Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

The gene mutation (Bacterial Reverse Mutation Assay/Ames test) was OECD 471/ GLP compliant (Klimisch score: 1) and was negative.

No studies on the in vitro mammalian cell cytogenicity or in vivo mammalian cell cytogenicity were available. A read-across approach was conducted with data from a chromosome aberration in vitro study and an in vivo mammalian erythrocyte micronucleus study from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde (EC No: 915-617-9)

  • Read-across justification of analogue approach to assess in vitro cytogenicity in mammalian cells for Ligustral/Cyclal C/Trigustral: see the attached documents

Read-across justification full report of in vitro cytogenicity study

Annex I Lead registration SIP of the source substance (HMPCC)

Annex II Read-across report of in vitro cytogenicity study

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 in Table 5 (see the attached documents).

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)

  • Read-across justification of analogue approach to assess in vivo mammalian erythrocyte micronucleus test for Ligustral/Cyclal C/Trigustral: see the attached documents

Read-across justification full report of micronucleus assay in vivo

Annex I Lead registration SIP of the source substance (HMPCC)

Annex II Read-across report of micronucleus assay in vivo

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 in Table.

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)


Justification for selection of genetic toxicity endpoint
Overall conclusion from the three studies: the substance is not mutagenic.

Short description of key information:
In vitro:
Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance Ligustral/Cyclal C/Trigustral did not induce mutagenicity using S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 102 in the presence or absence of Aroclor 1254 –induced rat liver S9 metabolic activation (OECD 471, GLP);

Read-across from 3 and 4-( 4-Hydroxy-4-methy lpenty 1 )-3 -cyclohexene-1-carboxaldehyde (EC No: 915-617-9):
Chromosome aberration (in vitro mammalian cell cytogenicity): the substance 3 and 4-( 4-Hydroxy-4-methy lpenty 1 )-3 -cyclohexene-1-carboxaldehyde (EC No: 915-617-9), an analogue of Ligustral/Cyclal C/Trigustral (CAS No. 68039-49-6), was concluded to be positive for the induction of structural chromosome aberrations in the presence of Aroclor 1254-induced rat liver S9, negative for the induction of structural aberrations in the absence of Aroclor 1254-induced rat liver S9, and negative for the induction of numerical chromosome aberrations in the presence and absence of Aroclor 1254-induced rat liver S9 in Chinese hamster ovary (CHO) cells. (OECD 473, GLP);

In vivo:

Read-across from 3 and 4-( 4-Hydroxy-4-methylpenty 1 )-3 -cyclohexene-1-carboxaldehyde (EC No: 915-617-9):

In vivo mammalian erythrocyte micronucleus test: the substance 3 and 4-( 4-Hydroxy-4-methy lpenty 1 )-3 -cyclohexene-1-carboxaldehyde (EC No: 915-617-9), an analogue of Ligustral/Cyclal C/Trigustral (CAS No. 68039-49-6), was concluded to be negative (OECD 474, GLP).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available information in the dossier, the substance Ligustral/Cyclal C/Trigustral (CAS No. 68039-49-6) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.