Registration Dossier

Administrative data

Description of key information

Read-across from 3 and 4-( 4-Hydroxy-4-methy lpentyl )-3 -cyclohexene-1-carboxaldehyde-subacute NOAEL (rat):  150 mg/kg bw/day (OECD 407, GLP)
Read-across from L-perillyl alcohol-subchronic NOAEL (rat): 120 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 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 metabolism route 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 for repeated dose toxicity study of Ligustral/Cyclal C/Trigustral is available. Therefore, read-across from the existing 28 days repeated dose oral toxicity test of the source substance is considered as an appropriate adaptation to the standard information requirements of Annex IX, 8.6.1 of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD® (SD) IGS BR strain rat
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: approximately 6 to 8 weeks old
- Weight at study initiation: the males weighed 145 to 184g, the females weighed 128 to 166g
- Housing: The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): Pellet diet 5LF2 (Certified diet) with certificate of analysis, ad libitum
- Water (e.g. ad libitum): Main drinking water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
Route of administration:
oral: gavage
Vehicle:
other: Arachis oil BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The test substance was administered by gavage using a stainless steel cannula attached to a disposable plastic syringe.


VEHICLE
- Concentration in vehicle: 3.75 mg/mL at 15 and 150 mg/kg bw/day, 250 mg/ml at 1000 mg/kg bw/day
- Amount of vehicle (if gavage): 4 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

The concentration of HMPCC prepared at an appropriate concentration as a solution in arachis oil BP was determined using GC. The concentrations assayed were 3.75, 37.5 and 250 mg/mL. Homogeneity and stability determinations indicated that formulations to be stable for at least 14 days. Formulations were prepared weekly and stored at +4°C in the dark.

Verification of HMPCC concentrations over the 4 week dosing period were determined and indicated that the prepared formulations were within± 13% of the nominal concentration. For more information refer to Appendix 15


Duration of treatment / exposure:
twenty-eight consecutive days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 15, 150 and 1000 mg/kg bw/day
Basis:
other: nominal conc
No. of animals per sex per dose:
5 female and 5 male per dose
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations will be performed immediately before dosing and one hour after dosing. An additional observation will be made five hours after dosing during the normal working week (not at weekend or on public holidays)

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 and at weekly intervals thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (Day 28)
- Animals fasted: No
- How many animals: all surviving animals from each test and control group
- The following parameters were examined.
Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Het), Erythrocyte indices (including mean corpuscular haemoglobin (MCH); mean corpuscular volume (MCV); mean corpuscular haemoglobin concentration (MCHC)), Total leucocyte count (WBC), Differential leucocyte count (including neutrophils (Neut) , lymphocytes (Lymph) , monocytes (Mono) , eosinophils (Eos) , basophils (Bas)), Platelet count (PL T), Reticulocyte count (Retic), Prothrombin time (CT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (Day 28)
- Animals fasted: No
- How many animals: all surviving animals from each test and control group
- The following parameters were examined:
Urea, Glucose, Total protein (Tot.Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASA T), Alanine aminotransf, rase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes


OTHER:
Functional Observations(including Behavioural Assessments, Functional Performance Tests, Sensory Reactivity);
Pathology (including Organ Weights and Histopathology )
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals were subjected to a full internal and external examination and any macroscopic abnormaliites were records (see Organ weight table below)

HISTOPATHOLOGY: Yes (see Histopathology table below)
Other examinations:
Functional Observations(including Behavioural Assessments, Functional Performance Tests, Sensory Reactivity)
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.

All data were summarised in tabular form. Where appropriate, quantitative data were analysed by the ProvantisTM Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for
parametric data or the Shir!ey Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control
group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
(See details on results)
Mortality:
mortality observed, treatment-related
Description (incidence):
(See details on results)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
(See details on results)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
(See details on results)
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
(See details on results)
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
(See details on results)
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
(See details on results)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
(See details on results)
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
(See details on results)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
(See details on results)
Gross pathological findings:
no effects observed
Description (incidence and severity):
(See details on results)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(See details on results)
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Clinical Observations: Animals of either sex treated with 1000 mg/kg/day showed transient increased salivation around the time of dosing from Day 3 onwads. Isolated incidents of red/brown staining around the mouth and scab formation (males only) were also evident between Days 13 and 25. Episodes of respiratory pattern changes and hunched posture were evident in animals of either sex treated with 1000 mg/kg/day during the final two weeks of the study. No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day (Table 1).

OTHER FINDINGS
Behavioural Assessment. No toxicologically significant changes were detected.
Functional Peiformance Tests. There were no treatment-related changes in the functional performance parameters measured.
Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity. (Tables 2-5)

Mortality: One male treated with 15 mg/kg/day was found dead on Day 20. While the aetiology of this death is uncertain, in isolation, it was considered not to be toxicologically significant. There were no further unscheduled deaths.

BODY WEIGHT AND WEIGHT GAIN
Males treated with 1000 mg/kg/day showed a reduction in bodyweight gain during Week 1. No significant effects were detected in females treated with 1000 mg/kg/day or animals of either sex treated with 150 or 15 mg/kg/day. Females treated with 1000 mg/kg/day did show a slight reduction in bodyweight gain during Week 1 and 4 only whilst males from this treatment group and 150 mg/kg/day showed a reduction in bodyweight gain throughout the remaining treatment period. (Tables 6-7)

FOOD CONSUMPTION
Males treated with 1000 mg/kg/day showed a reduction in food consumption during Week 1 only. No adverse effect on food consumption was detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 150 or 15 mg/kg bw/day. (Table 8)

FOOD EFFICIENCY
Males treated with 1000 mg/kg bw/day showed a reduction in food efficiency during Week 1 only. No adverse effect on food efficiency was detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 150 or 15 mg/kg bw/day.(Table 9)

HAEMATOLOGY
No treatment-related changes were detected.(Table 10)

CLINICAL CHEMISTRY
Animals of either sex treated with 1000 mg/kg bw/day showed an increase in plasma enzymes, albumin, and albumin/globulin ratio. In addition males also showed a reduction in plasma cholesterol, total protein and glucose. No such treatment-related effects were detected in animals of either sex treated with 15 mg/kg bw/day. In females treated with 150 mg/kg bw/day alanine aminotransferase and alkaline phosphatase were increased. Males treated with 150 mg/kg bw/day showed increased albumin levels and a reduction in cholesterol.(Table 11)

ORGAN WEIGHTS
Animals of either sex treated with 1000 mg/kg bw/day showed an increase in absolute and relative liver weight, with the effect extending to 150 mg/kg bw/day males. Males treated with 1000 mg/kg bw/day also showed an increase in absolute and relative kidney weight compared with controls. No such effects were detected in 150 mg/kg bw/day females or in animals of either sex treated with 15 mg/kg bw/day.(Table 12)

GROSS PATHOLOGY
Necropsy: No treatment-related macroscopic abnormalities were detected.(Table 13)

HISTOPATHOLOGY: NON-NEOPLASTIC

The following treatment-related changes were detected:
LIVER: Centrilobular or generalised hepatocyte enlargement, frequently with associated focal centrilobular inflantmatory cell infiltrates, were observed in relation to treatment for animals of either sex treated with 1000 mg/kg bw/day. In addition, centrilobular hepatocyte necrosis was seen for males treated with 1000 mg/kg bw/day. Three males treated with 150 mg/kg bw/day also demonstrated hepatocyte enlargement.

KIDNEY: The proximal tubular epithelium of males treated with 1000 mg/kg bw/day was observed to be generally denser than that for control rats. Changes in cytoplasmic density are occasionally observed as a consequence of test material administration and are frequently adaptive in nature in the absence of associated degenerative changes. Animals from the remaining treatment groups were not similarly affected (Table 14).

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no effects was observed
Critical effects observed:
not specified

Study report attachments:

Table 1 Summary incidence of daily clinical observations

Tables 2-5 Behavioural and functional assessments

Tables 6-9 Group mean weekly bodyweights and food consumption

Tables 10-12 Group mean haematological, blood chemistry and organ weights

Tables 13-14 Summary incidence necropsy and histopathological findings

Appendix 15 Chemical analysis of test material formulations, methods and results

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 3).

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 is 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 Ames test results (Refs. 9-14).

In the OECD QSAR Toolbox v3.1, the profilers ‘Toxic hazard classification by Cramer (original)’ and ‘Toxic hazard classification by Cramer (by extension)’ were applied to Ligustral/Cyclal C/Trigustral (CAS No. 68039-49-6) and HMPCC (CAS No. 51414-25-6/31906-04-4) as an indication of oral systemic toxicity. The target source substance is indicated as Cramer Class II and the source substance is indicated as Class III (Annex II Table 4). Class II contains substances which possess structures that are less innocuous than class I substances, but do not contain structural features suggestive of toxicity like those substances in class III. Class III contains substances with a chemical structure that permit no strong initial impression of safety and may even suggest a significant toxicity. The respective Classes indicate that read across from the source substance to the target substance should not represent an underestimation of the hazard associated with the oral repeated dose toxicity endpoint and the NOAEL value predicted for the target substance for this endpoint confirms this (see below).

For the source substance, a reliable (RL2) sub-acute repeated dose toxicity study (OECD 407/GLP) is available. In this key study, HMPCC was administered to four groups of Sprague-Dawley Crl:CD (SD) IGS BR male and female rats (5/sex/group) by gavage at dose levels of 0, 15, 150 and 1000 mg/kg bw/day. The following treatment-related effects were noted : bodyweight development, dietary intake and food efficiency were reduced in males in Week 1. Blood chemistry indicated increases in enzyme levels at 1000 mg/kg bw/day in males and females and in females and 150 mg/kg bw/day. Plasma alanine amintransferase, aspartate aminotransferase and alkaline phosphatase were elevated together wtih albumin and albumin/globulin ratio which suggests a hepatic effect. Absolute and relative liver weights were increased for animals of either sex treated with 1000 mg/kg bw/day, with effect extending to males at 150 mg/kg bw/day. Microscopic examination revealed centrilobular or generalised hepatocyte enlargement, frequently with associated focal centrilobular inflammatory cell infiltrates in animals of either sex treated with 1000 mg/kg bw/day together with single cell necrosis among centrilobular hepatocytes for males only. Three males treated with 150 mg/kg bw/day also demonstrated hepatocyte enlargement, however in the absence of associated inflammatory or degenerative changes at this dose level, this condition is almost certainly adaptive in nature. Histopathological changes were also identified in the kidneys but were confined to 1000 mg/kg bw/day males. The proximal tubular epithelium was observed to be generally denser than that for control rats and was associated with an increase in male absolute and relative kidney weights. In the absence of any degenerative kidney changes or any evidence of an effect on renal function, the changes in cytoplasmic density were probably adaptive in nature. The NOEL was 15 mg/kg bw/day for females and males. The NOAEL was 150 mg/kg bw/day for females and males. (Ref. 16) A NOAEL of 150 mg/kg bw/day is predicted for Ligustral/Cyclal C/Trigustral for the subacute repeated dose toxicity endpoint.

Classification and labeling

Based on available data in the IUCLID dossier, the target substance is classified according to CLP (1272/2008) as Skin Sensitization Category 1 and Skin Irritation Category 2 for human health hazards. 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 : 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 specific target organ toxicity – repeated (25-04-13).

The classification for specific target organ toxicity - repeated for Ligustral/Cyclal C/Trigustral is based on the results from the 28 day repeated dose toxicity data (read across from HMPCC; EC No. 915-617-9) and 90 day repeated dose toxicity data (read across from L-perillyl alcohol; EC No. 208-639-9). Based on the data presented from both of these studies, Ligustral/Cyclal C/Trigustral does not need to be classified as specific target organ toxicity - repeated when considering the criteria outlined in Annex I of 1272/2008/EC.

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 using the source substance for read across to the target substance will not underestimate the hazard associated with the repeated dose toxicity endpoint.

In the 28 days repeated dose toxicity study conducted with source substance, the histopathological changes noted in the liver in three male rats at the 150 mg/kg bw/day dose were considered adaptive changes and so a NOAEL of 150 mg/kg bw/day was derived from this study. Together with the results from the 90 day repeated dose toxicity data (read across from L-perillyl alcohol; EC No. 208-639-9), Ligustral/Cyclal C/Trigustral is not classified for specific target organ toxicity – repeated. 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. A NOAEL of 150 mg/kg bw/day is predicted for Ligustral/Cyclal C/Trigustral for the subacute repeated dose toxicity endpoint.

Uncertainty in the prediction: The main sources of uncertainty in the read across prediction are extrapolation uncertainty and parameter uncertainty (selection of the dose descriptor). The former relates to the choice of the source substance and the evidence we have discussed in the report decreases the uncertainty in the choice of source substance. We do not consider that the dose descriptor from the read across study (NOAEL: 150 mg/kg bw/day) underestimates the hazard associated with the target substance for the 28 day repeated dose toxicity endpoint and based on the overall classification conclusion from the repeated dose toxicity endpoint (28 day and 90 day read across studies), this decreases the uncertainty in the choice of dose descriptor.

Therefore, based on the considerations above, it can be concluded that the results of the 28 days repeated dose study conducted in the rats 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, 8.6.1.

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, and B: 3-(4-Hydroxy-4-methylpentyl)cyclohex-3-ene carboxaldehyde)

Conclusions:
In an oral 28-day repeated dose toxicity study in the rat, HMPCC was administed at dose levels of 15, 150, and 1000 mg/kg bw/day. HMPCC resulted in treatment-related effects at 1,000 mg/kg bw/day in both sexes and in certain endpoints at 150 mg/kg bw/day in both sexes. The NOEL value for males and females was 15 mg/kg bw/day. At 150 mg/kg bw/day, the histopathological changes in the liver noted in 3 males were considered adaptive changes. The NOAEL value for males and females was 150 mg/kg bw/day. These results are suitable for REACH purposes and using a read-across approach, a NOAEL value of 150 mg/kg bw/day is predicted for Ligustral/Cyclal C/Trigustral.

Executive summary:

In a subacute toxicity study (2153-0001), HMPCC was administered to four groups of Sprague-Dawley Crl:CD (SD) IGS BR male and female rats (5/sex/group) by gavage at dose levels of 0, 15, 150 and 1000 mg/kg bw/day.

The following treatment-related effects were noted :bodyweight development, dietary intake and food efficiency were reduced in males in Week 1. Blood chemistry indicated increases in enzyme levels at 1000 mg/kg bw/day in males and females and in females adn 150 mg/kg bw/day. Plasma alanine amintransferase, aspartate aminotransferase and alkaline phosphatase were elevated together wtih albumin and albumin/globulin ratio which suggests a hepatic effect. Absolute and relative liver weight were increased for animals of either sex treated with 1000 mg/kg bw/day, with effect extending to males at 150 mg/kg bw/day. Microscopic examination revealed centrilobular or generalised hepatocyte enlargement, frequently with associated focal centrilobular inflammatory cell infiltrates in animals of either sex treated with 1000 mg/kg bw/day together with single cell necrosis among centrilobular hepatocytes for males only. Three males treated with 150 mg/kg bw/day also demonstrated hepatocyte enlargement, however in the absence of associated inflammatory or degenerative changes at this dose level, this condition is almost certainly adaptive in nature. Histopathological changes were also identified in the kidneys but were confined to 1000 mg/kg bw/day males. The proximal tubular epithelium was obsesrved to be generally denser that that for control rats and was associated with an increase in male absolute and relative kidney weights. In the absence of any degenerative kidney changes or nay evidence of an effect on renal function, the changes in cytoplasmic density were probably adaptive in nature. The NOEL was15 mg/kg bw/day for females and males. The NOAEL was 150 mg/kg bw/day for females and males.

This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral study (OECD 407) in the rat.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key studies were given reliability Klimisch scores of 2. When assessed together the overall quality of the database is medium. This decreases the uncertainty in the prediction of the NOAEL for Ligustral/Cyclal C/Trigustral.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There is no repeated dose toxicity data available for Ligustral/Cyclal C/Trigustral (CAS 68039-49-6). A read-across approach was conducted with data from two repeated dose toxicity studies from 3 and 4-( 4-Hydroxy-4-methy lpentyl )-3 -cyclohexene-1-carboxaldehyde (HMPCC) EC No: 915-617-9) and L-perillyl alcohol (EC No. 208-639-9) .

  • Read-across justification of analogue approach to assess 28 days repeated dose toxicity for Ligustral/Cyclal C/Trigustral: see the attached documents

Read-across justification full report of 28 days repeated dose toxicity study

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

Annex II Read-across report of 28 days repeated dose toxicity 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 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 3).

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 is 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 Ames test results (Refs. 9-14).

In the OECD QSAR Toolbox v3.1, the profilers ‘Toxic hazard classification by Cramer (original)’ and ‘Toxic hazard classification by Cramer (by extension)’ were applied to Ligustral/Cyclal C/Trigustral (CAS No. 68039-49-6) and HMPCC (CAS No. 51414-25-6/31906-04-4) as an indication of oral systemic toxicity. The target source substance is indicated as Cramer Class II and the source substance is indicated as Class III (Annex II Table 4). Class II contains substances which possess structures that are less innocuous than class I substances, but do not contain structural features suggestive of toxicity like those substances in class III. Class III contains substances with a chemical structure that permit no strong initial impression of safety and may even suggest a significant toxicity. The respective Classes indicate that read across from the source substance to the target substance should not represent an underestimation of the hazard associated with the oral repeated dose toxicity endpoint and the NOAEL value predicted for the target substance for this endpoint confirms this (see below).

For the source substance, a reliable (RL2) sub-acute repeated dose toxicity study (OECD 407/GLP) is available. In this key study, HMPCC was administered to four groups of Sprague-Dawley Crl:CD (SD) IGS BR male and female rats (5/sex/group) by gavage at dose levels of 0, 15, 150 and 1000 mg/kg bw/day. The following treatment-related effects were noted : bodyweight development, dietary intake and food efficiency were reduced in males in Week 1. Blood chemistry indicated increases in enzyme levels at 1000 mg/kg bw/day in males and females and in females and 150 mg/kg bw/day. Plasma alanine amintransferase, aspartate aminotransferase and alkaline phosphatase were elevated together wtih albumin and albumin/globulin ratio which suggests a hepatic effect. Absolute and relative liver weights were increased for animals of either sex treated with 1000 mg/kg bw/day, with effect extending to males at 150 mg/kg bw/day. Microscopic examination revealed centrilobular or generalised hepatocyte enlargement, frequently with associated focal centrilobular inflammatory cell infiltrates in animals of either sex treated with 1000 mg/kg bw/day together with single cell necrosis among centrilobular hepatocytes for males only. Three males treated with 150 mg/kg bw/day also demonstrated hepatocyte enlargement, however in the absence of associated inflammatory or degenerative changes at this dose level, this condition is almost certainly adaptive in nature. Histopathological changes were also identified in the kidneys but were confined to 1000 mg/kg bw/day males. The proximal tubular epithelium was observed to be generally denser than that for control rats and was associated with an increase in male absolute and relative kidney weights. In the absence of any degenerative kidney changes or any evidence of an effect on renal function, the changes in cytoplasmic density were probably adaptive in nature. The NOEL was 15 mg/kg bw/day for females and males. The NOAEL was 150 mg/kg bw/day for females and males. (Ref. 16) A NOAEL of 150 mg/kg bw/day is predicted for Ligustral/Cyclal C/Trigustral for the subacute repeated dose toxicity endpoint.

Classification and labeling

Based on available data in the IUCLID dossier, the target substance is classified according to CLP (1272/2008) as Skin Sensitization Category 1 and Skin Irritation Category 2 for human health hazards. 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 : 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 specific target organ toxicity – repeated (25-04-13).

The classification for specific target organ toxicity - repeated for Ligustral/Cyclal C/Trigustral is based on the results from the 28 day repeated dose toxicity data (read across from HMPCC; EC No. 915-617-9) and 90 day repeated dose toxicity data (read across from L-perillyl alcohol; EC No. 208-639-9). Based on the data presented from both of these studies, Ligustral/Cyclal C/Trigustral does not need to be classified as specific target organ toxicity - repeated when considering the criteria outlined in Annex I of 1272/2008/EC.

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 using the source substance for read across to the target substance will not underestimate the hazard associated with the repeated dose toxicity endpoint.

In the 28 days repeated dose toxicity study conducted with source substance, the histopathological changes noted in the liver in three male rats at the 150 mg/kg bw/day dose were considered adaptive changes and so a NOAEL of 150 mg/kg bw/day was derived from this study. Together with the results from the 90 day repeated dose toxicity data (read across from L-perillyl alcohol; EC No. 208-639-9), Ligustral/Cyclal C/Trigustral is not classified for specific target organ toxicity – repeated. 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. A NOAEL of 150 mg/kg bw/day is predicted for Ligustral/Cyclal C/Trigustral for the subacute repeated dose toxicity endpoint.

Uncertainty in the prediction: The main sources of uncertainty in the read across prediction are extrapolation uncertainty and parameter uncertainty (selection of the dose descriptor). The former relates to the choice of the source substance and the evidence we have discussed in the report decreases the uncertainty in the choice of source substance. We do not consider that the dose descriptor from the read across study (NOAEL: 150 mg/kg bw/day) underestimates the hazard associated with the target substance for the 28 day repeated dose toxicity endpoint and based on the overall classification conclusion from the repeated dose toxicity endpoint (28 day and 90 day read across studies), this decreases the uncertainty in the choice of dose descriptor.

Therefore, based on the considerations above, it can be concluded that the results of the 28 days repeated dose study conducted in the rats 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, 8.6.1.

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, and B: 3-(4-Hydroxy-4-methylpentyl)cyclohex-3-ene carboxaldehyde)

  • Read-across justification of analogue approach to assess 90 days repeated dose toxicity study for Ligustral/Cyclal C/Trigustral: see the attached documents

Read-across justification full report of 90 days repeated dose toxicity study

Annex I Read-across report of 90 days repeated dose toxicity 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 side chains do not significantly influence the physicochemical properties of both substances, i.e. vapour pressure, partition coefficient and water solubility. The molecular weight of the target substance (Ligustral/Cyclal C/Trigustral) is 138 g/mol and the molecular weight of the source substance (L-perillyl alcohol) is 152 g/mol. The partition coefficient (Log Kow) of the target substance is 2.7 and for source substance is 3.2. The water solubility for both of the target and source substances is in the soluble range, (910 mg/L at 20 °C for target substance and 1250 mg/L at 25 °C for source substance). Neither of the substances is volatile with a vapour pressure 0.271 mmHg at 20°C for the target substance and 0.00628 mmHg at 20 °C for the source substance.

Toxicokinetics

Experimental data

No experimental data on absorption, distribution, metabolism or excretion is available for the target substance. For the source substance, several in vivo studies are available that investigated the metabolic/excretory pathways. In a pharmacokinetics study, L-perillyl alcohol was administrated to a male and females beagle dogs at a dose of 250 mg/kg bw; the results indicated that L-perillyl alcohol was rapidly absorbed from the gastrointestinal tract, followed by efficient first-pass metabolism to perillic acid and dihydroperillic acid (L.R. Phillips, 1995). The same plasma metabolites are identified four hours after female Wistar-Furth rats are administered a single dose of 1,000 mg/kg L-perillyl alcohol via gavage. No trace of the parent substance was found at any point, including 15 minutes post gavage (Haag and Gould, 1994). Another in vivo study in male Wistar rats confirmed the oxidation of L-perillyl alcohol to the carboxylic acid derivative involved the aldehyde form as an intermediate (Boon et al., 2000). In vitro, L-perillyl alcohol was oxidized by alcohol dehydrogenase and aldehyde dehydrogenase to perillaldehyde and perillic acid, respectively in rat hepatocytes. It was indicated that the major excretory pathways for perillyl alcohol (and perillaldehyde and perillic acid metabolites) were as ether and acyl glucuronides.

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), water solubility (900 - 1300 mg/L) and moderate log Kow values (Ligustral/Cyclal C/Trigustral: Log Kow 2.7; L-perillyl alcohol: Log Kow 3.2) 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 target substance is expected to be metabolized via oxidation of the aldehyde to a carboxylic acid group (see predicted metabolism below) and conjugated in Phase II reactions to facilitate excretion, mainly in the urine.

Other data in the literature

An endocyclic bond is present in both the target and source substances, so reduction of this bond by enzymes in the intestinal microflora may be possible. These enzymes have not been characterized and the reactions may take place either before absorption or after biliary excretion. The acid metabolites described above may also undergo aromatization of the ring to yield a hippuric acid derivative (JECFA Food Additives Series, 2012).

Predicted data for metabolism

Based on the SMARTCyp - Cytochrome P450 - Mediated Metabolism simulator (Toxtree v2.5.0), the primary site of metabolism of the target chemical is estimated to be oxidation of the aldehyde to a carboxylic acid group (Rank 1). The subsequent sites indicate aliphatic hydroxylation and possibly epoxidation as a possibility for the target chemical (Annex I – Table 3). Based on the experimental data from the source substance, an aldehyde metabolite was considered as an intermediate which was be oxidised rapidly to a carboxylic acid metabolite. It is expected that the source substance will also be oxidised to a carboxylic acid metabolite and conjugated in Phase II reactions to facilitate excretion.

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 L-perillyl alcohol a longer isopropenyl chain and lack of a methyl group beside the alcohol. The methyl group in Ligustral/Cyclal C/Trigustral may be hydroxylated which will enhance polarity and excretion in the urine. The isopropenyl substituent may be considered nonreactive in light of data available on proposed metabolic fate of terpenoid aldehydes e.g. perillaldehyde in mammals. The alcohol group may be conjugated directly to glucuronic acid which facilitates excretion in the urine. The terpenoid primary 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 based on current intake levels (JECFA Food Additives Series, 2012).

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

Comparison of data from human health endpoints

Toxicity data of the target and source substances

As is 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 was noted for both.

In the Toxtree v2.5.0, the profilers ‘Cramer rules’ and ‘Cramer rules, with extensions’ were applied to Ligustral/Cyclal C/Trigustral (CAS No. 68039-49-6) and L-perillyl alcohol (CAS No. 536-59-4) as an indication of oral systemic toxicity. Both of the target substance and source substance are indicated as Cramer Class I (Annex I – Table 4). The same Classes suggest that read across from the source substance to the target substance should not represent an underestimation of the hazard associated with the oral repeated dose toxicity endpoint and the NOAEL value predicted for the target substance for this endpoint confirms this (see below).

For the source substance a reliable (RL 2) sub-chronic repeated dose toxicity study is available. In this key study, L-perillyl alcohol was administered to three groups of Fischer F344 rats male and female rats (10/sex/group) by gavage at dose levels of 40, 120 and 400 mg/kg bw/day. No mortalities or clinical signs of toxicity were reported during the study. A significant decrease in body weight was noted in the high-dose group of males. No treatment-related effects on hematology or clinical chemistry parameters were noted. Absolute kidney, liver, and lung weights were increased in high-dose females, but there were no histopathological changes in these organs. The LOAEL is 400 mg/kg bw/day based on decreased body weight in males. The NOAEL is 120 mg/kg bw/day (Ref. 14). A NOAEL of 120 mg/kg bw/day is predicted for Ligustral/Cyclal C/Trigustral for the subchronic repeated dose toxicity endpoint.

Classification and labeling

Based on available data in the IUCLID dossier, the target substance is classified according to CLP (1272/2008) as Skin Sensitization Category 1 and Skin Irritation Category 2 for human health hazards. In ECHA’s C&L inventory database, the current self-classification and labelling for L-perillyl alcohol based on the CLP Regulation criteria indicate the following classification: Skin Irritation Category 2 (03-05-13), Eye Irritation Category 2 (03-05-13) and Skin sensitization Category 1 (03-05-13). It is not classified for specific target organ toxicity – repeated (03-05-13).

The classification for specific target organ toxicity - repeated for Ligustral/Cyclal C/Trigustral is based on the results from the 28 day repeated dose toxicity data (read across from HMPCC; EC No. 915-617-9) and 90 day repeated dose toxicity data (read across from L-perillyl alcohol; EC No. 208-639-9). Based on the data presented from both of these studies, Ligustral/Cyclal C/Trigustral does not need to be classified as specific target organ toxicity - repeated when considering the criteria outlined in Annex I of 1272/2008/EC.

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 using the source substance for read across to the target substance will not underestimate the hazard associated with the repeated dose toxicity endpoint.

In the 28 days repeated dose toxicity study conducted with source substance, the histopathological changes noted in the liver in three male rats at the 150 mg/kg bw/day dose were considered adaptive changes and so a NOAEL of 150 mg/kg bw/day was derived from this study. Together with the results from the 90 day repeated dose toxicity data (read across from L-perillyl alcohol; EC No. 208-639-9), Ligustral/Cyclal C/Trigustral is not classified for specific target organ toxicity – repeated. 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 L-perillyl alcohol. A NOAEL of 120 mg/kg bw/day is predicted for Ligustral/Cyclal C/Trigustral for the subchronic repeated dose toxicity endpoint.

Uncertainty in the prediction: The quality of the data is the main source of uncertainty in the read across prediction. The NOAEL value derived from the L-perillyl alcohol 90 day repeated dose toxicity study was 120 mg/kg bw/day. Based on this value and the lack of classification for the source substance for specific target organ toxicity – repeated, the read across prediction to the target substance should not represent an underestimation of the hazard for the 90 day repeated dose toxicity endpoint. These considerations decrease the uncertainty in the read across prediction of the NOAEL for Ligustral/Cyclal C/Trigustral.

Therefore, based on the considerations above, it can be concluded that the results of the 90 days repeated dose study conducted in the rats 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, 8.6.2.

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

L-perillyl alcohol;4-isopropenylcyclohex-1-en-1-ylmethanol;1-Cyclohexene-1-methanol, 4-(1-methylethenyl)-; (-)-perillyl alcohol

CAS No.

68039-49-6

536-59-4

Information on the physicochemical properties

Molecular weight

138

152

Physical state

liquid

liquid

Vapour pressure

36 Pa(0.271 mmHg) at 20°C

 

Ref.6

0.00628mmHg at 25°C(the chemical database: http://ull.chemistry.uakron.edu/erd/Chemicals/12000/11380.html)

Partition coefficient (Log Kow)

2.7

 

Ref. 7

3.17 (ChemID plus database)

Water solubility

910 mg/L at 20 °C

 

Ref. 8

1250mg/L at 25 °C(calculated)

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. 9

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. 10

Rat

Oral

LD50 = 2100 mg/kg bw

 

(JECFA Food Additives Series, 2012; ChemID plus database)

Acute dermal toxicity

rabbit

LD50: > 5000 mg/kg bw

2 (reliable with restrictions)

key study

 

Ref. 11

Rabbit

Dermal

LD50>5000 mg/kg bw

 

(ChemID plus database)

Skin irritation/corrosiveness

Rabbit

OECD Guideline 404(skin irritation)

2 (reliable with restrictions)

weight of evidence

skin irritant

 

Ref. 12

Rabbit

OECD Guideline 404skin irritation

2 (reliable with restrictions)

Skin irritant

 

(ECETOC TR066, 1995, page 50)

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. 12

rabbit

2 (reliable with restrictions)

weight of evidence

skin irritant

 

Ref. 12

Repeated dose toxicity

Read-across fromHMPCC

Rat male/female

Subacute (oral: gavage)

OECD Guidline 407

2 (reliable with restrictions)

NOAEL=150 mg/kg bw/day

Key study

 

Ref.14

Rat male/female

Subacute (oral: gavage)

Exposure: 28 days

NOEL=200 mg/kg bw/day

4 (Not assignable)

 

(National Cancer Institute 1996)

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

 

Ref.14

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.14

Environmental information

Hydrolysis

Nothydrolysis

 

Ref. 15

Be not expected to undergo hydrolysis in the environment due to a lack of hydrolysable functional groups

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. 16

log Koc = 2.0( Koc estimated from MCI, KOCWIN v2.00)

log Koc = 2.2( Koc estimated from Log Kow, KOCWIN v2.00)



Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The 90 days repeated dose read-across study from L-perillyl alcohol (EC No. 208-639-9) is of longer duration, but lacks detailed documentation about study method. The NOAEL value is 120 mg/kg bw/day.

The 28 day repeated dose read-across study from 3 and 4-( 4-Hydroxy-4-methy lpentyl )-3 -cyclohexene-1-carboxaldehyde (EC No: 915-617-9) was chosen as it was conducted according to OECD 407, is GLP compliant and is extensively documented. The NOAEL value is 150 mg/kg bw/day.

Justification for classification or non-classification

Based on available information in the dossier, the substance Ligustral/Cyclal C/Trigustral (CAS 68039-49-6) does not need to be classified as specific target organ toxicity (repeated) when considering the criteria outlined in Annex I of 1272/2008/EC based on the results of the read-across study from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde and L-perillyl alcohol.