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Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
700 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information as a whole meets the tonnage driven data requirement of REACH.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No study was located on Eugenol. Therefore a read-across approach on Isoeugenol was used to conclude on the reproductive toxicity potential of Eugenol. The justification for Read-across is in the Discussion field: "Toxicity to reproduction: other studies".

In a two-generation study conducted similarly to OECD 416 and in compliance with GLP, Isoeugenol was administered to Crl:CD(SD) rat/s daily via oral gavage at dose levels of 0, 70, 230 or 700 mg/kg bw/day from Study Day (SD) 1 until the day prior to necropsy. The F0 cohabitation began on SD 8. Mating pairs were allowed to produce three litters (F1a, F1b and F1c). Dosing of the F1 generation was initiated on post-natal day (PND) 21 of the F1c animals. On PND 81 ± 10, F1c animals were assigned to mating pairs and allowed to produce three litters (F2a, F2b and F2c).

Throughout the F0 and F1 generation, a dose-related decrease was measured in the mean body weight gain of all adult males compared to control (F0: -8%, -14%* and -34%*; F1: -12%, -25.5%* and -40%* during F1, at 70, 230 and 700 mg/kg bw/day, respectively). Although not dose related, all F0 females had a reduced mean body weight gain compared to control (-23%*, -24%*, -23%*, for 70, 230 and 700 mg/kg bw/day, respectively). Similar reduction was only observed in high-dose adult F1 females (-34%*).

Food consumption values were decreased in the high dose F0 males (-27%*, -7%, -12%*, during Week 1, 12 and 14 respectively). During Week 1, mid- and high-dose F0 females had decreased food consumption (-8%* and -20%*). Combined male and female food consumption was not reduced during Week 6. Food consumption values were decrease in all F1 males during Weeks 12 (-12%*, -13.5* and -15%*, for 70, 230 and 700 mg/kg bw/day, respectively) and Week 14 (-6%, -15% and -23%*, for 70, 230 and 700 mg/kg bw/day, respectively). Combined male and female food consumption was also reduced during Week 3 (-6%, -8%* and -8%*, for 70, 230 and 700 mg/kg bw/day, respectively) and Week 6 (-2%, -5% and --9%*, for 70, 230 and 700 mg/kg bw/day, respectively).

The aggregate number of live male pups born during all litters to the F0 parents was decreased by 21%* in the high-dose group (F1a: -22%, F1b: -13%, F1c: -22%). In an outbreeding study, a 42%* increase in the number of live females per litter and a 34%* increase in the number of implantation sites was observed when naïve females were mated with high-dose males. This finding was not reproduced during all litters to the F1 parents and in the outbreeding study, i.e.naïve males mated with high-dose females. Moreover, it occurred only at concentrations greater than parental toxicity. Historical control data are not available, however, in the absence of obvious dose-response reliationship and reproducibility, this finding is considered to be of very low toxicological concern.

A decreased body weight was observed in all high-dose litters to the F1 parents compared to control (Combined: -5%, Males: -5.5%, Females: -4%). This effect occurred only at concentrations greater than parental toxicity.

There were no effects on any other reproductive parameters throughout both generations. Sperm parameters and vaginal cytology were unchanged in the F0 and F1 generations.

At the F0 and F1 necropsies, some decrease in absolute organ weights and increases in organ-to-body weight ratios were seen in the high-dose males and females. These differences may be attributed to decreased terminal body weights. Treatment-related findings included hyperkeratosis and hyperplasia in the non-glandular stomach at all dose levels and in both sexes of the F0 and F1 animals.

It should be pointed out that the appearance of hyperkeratosis and hyperplasia in non-glandular stomachs and decreased body weight may be due to the route of administration whereby the test material is administered by intubation thereby delivering a bolus of a substance that is shown to have clearly irritant properties at high concentrations. (HERA, 2005).

A NOAEL was not reported by the study authors for this study. However, based on expert judgment the following NOAELs/LOAELs are proposed:

NOAEL fertility ≥ 700 mg/kg bw/day.

NOAEL development ≥ 230 mg/kg bw/day / LOAEL = 700 mg/kg bw/day, as a worst-case based on decreased male and female F2 pup weights.

LOAEL parental toxicity ≤ 70 mg/kg bw/day, based on decreased body weight gains at all dose-levels, although probably linked to the bolus effect.

* Statistically significant

Reference:

HERA (2005). Risk assessment of Isoeugenol: 4-Hydroxy-3-methoxy-1-propen-1-yl benzene CAS 97-54-1. Human and Environmental Risk Assessment on ingredients of Household Cleaning Products. February 2005.


Short description of key information:
2-generation study on Isoeugenol (similar to OECD 416, GLP, Rel. 2, K):
- NOAEL fertility ≥ 700 mg/kg bw/day.
- NOAEL development ≥ 230 mg/kg bw/day / LOAEL = 700 mg/kg bw/day, as a worst-case based on decreased male and female F2 pup weights.
- LOAEL parental toxicity ≤ 70 mg/kg bw/day, based on decreased body weight gains at all dose-levels, although probably linked to the bolus effect.

Justification for selection of Effect on fertility via oral route:
No study was located on Eugenol. A read-across to Isoeugenol was used. The key study is GLP-compliant and of high quality (Klimisch score = 2).

Effects on developmental toxicity

Description of key information
Developmental toxicity study on Isoeugenol (similar to OECD 414, GLP, Rel. 2, K):
- LOAEL maternal toxicity = 250 mg/kg bw/day, based on dose-dependent reduced body weight gain.
- NOAEL developmental toxicity = 500 mg/ kg bw/day, based on intra-uterine growth retardations mildly delayed skeletal ossification observed at 1000 mg/kg bw/day. However, this finding is likely secondary to maternal toxicity and not indicative of a teratogenic effect.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information as a whole meets the tonnage driven data requirement of REACH.
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

The teratogenic potential of eugenol was investigated in a key study with the read-across compound isoeugenol and a supporting study with eugenol in rats (NTP, 1999) and in mice (Amini et al., 2003).The justification for Read-across is in the Discussion field: "Toxicity to reproduction: other studies".

Both studies were evaluated against OECD Guideline No. 414 and neither study was conducted according to Good Laboratory Practices (GLP).

In a key embryo-foetal developmental study with isoeugenol, the oral (gavage) administration of 0 (control), 250, 500, or 1,000 mg /kg body weight/day to female CD rats on gestation day (GD) 6 to 19 resulted in a number of test article-related maternal effects at all dose levels (NTP, 1999). These findings included lower maternal body weight (4.6%, 11.6%, and 9.9% lower at dose levels of 250, 500, and 1,000 mg/kg body weight/day, respectively) when measured on GD 20, and statistically significant and dose-dependent lower gestation body weight gain (13.6%, 22.6%, and 30.5% lower at dose levels of 250, 500, and 1,000 mg/kg body weight/day, respectively) when measured over GD 0 to 20 compared to control group.

Other effects in maternal animals included clinical signs of piloerection and sedation at 500 and 1,000 mg/kg body weight/day, and lower gravid uterine weight (statistically significant values of 8.54% and 10.5% at 500 and 1,000 mg/kg body weight/day) and lower maternal relative food intake (statistically significant values of 15.3% at 1,000 mg/kg body weight/day) compared to control group.

There were no treatment-related group mean differences for the following reproductive parameters: number of corpora lutea, number of implantation sites, percent preimplantation loss, percent preimplantation loss, resorptions, late foetal deaths, average litter size, and percentage of male foetuses per litter.

The only developmental effect noted was 7 to 9% lower body weight in high-dose foetuses when compared with Controls. There were no statistically significant differences among groups for the incidences of foetal malformations (i.e., total, external, or skeletal); however, the incidence of skeletal variations (i.e., unossified sternebrae) was significantly increased in high-dose foetuses. Incidental occurrences of visceral malformations were noted in one mid-dose and one high-dose foetus; however, these did not occur in a dose-related manner and were therefore considered not to be related to the test article; these variations included enlarged lateral ventricle, enlarged nasal sinus, agenesis of the innominate artery, and distended ureter.

The findings of unossified sternebra(e) at the high dose of 1,000 mg isoeugenol/kg body weight/day in the embryo-foetal developmental study are likely secondary to maternal toxicity and are not indicative of a teratogenic effect.  It is widely accepted that findings of unossified sternebra(e) are strongly influenced by alterations in maternal factors such as body weight, food consumption, and physiology (Carney and Kimmel, 2007).  In this particular study, the findings of unossified sternebra(e) were only reported at a maternally toxic dose.  Furthermore, even in the absence of maternal toxicity, delayed ossification is considered to be indicative of a foetotoxic effect, but not a teratogenic effect, as ossification usually will complete in the postnatal period and this finding has a relatively high background incidence.

According to the NTP, a maternal NOAEL could not be established; however, the lowest-observed-adverse-effect level (LOAEL) was reported to be the low dose level of 250 mg/kg body weight/day. The NOAEL for developmental toxicity was reported to be 500 mg/kg body weight/day, based on findings of growth delay and mildly delayed skeletal ossification in the high-dose foetuses.

In a supporting study, the oral (gavage) administration of a single dose level of 100 mg eugenol/kg body weight/day to NMRI mice on GD 5 to 15 did not result in any teratogenicity (i.e., there were no effects on crown rump length and no defects of the craniofacial region, hands, feet, or tail) and no significant effects on number of live foetuses (Amini et al., 2003).

Reference:

Carney EW, Kimmel CA (2007). Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs. Birth Defects Res B Dev Reprod Toxicol. 2007 Dec;80(6):473-96.


Justification for selection of Effect on developmental toxicity: via oral route:
No study was located on Eugenol. A read-across to Isoeugenol was used. The key study is GLP-compliant and of high quality (Klimisch score = 2).

Toxicity to reproduction: other studies

Additional information

Justification for Read-Across:

The use of isoeugenol (CAS No. 97-54-1) as a structural surrogate for eugenol (CAS No. 97-53-0) is justified on the basis of structural similarity; both substances are 2-methoxy-4-propenylphenol isomers, whose chemical structures are identical except for the position of the double bond in the propenyl substituent. The difference in the position of the double bond is expected to exert little or no effect on the physical-chemical properties of the two substances with respect to biological activity, and their absorption and distribution profiles are therefore also expected to be essentially identical.

Absorption, Distribution, Metabolism, and Excretion of Eugenol and Isoeugenol:

Rapid and extensive urinary excretion of eugenol (i.e.,75 to 80% of the administered dose) was reported 24 hours following the oral administration of 0.5, 5.0, 50, or 1,000 mg eugenol/kg body weight to female Wistar albino rats while faecal excretion accounted for 10% of the administered dose (Sutton et al.,1985). The major metabolites of eugenol were reported to be the glucuronic acid and sulphate conjugates, while O-demethylation and reduction of the allylic double bond were reported to be minor metabolites. The administration of a single, oral dose of 156 mg isoeugenol/kg body weight to male Fischer rats resulted in > 85% urinary excretion of the sulphate or glucuronide metabolites by 72 hours (EFSA, 2009). Approximately 10% of the administered dose was recovered in the faeces and < 0.1% was recovered as CO2or expired organic material. No parent isoeugenol was detected in the blood at any of the time points analysed (i.e., 0.25 to 72 hours). The total amount of radioactivity remaining in selected tissues (i.e., heart, kidneys, liver, muscle, subcutaneous adipose tissue and testicular adipose tissue) by 72 hours was less than 0.25% of the administered dose. Based on the findings of this study, it can be concluded that isoeugenol is rapidly metabolised in the rat and is excreted predominantly in the urine as phase II conjugates of the parent compound. In conclusion, the pharmacokinetic profiles of isoeugenol and eugenol appear to be similar; the major route of elimination of these compounds is via the urine and the major metabolites are glucuronic acid and sulphate conjugates.

References:

EFSA. Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from Commission on Consideration of eugenol and related hydroxyallylbenzene derivatives evaluated by JECFA (65th meeting) structurally related to ring-substituted phenolic substances evaluated by EFSA in FGE.22 (2006) (question no EFSA-2008-32L, published on 12 February 2009 by European Food Safety Authority).

EFSA J. 2009:ON-965:1-54. Available at:http://www.efsa.europa.eu/en/efsajournal/pub/965.htm.

Sutton D, Sangster S, Caldwell J (1985). Dose-dependent variation in the disposition of eugenol in the rat. Biochem Pharmacol 34(3):465-466.

Justification for classification or non-classification

Reproductive toxicity was only reported at concentrations greater than maternal toxicity for the surrogate isoeugenol. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.7.

Additional information

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