Eugenol

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

November 19, 1999 to July 3, 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study design is considered to follow OECD guideline Test Guideline No 416 (2001) with insignificant deviations (details on mating procedures lacking, gross observation and histopathology of randomly selected offspring not conducted)

Data source

Reference

Reference 1

Reference Type:

other: revised final report

Title:

Unnamed

Year:

2003

Report date:

2003

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

details on mating procedures lacking, gross observation and histopathology of randomly selected offspring not conducted

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isoeugenol

EC Number:

202-590-7

EC Name:

Isoeugenol

Cas Number:

97-54-1

Molecular formula:

C10H12O2

IUPAC Name:

2-methoxy-4-prop-1-en-1-ylphenol

Details on test material:

- Name of test material (as cited in study report): Isoeugenol
- Storage condition of test material: 5C protected from light

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: (P) 11 wks; (F1) 11 wks
- Weight at study initiation: (P) Males: 355.6-420.2 g; Females: 220.3-281.6 g; (F1) not reported
- Fasting period before study: no
- Housing: cohoused, 2/cage, polycarbonate cages suspended on stainless-steel racks
- Diet (e.g. ad libitum): Pelleted Harlan Teklad ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23.3
- Humidity (%): 30-70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: July 13, 1999 To: January 13, 2000

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported
- Concentration in vehicle: 14, 46, or 140 mg/mL
- Amount of vehicle (if gavage): 1500 mL (total volume)
- Lot/batch no. (if required): not reported
- Purity: 94.9-124.8% (nominal)

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 9 weeks
- Proof of pregnancy: not reported, animals mated on day 8 of dosing
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.: not reported
- Further matings after two unsuccessful attempts: not reported
- After successful mating each pregnant female was caged (how): in cages
- Any other deviations from standard protocol: mating pairs allowed to produced 3 litters, individuals selected from third litter for F1 mating

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosages were prepared and samples were verified for the first, second, and twenty-sixth mixes. Selected samples were analyzed by high performance liquid chromatography. All dose formulations were within 94.9-124.8% of the nominal concentration

Duration of treatment / exposure:

From 8 days before cohabitation until post natal day 21 after the third litter was born (F1c). F1c selected for F1 cohabitation were administered dosing starting on post natal day 21, with cohabitation at post natal day 81 and the mating pairs were allowed to produce 3 litters

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until 61 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 81 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
70 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
230 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
700 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

20

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range finding study
- Rationale for animal assignment (if not random): random

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once weekly

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and at littering, as well as Post natal day 4, 7, 14, 18, and 21

FEED CONSUMPTION: Yes
- Time schedule for examinations: weeks 1, 6, 12 (males only), and 14 (males only)

Oestrous cyclicity (parental animals):

Vaginal cytology was performed at 14 days after weaning

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, sperm motility, epididymal sperm density, epididymal sperm morphology, spermatids/testis, total spermatids/testis, total spermatid/cauda, epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, post partum day 16
- If yes, maximum of 4 pups/litter (2/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
number and sex of pups, stillbirths, live births, body weight, sexual development

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals at study day 178 or 179
- Maternal animals: All surviving animals at study day 178 or 179

GROSS NECROPSY
- Gross necropsy consisted of external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ /tissue histopathology: liver, kidneys, ventral and dorsolateral prostates, seminal vesicles with coagulating glands, spleen, stomach, ovaries, uterus, cervix, vagina, left testis and epididymis, and gross lesions
Organ weights: Liver, spleen, right epididymis, ventral prostate, ovaries, uterus/vagina/cervix, kidneys, right testis, right cauda epididymis

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
-not examined

Statistics:

Statistical analysis by Analytical Science Inc.

Reproductive indices:

Cochran-Armitage test

Offspring viability indices:

Shirley's or Dunn's test

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Description (incidence and severity):

Test substance intake: (gavage study)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
low to moderate (0-39%) in incidence and not considered to be treatment-related

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
decreases in body weight (statistically significant decreases of 7.28% and 18.0% at male dose levels of 230 and 700 mg/kg body weight/day and of 9.61%, 11.3%, and 10.8% at female dose levels of 70, 230, and 700 mg/kg body weight/day as measured at Week 26) beginning at week 2 of the study. Decreases in food consumption in mid- and high-dose

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
gavage

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No changes in number of females with regular cycles, cycle length, number of cycles, and in number of cycling females

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
no changes in sperm motility, epididymal sperm density, percent abnormal sperm, number of spermatids per cauda epididymis, and total number of spermatids per testis

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
decrease in live male pups, dam body weights at delivery, sire body weights at delivery, absolute female anogenital distance, dam body weights during lactation in high-dose groups; increases in female anogenital distance to body weight ratio in high dose groups

ORGAN WEIGHTS (PARENTAL ANIMALS)
increases in relative weights of right epididymis, right testis, and kidneys in the high dose group; increases in absolute spleen weights in high-dose males; decrease in ovary weights in mid-dose group; increase in relative liver weights in low-, mid-, and high-dose females; increase in relative kidney weights in low and high-dose females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
incidental observations spread across dose groups and not related to treatment

HISTOPATHOLOGY (PARENTAL ANIMALS)
hyperkeratosis and hyperplasia of the squamous epithelium of the non-glandular stomach

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

>= 700 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect observed. Expert judgment

Remarks on result:

other: Generation: P, F1 (migrated information)

2

Dose descriptor:

LOAEL

Remarks:

Parental toxicity

Effect level:

<= 70 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on decreased body weight gains at all dose-levels, although probably linked to the bolus effect.

Remarks on result:

other: Generation: P, F1 (migrated information)

3

Dose descriptor:

NOAEL

Effect level:

>= 230 - < 700 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Expert judgment

Remarks on result:

other: Generation: F1, F2 (migrated information)

4

Dose descriptor:

LOAEL

Effect level:

700 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on decreased male and female F2 pup weights. Expert judgment. Please see 'Basis for effect level / Remarks".

Remarks on result:

other: Generation: F1, F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
1 death in mid-dose adolescent male, 2 deaths in high-dose adolescent males, 1 death in high-dose adolescent female. Possible aspiration of compound as cause of deaths.

CLINICAL SIGNS (OFFSPRING)
during the study incidence of observations was low and not considered to be treatment related; At post dose clinical observations languid and prostrate behavior and salivation was observed in the mid- and high-dose groups

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 230 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Expert judgement

2

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Expert judgement

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

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.

Executive summary:

A read-across strategy is used for the endpoint of toxicity to reproduction, with justification provided below.

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. Based on data from studies in rats, the pharmacokinetic profiles of isoeugenol and eugenol appear to be similar; the major route of elimination of these compounds isviathe urine and the major metabolites are glucuronic acid and sulphate conjugates.

Study Summary

In a two-generation study conducted similarly to OECD 416 and in compliance with GLP, Isoeugenol was administered to 20 F0 Crl:CD(SD) rat/sex/dose 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).

Endpoints evaluated included body weight, feed consumption, clinical signs, number and weight of pups, anogenital distance, sperm parameters, vaginal cytology, organ weights, and gross and microscopic pathology.

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). Similarreduction 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.