for both the non-activated and S9 activated exposure groups.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical data is presented in Appendix I Historical Control data (1995-1997).
Remarks on result:
other: other: preliminary toxicity test 4 hours
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 10 Summary of Chromosome Aberration Assay

Treatment S9 Activation Treatment time1
(Hours)
Mitotic Index Cells Scored Aberrations per Cell2
(Mean ± SD)
Cells with Aberrations3
(%)
Numerical  Structural
DMSO - 4 9.1 200 0.000 ± 0.000 1.5 0.0
   
3 and 4-(4-Hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde
200 µg/mL - 4 8.9 200 0.000 ± 0.000 0.5 0.0
400 µg/mL - 4 8.7 200 0.120 ± 0.507 2.0 7.0**
600 µg/mL - 4 1.1 200 0.005 ± 0.071 3.5 0.5
   
MMC,  
0.2 µg/mL - 4 8.0 200 0.260 ± 1.117 3.5 12.0**
               
DMSO + 4 10.1 200 0.005 ± 0.071 3.5 0.5
   
3 and 4-(4-Hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde
200 µg/mL + 4 4.3 200 0.105 ± 0.393 4.5 8.5**
800 µg/mL + 4 5.9 200 0.205 ± 0.689 4.0 11.0**
900 µg/mL + 4 1.1 200 0.665 ± 1.608 5.5 24.0**
   
CP,  
10 µg/mL + 4 8.0 200 0.455 ± 1.016 4.5 22.5**
   
DMSO - 20 5.0 200 0.000 ± 0.000 2.0 0.0
   
3 and 4-(4-Hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde
100 µg/mL - 20 8.5 200 0.005 ± 0.071 2.5 0.5
200 µg/mL - 20 8.8 200 0.015 ± 0.122 1.0 1.5
400 µg/mL - 20 1.1 200 0.045 ± 0.271 2.0 3.5**
   
MMC,  
0.1 µg/mL - 20 5.9 200 0.140 ± 0.376 2.5 13.0**

 1 Cells from all treatment conditions were harvested at 20 hours after the initiation of the treatments.

 2 Severely damaged cells were counted as 10 aberrations.

 3 *, p≤0.05; **, p≤0.01; Fisher's exact test.

Study report attachments:

Tables 1-3 Preliminary toxicity tests

Tables 4-5 Chromosome aberration assay without S9 (4 hours)

Tables 6-7 Chromosome aberration assay without S9 (4 hours)

Tables 8-9 Chromosome aberration assay without S9 (20 hours)

Appendix I 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 substance. The toxicokinetic assessment is based on physicochemical properties of the substances and predicted data.

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.

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).

In the OECD QSAR Toolbox v3.1, the following metabolism simulators were applied with the primary profilers (‘DNA binding by OECD’, ‘DNA binding by OASIS’, ‘protein binding by OASIS’) and endpoint specific profilers (DNA alerts for AMES, MN and CA by OASIS v.1.1 and in vivo mutagenicity (Micronucleus) alerts by ISS): Microbial Metabolism Simulator, rat liver S9 Metabolism Simulator and skin metabolism simulator (Annex II - Table 3). For the Microbial Metabolism Simulator, 48 metabolites were identified for Ligustral/Cyclal C/Trigustral (CAS No. 68039-49-60) and 73/74 for HMPCC (CAS No. 51414-25-6/31906-04-4) respectively. In vivo, the enzymes in the intestinal microflora have not been characterized, and the reactions may take place either before absorption or after biliary excretion. The reduction of endocyclic bonds (present in both target and source substance) may involve these enzymes. It is noted that these simulators only identify potential metabolites and not stable metabolites and though metabolism prior to absorption may occur, both substances are well absorbed orally so the impact of the metabolites predicted by the Microbial Metabolism Simulator are not further considered. For the rat liver S9 Metabolism Simulator, both the target and source substances predicted one metabolite produced by oxidation of the aldehyde to a carboxylic acid group (Annex II - Table 3). No mechanistic alert was identified for this metabolite. For the skin metabolism simulator, both the target and source substances predicted four metabolites. The primary and endpoint specific profilers indicated the same results for the metabolites as for the parent substances (i.e. mechanistic alert for Schiff base formation with aldehydes and a structural alert for aldehydes; Section 2.2 a).

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 chemicals 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 (US EPA, March 2010).

In the in vitro mammalian cell cytogenetics assay (Chromosome aberration) conducted in accordance with OECD 473/GLP, CHO-K1 cell cultures were exposed to HMPCC in DMSO at concentrations of 100, 200, 400, 600, 700, 800, 900 and 1000 µg/mL [4 hrs and 20 hrs; with and without metabolic activation (Aroclor 1254-induced rat liver S9)]. HMPCC was tested up to cytotoxic concentrations in each exposure group. Positive controls induced the appropriate response. There was a concentration-related positive increase in structural chromosome aberrations, but not numerical aberrations, induced over background in the 4 hrs (without activation) treatment group, but this was not considered biologically significant based on historical laboratory data. 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. There was a concentration-related positive increase in structural chromosome aberrations, but not numerical aberrations, induced over background in the 20 hrs (without activation) treatment group, but this was not considered biologically significant based on historical laboratory data.

The biologically significant positive result in the in vitro assay was noted in the 4 hrs (with Aroclor 1254-induced rat liver S9 activation) treatment group. The rat liver S9 metabolism simulator predicted one metabolite produced by oxidation of the aldehyde to a carboxylic acid group for both the target and source substances but no mechanistic alert was identified for this metabolite. It is noted that these simulators only identify potential metabolites and not stable metabolites and this may explain the disconcordance between the experimental and predicted data.

Considering all the information, Ligustral/Cyclal C/Trigustral is predicted to have a comparable level of cytogenicity in vitro to HMPCC, so using a read-across approach, a positive response (structural aberrations) in the presence of metabolic activation may be expected and a negative response in the absence of metabolic activation is predicted for Ligustral/Cyclal C/Trigustral in the in vitro mammalian cell cytogenetics assay (Chromosome aberration).

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 Uncertainty in the prediction in 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 toxicokinetics 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 vitro mammalian cell cytogenetics assay (Chromosome aberration).

In the in vitro mammalian cell cytogenetics assay (Chromosome aberration), HMPCC was concluded to be positive for the induction of structural chromosome aberrations in the S9 activated test system, negative for the induction of structural aberrations in the non-activated test system, and negative for the induction of numerical chromosome aberrations in both the non-activated and S9 activated test systems in Chinese hamster ovary (CHO) cells. (OECD 473, GLP).

Uncertainty in the prediction: The main source of uncertainty is that the read across prediction indicates that the target substance is positive in the in vitro cytogenicity study in mammalian cells, when it may be negative. However, this read across justification is presented as an appropriate adaptation to the standard information requirements of Annex VIII, 8.4.2 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 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 the 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. As Ligustral/Cyclal C/Trigustral is not genotoxic, the uncertainty in the prediction does not affect the overall conclusion.

Therefore, based on the considerations above, it is concluded that the results of the in vitro mammalian cell cytogenetics assay (Chromosome aberration) conducted with the source substance is likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.4.2.

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):
ambiguous with metabolic activation
negative without metabolic activation

Under the conditions of the assay described in this report, 3 and 4-(4-Hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde (EC No. EC No. 915-617-9) demonstrated that it was positive for induction of structural chromosome aberrations in Chinese hamster ovary (CHO) cells (with S9 metabolic activation) and negative both for structural aberrations (without S9 metabolic activation) and numerical chromosome aberrations (with and without S9 metabolic activation) in CHO cells. These results are suitable for REACH purposes and using a read-across approach, and a positive response is predicted for Ligustral/Cyclal C/Trigustral.
Executive summary:

In a mammalian cell cytogenetics assay [Chromosome aberration] (AA10BX.331.BTL), CHO-K1 cell cultures were exposed to HMPCC in DMSO at concentrations of 100, 200, 400, 600, 700, 800, 900 and 1000 µg/mL [4 hrs and 20 hrs; with and without metabolic activation (Aroclor 1254-induced rat liver S9)].

HMPCC was tested up to cytotoxic concentrations in each exposure group. Positive controls induced the appropriate response. There was a concentration related positive increase in structural chromosome aberrations, but not numerical aberrations, induced over background in the 4 hrs (without activation) treatment group, but this was not considered biologically significant based on historical laboratory data. 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. There was a concentration related positive increase in structural chromosome aberrations, but not numerical aberrations, induced over background in the 20 hrs (without activation) treatment group, but this was not considered biologically significant based on historical laboratory data.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 (In vitro mammalian cytogenetics - chromosome aberration).