Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information
Short description of key information:
Toxicity to Reproduction (Screening study):
In accordance with Column 2 of ANNEX VIII of the REACH regulation, screening for reproductive/developmental toxicity study does not need to be conducted as an oral developmental toxicity study is available.(Read-across study from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde).

Toxicity to Reproduction (Two generation study):
In accordance with Column 2 of ANNEX IX of REACH regulation, a two generation reproductive toxicity study does not need not to be conducted as the existing 28 day study (Read-across study from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde) does not indicate adverse effects on reproductive organs or tissues.

Justification for selection of Effect on fertility via oral route:
In accordance with Column 2 of ANNEX VIII of the REACH regulation, screening for reproductive/developmental toxicity study does not need to be conducted as a developmental toxicity study is available.(Read-across study from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde).


In accordance with Column 2 of ANNEX IX of REACH regulation, a two generation reproductive toxicity study does not need not to be conducted as the existing 28 day study (Read-across study from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde) does not indicate adverse effects on reproductive organs or tissues.

Justification for selection of Effect on fertility via inhalation route:
In accordance with Column 2 of ANNEX VIII of the REACH regulation, screening for reproductive/developmental toxicity study does not need to be conducted as an oral developmental toxicity study is available.(Read-across study from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde).

In accordance with Column 2 of ANNEX IX of REACH regulation, a two generation reproductive toxicity study does not need not to be conducted as the existing 28 day study (Read-across study from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde) does not indicate adverse effects on reproductive organs or tissues.

Justification for selection of Effect on fertility via dermal route:
In accordance with Column 2 of ANNEX VIII of the REACH regulation, screening for reproductive/developmental toxicity study does not need to be conducted as an oral developmental toxicity study is available.(Read-across study from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde).

In accordance with Column 2 of ANNEX IX of REACH regulation, a two generation reproductive toxicity study does not need not to be conducted as the existing 28 day study (Read-across study from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde) does not indicate adverse effects on reproductive organs or tissues.

Effects on developmental toxicity

Description of key information
Developmental toxicity study (OECD 415, GLP; read-across from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde):
NOAEL (developmental): 25 mg/kg bw/day
NOEL (maternal): 100 mg/kg bw/day
Link to relevant study records
Reference
Endpoint:
developmental toxicity
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 toxicokinetics of the substances. The target substance (Ligustral/Cyclal C/Trigustral) is a multi-constituent substance with 2 main constituents which are diastereomers (3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-, (1R,2R)-rel- and 3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-, (1R,2S)-rel-; Table 1). The source substance (HMPCC) is also a multi-constituent substance with 2 main constituents with a major (1,4-disubstituted-cyclohexenyl) and minor (1,3-disubstituted cyclohexenyl substance; Table 2) isomer. The target substance (Ligustral/Cyclal C/Trigustral) and source substance (HMPCC) have structurally similar backbones which are an unsaturated six member cyclohexyl ring with an attached ethylaldehyde group and the alkyl chain linked to the double bond. The structural differences consist of an alkyl chain which is methyl in Ligustral/Cyclal C/Trigustral and in HMPCC a longer 4-hydroxy-4-methylpentyl chain and lack of a methyl group beside the ethylaldehyde. There is also a methyl group at the meta position in Ligustral/Cyclal C/Trigustral. These differences are chemically simple structures and their impact on the read across is discussed further below. Similar expected metabolism is likely between the target and source substances. No reliable data on developmental toxicity of Ligustral/Cyclal C/Trigustral is available. Therefore, read-across from an existing one generation reproduction toxicity study of the source substance and 2 supporting studies: (i) Oral (Gavage) Repeated-Dose Toxicity Study of HMPCC in Adult Rats, Including Neonatal Evaluation and (ii) Oral (Gavage) Repeated-Dose Toxicity Study of HMPCC in Adult Rats With Determination of Milk Levels, Including Neonatal Evaluation and Recovery Period of the source substance are considered as an appropriate adaptation to the standard information requirements of Annex IX, 8.7.2 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:
other: OECD Guideline 415 (One Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD( (SD) IGS BR strain
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, UK.
- Age at study initiation: males: six weeks old, females: ten weeks old
- Weight at study initiation: males: 191 to 255g, females: 211 to 271g
- Housing: Initially, all animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase, animals were transferred to similar cages on a one male: one female basis.
Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually, during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes.
- Diet : Pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK) ad libitum
- Water : Mains drinking water, ad libitum
- Acclimation period: thirteen 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 f1uorescent 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 exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil BP (Lab 3 limited, Unit 1, Rossroad Business Centre, Northampton,
UK)
- Concentration in vehicle: 0, 6.25, 25 and 125 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Verification of HMPCC concentrations over the dosing period were determined and indicated that the prepared formulations were within acceptable limits of the nominal concentration. For more information refer to Appendix 29 (Chemical Analysis of Test Material Formulations, Methods and Results).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: yes
- M/F ratio per cage: 1:1
- Length of cohabitation: twenty-one days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages.
Duration of treatment / exposure:
1. Before mating, all animlas were dosed for 76 days.
2. All animals were paired within administration for 21 days.
3. Pregnant females were dosed until day 21 post partum, except during littering/parturition. Non-pregnant females were dosed after Day 25 post coitum.
Frequency of treatment:
daily
Duration of test:
Nineteen consecutive weeks, including mating, gestation and lactation.
No. of animals per sex per dose:
24 males and 24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Preliminary fourteen day repeated dose oral range-finder was conducted at dose of 0, 500 and 1000 mg/kg bw/day in ratsfor selection of dose levels for use in an oral OECD 415 rat study. There were no unscheduled deaths. At dose of 1000 mg/kg bw/day, different clinical observations, body weight and necropy observation were found. There were no effects were detected at the dose of 500 mg/kg bw/day. Therefore, the dose levels for the main stude were: 25, 100, 500 mg/kg bw/day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, in pregnancy and parturition
- Time schedule: approximately 08:30, 12:30 and 16:30 hours

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours, after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends or public holiday.

BODY WEIGHT: Yes
- Time schedule for examinations: lndividual body weights were recorded on Day 0 (the day before the start of treatment) and then weekly for males until termination. Females were weighed weekly during maturation and daily during mating. Once mating was evident body weights were then recorded on Day 1, 4, 7, 14 and 21 of post coitum and post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- 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: Yes

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

POST-MORTEM EXAMINATIONS: Yes
- Organs examined: the following organs weighed before fixation: epididymides, seminal vesicles (with coagulating gland), ovaries, uterus (with cervix), pituitary gland, testes, and prostate

OTHER: Histopathology and Sperm Assessment
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [all per litter ]
- Skeletal examinations: No
- Head examinations: No
Statistics:
Organ weight (absolute and relative to terminal body weight), weekly body weight gain, litter weights, offspring body weights were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunne's test. Where Levene' s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOV A and Mann-Whitney ‘ U' test.

The non-parametric methods were also used to analyse implantation loss, offspring sex ratio, litter size and landmark developmental markers.

Histopathological data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Indices:
1. Mating performance and Fertility
Pre-coital lnterval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of matmg.
Fertility lndices:
mating index (%) = (number of animals mated/ number of animals paired) x 100
pregnancy index (100%) = (Numberof pregnant females/Number of animals mated) x 100

2. Gestation and Parturition Data
Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. Where the start of parturition occurred ovemight, the total was adjusted by subtracting half a day.
Parturition lndex (%) = (number of females delivering live offspring/number of pregnant females) x 100

3. Lactation Data

Live Birth and Viability lndices (%)
Live Birth Index = (Number of offspring alive on Day 1/Number of offspring born) x 100
Viabilitylndex 1 = (Number of offspring alive on Day 4/Numberof offspring alive on Day 1) x 100
Viability lndex 2= (Number of offspring alive on Day 7/Numberof offspring alive on Day 4) x 100
Viability lndex 3= (Number of offspring alive on Day 14/Numberof offspring alive on Day 7) x 100
Viability lndex 4= (Number of offspring alive on Day 21/Numberof offspring alive on Day 14) x 100
Viability lndex 5= (Number of offspring alive on Day 21/Numberof offspring alive on Day 1) x 100

Sex Ratio (% males)
Group mean values calculated from each litter value on Day 1 and 4 using the following formula: (Number of male offspring/Total number of offspring) x100

Implantation Losses (%)
pre-implantation loss = (Number of Corpora Lutea -Number of implantation sites/Number of Corpora Lutea)x 100
post-implantation loss = (Number of implantation sites - number of offspring/Number of implantation sites)x 100


Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No signification treatment related effects were observated in clinical observation, body weight and food/water consumption. Reproductive screening also showed no related effects in Oestrous Cycle Assessment, Mating and Gestation. In addition , there were no significant differences in Necropsy, Uterine examination, Organ Weights and Histopathology at dose of 100 mg/kg bw/day.

At the dose of 500 mg/kg bw/day, one male treated was killed in extremis, one female was dead and another two females were killed in extremis during parturition. Episodes of hunched posture, pilo-erection and tiptoe gait were evident in 500 mg/kg bw/day females during the fina1 week of gestation. Body weight gain for males treated was lower than control animals. A notable reduction in food consumption throughout lactation in females were observed. The adult rnale treated with 500 mg/kg bw/day that was killed in extremis showed gaseous distension in the gastro-intestinal tract. The fernale treated with 500 mg/kgbw/day that was found dead around parturition had twenty-one foetuses in-utero. The two females from this treatment group that were killed in extremis both had dead/inactive foetuses in-utero and red/brown staining around the anogenital region and dark contents in the stornach or enlarged adrenals and an absent rougae on the non-glandular region of the stomach.
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Basis for effect level:
other: reproductive and developmetal toxicity
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Offspring Litter Size and Viabiliy: Live birth index for 500 mg/kg bw/day fema1es was significantly lower (-15%) than control animals with litter size continuing to be statistically lower than control animals throughout lactation. Of the high dose females that gave birth to live litters, six females had a total litter loss, predominantly between birth and Day 1.

Offspring Growth and Development: Body weight gain at 500 mg/kg bw/day was lower (-58%) than control animals for the first week of age and again from Day 14 to weaning (Day 21 of age) (-7%). Litter weigt, at this treatment group, was notably lower than control animals throughout lactation. A delay in the onset (+ 2.1 days) and completion (+2.8 days) of pinna unfolding was evident in 500 mg/kg bw/day offspring together with a reduction in the number of offspring passing surface righting, air righting and pupil reflex. A total of eight 500 mg/kg bw/day litters had not fully completed eye opening by weaning.

Litter observations. Skin sloughing was detected in offspring during the first week of lactation in all treatrnent groups (more pronounced in the high dose group) together with rnultiple ridges along the tail in 500 and 100 mg/kg bw/day litters. Swollen ears became apparent in 500 and 100 mg/kg bw/day litters together with the premature opening of eyes and sparse fur coverage in 500 mg/kg bw/day litters. No such effects were detected in 25 mg/kg bw/day litters.
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Basis for effect level:
other: offspring viability and growth
Abnormalities:
not specified
Developmental effects observed:
not specified

Study report attachments:

Tables 1-2: Adult Clinical Observations (Group incidence)

Tables 3-5: Adult Body Weights and Body Weight Changes (Group Means)

Tables 6 -9: Adult Food consumption/efficiency (Group Means)

Table 10 Oestrus Cycles - GroupValues

Table 11 Mating Performance and Fertility - GroupValues

Table 12 Surnmary Incidence of Gestation Lengths

Table 13 Litter Data and Litter Weights - GroupMean Values

Table 14 Offspring Body Weight and Body Weight Change - Group Mean Values

Table 15 lmplantation Losses and Offpring Survival lndices - Group Mean Litter Values

Table 16 Sex Ratio -Group Mean Litter Values

Table 17 Offspring Clinical Signs -Group lncidences

Table 18 Developmental Markers - Group Mean Values

Table 19 Ref1exological Responses - Group Mean Litter Values

Tables 20 -22 Necropsy Findings - Group lncidences

Tables 23 -26 Absolute/Relative Organ Weights - GroupMean Values

Tables 27 -28 Histopathological Findings

Tables 29 Sperm Concentration and Motility - Group Mean Values

Table 30 Sperm Morphology - Group Mean Values

Table 31 Homogenisation Resistant Spermatid Counts - Group Mean Values

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 6). 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 6): 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 chain of 4-hydroxy-4-methylpentyl 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 suggest that read across from the source substance to the target substance should not represent an underestimation of the hazard associated with the oral developmental 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) one generation reproductive toxicity study (OECD 415/GLP) is available. In this key study, HMPCC was administrated to Sprague-Dawley Crl:CD( (SD) IGS BR rats (24/sex/dose) in arachis oil BP at dose levels of 0, 25, 100 and 500 mg/kg bw/day for nineteen consecutive weeks, including mating, gestation and lactation. At 500 mg/kg bw/day the effects that were considered a result of treatment included four deaths, a reduction in body weight gain (males only). All other findings were either incidental or considered not a treatment related effect. At 500 mg/kg bw/day the number of implantation sites was reduced. Although this was slight, a similar finding at 100 mg/kg bw/day means that an effect of treatment cannot be discounted. Live birth index was significantly lower than control animals, with litter size and subsequent litter weight continuing to be lower throughout lactation. Normal body weight gain was also reduced between Days 1 and 7 and 14 and 21 of lactation. Of the females that gave birth to live litters, six females had a total litter loss, predominantly between birth and Day 1 post partum. A delay in the onset and completion of pinna unfolding was evident together with a reduction in the number of offspring passing surface righting, air righting, pupil reflex and startle reflex. A total of eight litters had not fully completed eye opening by weaning. These findings are again indicative of the delay in the overall development of the offspring. Skin sloughing was detected during the first week of lactation together with multiple ridges along the tail, swollen ears and sparse fur coverage. Microscopic examination of skin samples revealed acanthosis and hyperkeratosis. At 100 mg/kg bw/day litter size was also reduced throughout lactation. Skin sloughing, multiple ridges along the tail and swollen ears were also evident together with microscopic skin changes revealed as acanthosis and hyperkeratosis. At 25 mg/kg bw/day skin sloughing was restricted to a few offspring from three litters. This clinical sign was not persistent, it occurred at a low incidence and was not associated with the histopathological findings seen in the skin of offspring at higher dose levels. There were no effects on survival, growth or development of the offspring. It is therefore considered that 25 mg/kg bw/day represents the NOAEL for developmental toxicity. The NOEL for adult toxicity was therefore considered to be 100 mg/kg bw/day and the NOAEL for reproductive and developmental toxicity was considered to be 25 mg/kg bw/day. (Ref. 17)

A follow-up study (Oral repeated dose toxicity study including neonatal evaluation; GLP, RL2) of the source substance was conducted to determine if the effects observed in the offspring in the OECD 415 one-generation study are related to the test substance and if so, whether the effect was a result of prenatal or postnatal exposure. This supporting study also investigated whether HMPCC produced a functional zinc deficiency in the dams, thereby producing the skin peeling/flaking in pups (as opposed to affecting dermal development directly or by altering nutrition). In this study, when pregnant rats were treated with HMPCC at 500 mg/kg bw/day during the gestation period, transient clinical signs of flaking were noted in the F1 generation pups with relatively few observations of skin peeling. This effect occured at the maternally toxic doses. Conversely, treatment of the dams with HMPCC at 500 mg/kg bw/day during the lactation period resulted in skin peeling in all F1 generation pups, without resolution, and with minimal observations of flaking prior to sacrifice. Treatment of dams with HMPCC during gestation or lactation had no biologically important effect on zinc or metallothionein levels (Ref. 17)

A second supporting study (Oral (Gavage) Repeated-Dose Toxicity Study of HMPCC in Adult Rats With Determination of Milk Levels, Including Neonatal Evaluation and Recovery Period; GLP, RL2) was performed to confirm that residual HICC in the maternal milk is responsible for the skin effects observed in the pup. No source substance was detected in the milk of rats treated with 10 or 25 mg/kg bw/day on either postpartum 14 or 21 days. At dosage of 500 mg/kg bw/day, HMPCC was quantifiable in the milk of two of 10 rats on DP 14 (73.5 ng/mL) and four of five rats on DP 21 (59.85 ng/mL) and observations in the F1 generation included mortality, reductions in pup body weights and observations of skin peeling. The skin peeling resolved two weeks into the postweaning recovery period. A NOAEL of 25 mg/kg bw/day based on pups viability and growth was derived from this study (Ref. 17)

A clear mechanism for the skin peeling effect of HMPCC on the pups was not identified, but the route of exposure (through maternal milk) was identified. A NOAEL value for developmental toxicity/ pup viability and growth was determined from this series of studies from the source substance. A NOAEL of 25 mg/kg bw/day is predicted for Ligustral/Cyclal C/Trigustral for developmental toxicity.

Classification and labelling

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 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 reproductive toxicity.

Based on the series of developmental studies presented for the source substance and other information in the dossier, the conclusion for classification of reproductive toxicity for Ligustral/Cyclal C/Trigustral is inconclusive. The hypothesis of HMPCC producing a functional zinc deficiency in the dams, thereby producing the skin peeling/flaking in pups was tested but was not found to be true. The dossier may be updated if any further information becomes available to clarify the proposed mechanism and to discuss how it may affect the conclusions on the target substance. Therefore, the target substance should be assigned as ‘no classification’ in reproductive toxicity with reason of ‘inconclusive’, when the criteria outlined in Annex I of 1272/2008/EC is applied.

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 developmental endpoint.

In the one-generation reproductive toxicity study, offspring adverse effects were observed at 100 and 500 mg/kg bw/day with a of NOAEL (developmental) 25 mg/kg bw/day. A subsequent oral repeated dose toxicity study, including neonatal evaluation, indicated offspring skin peeling was mainly due to postpartum exposure. The second supporting study (Oral (Gavage) Repeated-Dose Toxicity Study of HMPCC in Adult Rats With Determination of Milk Levels, Including Neonatal Evaluation and Recovery Period), confirmed that residual HMPCC in the maternal milk is responsible for the skin effects observed in the pup. No source substance was detected in the milk of rats treated with 10 or 25 mg/kg bw/day but HMPCC was quantifiable in the milk of 2/10 rats on DP 14 and 4/5 rats on DP 21 with mortality, reductions in pup body weights and observations of skin peeling in the F1 generation. A clear mechanism for the skin peeling effect of HMPCC on the pups was not identified, but the route of exposure (through maternal milk) was identified. A NOAEL value for developmental toxicity/ pup viability and growth of 25 mg/kg bw/day was determined from this series of studies from the source substance. 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 25 mg/kg bw/day is predicted for Ligustral/Cyclal C/Trigustral for developmental toxicity.

The dose descriptor obtained from the existing OECD 415 one generation reproductive toxicity study performed on the source substance is considered as an appropriate starting point for deriving a DNEL. The remaining uncertainty associated with this read-across approach is accounted for by using the appropriate assessment factors. A qualitative uncertainty evaluation is completed on the sources of uncertainty in the following table.

The predicted NOAEL value for Ligustral/Cyclal C/Trigustral is 25 mg/kg bw/day based on read-across from the HMPCC studies. The default assessment factor ‘quality of the whole database’ used in the hazard assessment of 1 is increased to 2 to account for uncertainty, based on the data provided in the available developmental studies and supporting expected toxicokinetics. This modification should be sufficient to account for any uncertainty in the read across approach. The derivation of the DNEL from the read across NOAEL value and application of conservative assessment factors should not represent an underestimation of the hazard associated with the oral developmental toxicity endpoint.

Therefore, based on the considerations above, it can be concluded that the developmental toxicity study conducted in 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, 8.7.2.

Data matrix

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

Corresponding standard information required

Target substance

Source substance

Synonyms

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

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

CAS No.

68039-49-6

31906-04-4 /51414-25-6

Information on the physicochemical properties

Molecular weight

138.21

210.32

Physical state

liquid

liquid

Vapour pressure

36 Pa (0.271 mmHg) at 20°C

 

Ref. 5

0.0012 mm Hg at 25°C(US EPA, 2010)1

< 0.001 mm Hg at 20 °C(SCCS, 2011)2

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

Partition coefficient (Log Kow)

2.7

 

Ref. 6

2.1(US EPA, 2010)1

 

Water solubility

0.91g/l at 20 °C

 

Ref. 7

184.6 mg/l at 25 °C(SCCS, 2011)2

Toxicological information

Toxicokinetics

Assessment based on phys-chem properties

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

Widely distribution, low bioaccumulation;

Excretion via urine

Ref. 8

Assessment based on phys-chem properties:

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

Widely distribution, low bioaccumulation;

Excretion via urine

Acute oral toxicity

rat

oral

LD50:3900mg/kg bw

2 (reliable with restrictions)

key study

 

Ref. 9

Rat

Oral: gavage

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

Acute dermal toxicity

rabbit

LD50: > 5000 mg/kg bw

2 (reliable with restrictions)

key study

 

Ref. 10

Rabbit

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

Skin irritation/corrosiveness

 

 

 

 

Rabbit

OECD Guideline 404(skin irritation)

2 (reliable with restrictions)

weight of evidence

skin irritation

 

Ref. 11

 

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

 

 

guinea pig (albino Dunkin Hartley guinea pig)

Coverage: occlusive (clipped)

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

OECD Guidline 406 (skin sensitisation)

2 (reliable with restrictions)

weight of evidence

skin irritant

 

Ref. 11

rabbit

2 (reliable with restrictions)

weight of evidence

skin irritant

 

Ref. 11

Eye irritation

rabbit (New Zealand White albino rabbit)

Vehicle: unchanged (no vehicle)

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

2 (reliable with restrictions)

key study

Not irritation (Naarden test report, 1981)

 

Ref. 12

Eyeirritation (rabbit, GLP) (HMPCC full dossier)

 

Sensitization

guinea pig (albino Dunkin Hartley guinea pig) female

Guinea pig maximisation test

 

OECD Guideline 406 (Skin Sensitisation)

Sensitizing to skin (Symrisetest report, 1998)

 

1 (reliable without restriction)

key study

 

Ref. 13

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

moderate skin sensitizer(SCCS, 2011)2

Genotoxicity – Ames test

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

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Negative

1 (reliable without restriction)

key study

 

Ref. 14

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

Genotoxicity in vitroChromosomal aberration

Read-across from HMPCC

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

 

Ref. 14

in vitro mammalian chromosome aberration test (chromosome aberration)

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

OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test)

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

2 (reliable with restrictions)

key study

 

Ref. 14

Mammalian Erythrocyte Micronucleus Test

Read-across from HMPCC

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

 

Ref. 15

micronucleus assay (chromosome aberration)

mouse (ICR) male/female

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Negative (OECD 474)

2 (reliable with restrictions)

key study

 

Ref. 15

Repeated dose toxicity

Read-across from HMPCC

NOAEL=150 mg/kg bw/day

 

Ref.16

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

subacute (oral: gavage)

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

Vehicle: Arachis oil BP

Exposure: twenty-eight consecutive days (daily)

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

NOAEL=150 mg/kg bw/day

2 (reliable with restrictions)

key study

 

Ref.16

 

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

 

Ref.16

rat (Fischer 344) male/female

subchronic (oral: gavage)

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

Vehicle: soybean oil

Exposure: 90 consecutive days (daily)

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

2 (reliable with restrictions)

key study

 

Ref.16

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

Developmental: Read-across from HMPCC

NOAEL=25 mg/kg bw/day

 

Ref.17

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

oral: gavage

OECD Guideline 415 (One Generation Reproduction Toxicity Study)

Developmental: NOAEL=25 mg/kg bw/day

2 (reliable with restrictions)

key study

 

Ref.17

Environmental information

Hydrolysis

No hydrolysis

 

Ref. 18

Stable under environmental conditions(US EPA, 2010)1

Biodegradation

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

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

not readily biodegradable

 

1 (reliable without restriction)

key study

 

Ref. 19

notreadily biodegradable(US EPA, 2010)1

bioaccumulation

a low potential for bioaccumulation

 

Ref. 20

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

Adsorption coefficient (Koc)

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

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

 

log Koc = 2.2 (Givaudan, 2010)

 

1 (reliable without restriction)

key study

 

Ref. 21

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

Note:

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

 

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

Conclusions:
The oral administration of HMPCC to rats by gavage at a maximum dose level of 500 mg/kg bw/day for a maximum of nineteen consecutive weeks, including the mating, gestation and lactation phases resulted in four adult deaths at 500 mg/kg bw/day and toxicologically significant changes in F1 offspring at 500 and 100 mg/kg bw/day. The NOEL for adult toxicity was therefore considered to be 100 mg/kg bw/day and the NOAEL for reproductive and developmental toxicity was considered to be 25 mg/kg bw/day.

These results are suitable for REACH purposes and using a read-across approach, a NOAEL value of 25 mg/kg bw/day for developmental toxicity is predicted for Ligustral/Cyclal C/Trigustral.
Executive summary:

In a one generation reproduction toxicity study (1543/140), HMPCC was administrated to Sprague-Dawley Crl:CD( (SD) IGS BR rats (24/sex/dose) in arachis oil BP at dose levels of 0, 25, 100 and 500 mg/kg bw/day for nineteen consecutive weeks, including mating, gestation and lactation.

At 500 mg/kg bw/day the effects that were considered a result of treatment included four deaths, a reduction in body weight gain (males only). All other findings were either incidental or considered not a treatment related effect.

At 500 mg/kg bw/day the number of implantation sites was reduced. Although this was slight, a similar finding at 100 mg/kg bw/day means that an effect of treatment can not be discounted. Live birth index was significantly lower than control animals, with litter size and subsequent litter weight continuing to be lower throughout lactation. Normal body weight gain was also reduced between Days 1 and 7 and 14 and 21 of lactation. Of the females that gave birth to live litters, six females had a total litter loss, predominantly between birth and Day 1 post partum.

A delay in the onset and completion of pinna unfolding was evident together with a reduction in the number of offspring passing surface righting, air righting, pupil reflex and startle reflex. A total of eight litters had not fully completed eye opening by weaning. These findings are again indicative of the delay in the overall development of the offspring. Skin sloughing was detected during the first week of lactation together with multiple ridges along the tail, swollen ears and sparse fur coverage. Microscopic examination of skin samples revealed acanthosis and hyperkeratosis. At 100 mg/kg bw/day litter size was also reduced throughout lactation. Skin sloughing, multiple ridges along the tail and swollen ears were also evident together with microscopic skin changes revealed as acanthosis and hyperkeratosis. At 25 mg/kg bw/day skin sloughing was restricted to a few offspring from three litters. his clinical sign was not persistent, it occurred at a low incidence and was not associated with the histopathological findings seen in the skin of offspring at higher dose levels.There were no effects on survival, growth or development of the offspring. It is therefore considered that 25 mg/kg bw/day represents the NOAEL for developmental toxicity.

The NOEL for adult toxicity was therefore considered to be 100 mg/kg bw/day and the NOAEL for reproductive and developmental toxicity was considered to be 25 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The predicted NOAEL value for Ligustral/Cyclal C/Trigustral is derived from a read-across key study of HMPCC (RL2) together with 2 read-across supporting studies (RL2). The default assessment factor of 1 is increased to 2 to account for uncertainty in the quality of the whole database, based on the available developmental study. This modification should be sufficient to account for any uncertainty. The overall quality of the database is high.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There is no reproductive toxicity data available for Ligustral/Cyclal C/Trigustral (CAS 68039-49-6). A read across approach was conducted with a one generation reproductive toxicity study (OECD 415) from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde and 2 supporting studies from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde: Oral (Gavage) Repeated-Dose Toxicity Study of HMPCC in Adult Rats, Including Neonatal Evaluation) and Oral (Gavage) Repeated-Dose Toxicity Study of HMPCC in Adult Rats With Determination of Milk Levels, Including Neonatal Evaluation and Recovery Period).

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

Read-across justification full report of developmental toxicity

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

Annex II Read-across report of developmental 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 6). 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 6): 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 chain of 4-hydroxy-4-methylpentyl 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 suggest that read across from the source substance to the target substance should not represent an underestimation of the hazard associated with the oral developmental 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) one generation reproductive toxicity study (OECD 415/GLP) is available. In this key study, HMPCC was administrated to Sprague-Dawley Crl:CD( (SD) IGS BR rats (24/sex/dose) in arachis oil BP at dose levels of 0, 25, 100 and 500 mg/kg bw/day for nineteen consecutive weeks, including mating, gestation and lactation. At 500 mg/kg bw/day the effects that were considered a result of treatment included four deaths, a reduction in body weight gain (males only). All other findings were either incidental or considered not a treatment related effect. At 500 mg/kg bw/day the number of implantation sites was reduced. Although this was slight, a similar finding at 100 mg/kg bw/day means that an effect of treatment cannot be discounted. Live birth index was significantly lower than control animals, with litter size and subsequent litter weight continuing to be lower throughout lactation. Normal body weight gain was also reduced between Days 1 and 7 and 14 and 21 of lactation. Of the females that gave birth to live litters, six females had a total litter loss, predominantly between birth and Day 1 post partum. A delay in the onset and completion of pinna unfolding was evident together with a reduction in the number of offspring passing surface righting, air righting, pupil reflex and startle reflex. A total of eight litters had not fully completed eye opening by weaning. These findings are again indicative of the delay in the overall development of the offspring. Skin sloughing was detected during the first week of lactation together with multiple ridges along the tail, swollen ears and sparse fur coverage. Microscopic examination of skin samples revealed acanthosis and hyperkeratosis. At 100 mg/kg bw/day litter size was also reduced throughout lactation. Skin sloughing, multiple ridges along the tail and swollen ears were also evident together with microscopic skin changes revealed as acanthosis and hyperkeratosis. At 25 mg/kg bw/day skin sloughing was restricted to a few offspring from three litters. This clinical sign was not persistent, it occurred at a low incidence and was not associated with the histopathological findings seen in the skin of offspring at higher dose levels. There were no effects on survival, growth or development of the offspring. It is therefore considered that 25 mg/kg bw/day represents the NOAEL for developmental toxicity. The NOEL for adult toxicity was therefore considered to be 100 mg/kg bw/day and the NOAEL for reproductive and developmental toxicity was considered to be 25 mg/kg bw/day. (Ref. 17)

A follow-up study (Oral repeated dose toxicity study including neonatal evaluation; GLP, RL2) of the source substance was conducted to determine if the effects observed in the offspring in the OECD 415 one-generation study are related to the test substance and if so, whether the effect was a result of prenatal or postnatal exposure. This supporting study also investigated whether HMPCC produced a functional zinc deficiency in the dams, thereby producing the skin peeling/flaking in pups (as opposed to affecting dermal development directly or by altering nutrition). In this study, when pregnant rats were treated with HMPCC at 500 mg/kg bw/day during the gestation period, transient clinical signs of flaking were noted in the F1 generation pups with relatively few observations of skin peeling. This effect occured at the maternally toxic doses. Conversely, treatment of the dams with HMPCC at 500 mg/kg bw/day during the lactation period resulted in skin peeling in all F1 generation pups, without resolution, and with minimal observations of flaking prior to sacrifice. Treatment of dams with HMPCC during gestation or lactation had no biologically important effect on zinc or metallothionein levels (Ref. 17)

A second supporting study (Oral (Gavage) Repeated-Dose Toxicity Study of HMPCC in Adult Rats With Determination of Milk Levels, Including Neonatal Evaluation and Recovery Period; GLP, RL2) was performed to confirm that residual HICC in the maternal milk is responsible for the skin effects observed in the pup. No source substance was detected in the milk of rats treated with 10 or 25 mg/kg bw/day on either postpartum 14 or 21 days. At dosage of 500 mg/kg bw/day, HMPCC was quantifiable in the milk of two of 10 rats on DP 14 (73.5 ng/mL) and four of five rats on DP 21 (59.85 ng/mL) and observations in the F1 generation included mortality, reductions in pup body weights and observations of skin peeling. The skin peeling resolved two weeks into the postweaning recovery period. A NOAEL of 25 mg/kg bw/day based on pups viability and growth was derived from this study (Ref. 17)

A clear mechanism for the skin peeling effect of HMPCC on the pups was not identified, but the route of exposure (through maternal milk) was identified. A NOAEL value for developmental toxicity/ pup viability and growth was determined from this series of studies from the source substance. A NOAEL of 25 mg/kg bw/day is predicted for Ligustral/Cyclal C/Trigustral for developmental toxicity.

Classification and labelling

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 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 reproductive toxicity.

Based on the series of developmental studies presented for the source substance and other information in the dossier, the conclusion for classification of reproductive toxicity for Ligustral/Cyclal C/Trigustral is inconclusive. The hypothesis of HMPCC producing a functional zinc deficiency in the dams, thereby producing the skin peeling/flaking in pups was tested but was not found to be true. The dossier may be updated if any further information becomes available to clarify the proposed mechanism and to discuss how it may affect the conclusions on the target substance. Therefore, the target substance should be assigned as ‘no classification’ in reproductive toxicity with reason of ‘inconclusive’, when the criteria outlined in Annex I of 1272/2008/EC is applied.

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 developmental endpoint.

In the one-generation reproductive toxicity study, offspring adverse effects were observed at 100 and 500 mg/kg bw/day with a of NOAEL (developmental) 25 mg/kg bw/day. A subsequent oral repeated dose toxicity study, including neonatal evaluation, indicated offspring skin peeling was mainly due to postpartum exposure. The second supporting study (Oral (Gavage) Repeated-Dose Toxicity Study of HMPCC in Adult Rats With Determination of Milk Levels, Including Neonatal Evaluation and Recovery Period), confirmed that residual HMPCC in the maternal milk is responsible for the skin effects observed in the pup. No source substance was detected in the milk of rats treated with 10 or 25 mg/kg bw/day but HMPCC was quantifiable in the milk of 2/10 rats on DP 14 and 4/5 rats on DP 21 with mortality, reductions in pup body weights and observations of skin peeling in the F1 generation. A clear mechanism for the skin peeling effect of HMPCC on the pups was not identified, but the route of exposure (through maternal milk) was identified. A NOAEL value for developmental toxicity/ pup viability and growth of 25 mg/kg bw/day was determined from this series of studies from the source substance. 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 25 mg/kg bw/day is predicted for Ligustral/Cyclal C/Trigustral for developmental toxicity.

The dose descriptor obtained from the existing OECD 415 one generation reproductive toxicity study performed on the source substance is considered as an appropriate starting point for deriving a DNEL. The remaining uncertainty associated with this read-across approach is accounted for by using the appropriate assessment factors. A qualitative uncertainty evaluation is completed on the sources of uncertainty in the following table.

The predicted NOAEL value for Ligustral/Cyclal C/Trigustral is 25 mg/kg bw/day based on read-across from the HMPCC studies. The default assessment factor ‘quality of the whole database’ used in the hazard assessment of 1 is increased to 2 to account for uncertainty, based on the data provided in the available developmental studies and supporting expected toxicokinetics. This modification should be sufficient to account for any uncertainty in the read across approach. The derivation of the DNEL from the read across NOAEL value and application of conservative assessment factors should not represent an underestimation of the hazard associated with the oral developmental toxicity endpoint.

Therefore, based on the considerations above, it can be concluded that the developmental toxicity study conducted in 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, 8.7.2.

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

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 Effect on developmental toxicity: via oral route:
Only 1 key study is available.

Justification for selection of Effect on developmental toxicity: via inhalation route:
An oral developmental toxicity study is available.

Justification for selection of Effect on developmental toxicity: via dermal route:
An oral developmental toxicity study is available.

Justification for classification or non-classification

Based on the available information in the dossier, the substance Ligustral/Cyclal C/Trigustral (CAS 68039-49-6) does not need to be classified for reproductive toxicity when the criteria outlined in Annex I of 1227/2008/EC are applied based on the results of the read-across study from 3 and 4-( 4-Hydroxy-4-methylpentyl)-3 -cyclohexene-1-carboxaldehyde.