Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

2-generation study on Trans-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.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 19, 1999 to July 3, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
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)
Qualifier:
equivalent or similar to
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. certificate)
Limit test:
no
Specific details on test material used for the study:
- Storage condition of test material: 5°C protected from light
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals 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
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
Dose / conc.:
70 mg/kg bw/day (actual dose received)
Dose / conc.:
230 mg/kg bw/day (actual dose received)
Dose / conc.:
700 mg/kg bw/day (actual dose received)
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
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
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs noted during the study included abrasions, alopecia, discharge from the eyes and nose, redded area, urine stains, few feces, rales, languid behavior, ulcer, dyspnea, paleness, rough hair coat, swelling, paralysis, malocclusion, small and large stationary tissue masses, and thinness. The incidence of these observations was low to moderate (0-39%), but was not considered to be treatment related. Post-dose observations for clinical signs were performed on all animals. Most observations seen were in the high dose animals and included languid and prostrate behavior and salivation. These observations would continue to be seen until the 1 hour post-dose observation timepoint. At the 2 hour timepoint, all animals would appear normal.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality was observed in four F0 males and ten F0 females. One low dose male was killed moribund on SD138 because of severe clinical signs including dyspnea, rales, few feces, rough haircoat, and languid behavior. Three males (one in each of low-, mid- and high- dose groups) were found dead on SD179, SD10, and SD154, respectively. Each animal appeared normal prior to death. Two mid-dose females were found dead, one on SD114 and one on SD 133. One female appeared normal prior to death. The other female apparently died as a result of gavage error. Eight high dose females were found dead. Two deaths appear to be the result of gavage errors. Three deaths occurred due to parturition difficulties. Three females appeared normal prior to their deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
Throughout the F0 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%* 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).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decreases in food consumption in mid- and high-dose. 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 efficiency:
not examined
Description (incidence and severity):
Gavage
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
hyperkeratosis and hyperplasia of the squamous epithelium of the non-glandular stomach at all dose levels and in both sexes of the F0 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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No changes in number of females with regular cycles, cycle length, number of cycles, and in number of cycling females
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
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:
effects observed, treatment-related
Description (incidence and severity):
Decrease in live male pups (21% lower at the high dose compared to controls), decreases in dam body weights at delivery (-8 to -13%), decreases in sire body weights at delivery (-12 to -14%), decrease in absolute female anogenital distance in F1c litter (-8%), dam body weights during lactation in high-dose groups (-7 to -13%); increases in female anogenital distance to body weight ratio in high dose F1a litter (+10%).
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.
Key result
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
Key result
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:
body weight and weight gain
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)
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)
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs noted during the study included abrasions, alopecia, discharge from the eyes, diarrhea, urine stains, few feces, rales, anorexia, hypothermia, dyspnea, paleness, rough hair coat, swelling, hunched posture, malocclusion, small stationary tissue masses, and thinness. The incidence of these observations was low (0-25%), except for alopecia (10-45%), and they were not considered to be treatment related in incidence and severity. Post-dose observations for clinical signs were performed on all animals. Most observations seen were in the mid and high dose animals and included languid and prostrate behavior and salivation. These observations would continue to be seen up to the thirty minute timepoint.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality was observed. One low dose male was killed moribund on PND171 because of severe clinical signs including rouh haircoat, thinness, hunched posture, diarrhea, few feces, red discharge from the mouth, and anorexia. One mid dose male was killed moribund on PND 193 as a result of a gavage error. One high dose male was found dead on PND 171. The animal appeared normal prior to its death. One mid dose female was found dead on PND107 as a result of a gavage error and one on PND124. The second animal appeared normal prior to its death. Two high dose females were found dead on PND109 and PND153. the first animal died immediately after dosing of possible aspiration of compound. The second animal appeared normal prior to its death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower absolute body weights in dams and sires (statistically significant values of 5.39% and 10.0% in mid- and high-dose males; female decreases were not statistically significant).
Throughout the F1 generation, a dose-related decrease was measured in the mean body weight gain of all adult males compared to control (F1: -12%, -25.5%* and -40%* at 70, 230 and 700 mg/kg bw/day, respectively). Similar reduction was only observed in high-dose adult F1 females (-34%*).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
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).
Food efficiency:
not examined
Description (incidence and severity):
gavage
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Organ weight changes included increases in relative weights of the liver, right testis, kidneys, spleen, and cauda epididymis in high-dose males and increases in relative liver and kidney weights in high-dose females compared to controls. 
At the 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.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross findings observed in the males were considered incidental ias not dose-related in incidence and severity with findings spread over the dose groups. There were no gross findings in the females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings included hyperkeratosis and hyperplasia in the non-glandular stomach at all dose levels and in both sexes of the 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.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Decreases in dam body weights at delivery of F2 litters (-13 to -16%), decreases in sire body weights at delivery of F2 litters (-18 to -21%), Increase in the female anogenital distance to body weight ratio during the F2a litter by 12%, Decreases in the average absolute F2 combined live male pup weight (-5 to -7%), decreases in the average absolute F2 combined live female pup weight (-4 to -5%), decreases in the average absolute F2 combined live male and female pup weight (-5 to -6%), decreases in the average F2 combined live male pup weight (adjusted for litter size) (-5 to -6%), decreases in the average of F2a litter live female pup weight (adjusted for litter size) (-4 to -5%), decreases in the average combined live male and female pup weight (adjusted for litter size) (-4 to -5%).
Key result
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.
Key result
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:
body weight and weight gain
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs noted during the study included abrasions, alopecia, urine stains, rough hair coat, hunched posture, cyanosis, and thinness. The incidence of these observations was low (0-25%) and they were not considered to be treatment related in incidence or severity. Post-dose clinical observations were performed on all animals. Most observations seen were in the mid and high dose animals and included languid and prostrate behavior and salivation. These observations would continue to be seen up to the thirty minute timepoint.

Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
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.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Description (incidence and severity):
Gavage
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No changes were noted in sexual developmental parameters (including testicular descent, preputial separation, and vaginal opening) in any generation. 
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Increases in the female anogenital distance to body weight ratio in the F1a litter compared to control group.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower (i.e., 4 to 5%) pup body weights
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No changes were noted in sexual developmental parameters (including testicular descent, preputial separation, and vaginal opening). 
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
 
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Increases in female F2a litter anogenital distance to body weight ratio compared to controls.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 230 - < 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOAEL
Generation:
F2
Effect level:
ca. 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Reproductive effects observed:
no
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:

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). 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.

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

A key study was identified on the registered substance (NTP, 2003, Rel.2). In this two-generation study conducted similarly to OECD 416 and in compliance with GLP, Trans-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 in the mean body weight gain was seen in 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 decreased 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 that which caused parental toxicity. Historical control data are not available, however, in the absence of an obvious dose-response relationship 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 that which caused 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.

Effects on developmental toxicity

Description of key information

Developmental toxicity study on Trans-Isoeugenol (similar to OECD 414, GLP, Rel. 1, 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.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 16, 1998 to August 18, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Study design is considered to follow OECD guideline Test Guideline No 414 (2001).
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Storage condition of test material: 5°C protected from light
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: Not reported
- Weight at study initiation: 232-275 g on gestational day 0
- Fasting period before study: Not reported
- Housing: Sani-Chip hardwood cage litter
- Diet (e.g. ad libitum): Purina Certified Roden Chow ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-23.3
- Humidity (%): 39.8-59.9
- Air changes (per hr): Not reported environmental conditions monitored and controlled by computer
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: Not reported
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: Not reported
- Amount of vehicle (if gavage): 5 ml/kg (administered dose volume)
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- 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
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy:sperm found in vaginal lavage referred to as day 0 of pregnancy
- Any other deviations from standard protocol: not reported
Duration of treatment / exposure:
Day 6 through day 19 of gestation
Frequency of treatment:
Daily
Duration of test:
13 days
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 pregnant females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on screening study

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily at dosing, and at 0.5 and 2 hours after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Gestational Day 0, 6 through 19, 20 and immediately following termination at day 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Gestational Day 0, 6, 9, 12, 15, 18, 19, and 20

WATER CONSUMPTION: Yes
- Time schedule for examinations: Gestational Day 0, 6, 9, 12, 15, 18, 19, and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: liver, gravid uterus, thoracic and abdominal cavities
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: Yes
- Number of late resorptions: Yes
- Other: number of dead and live fetuses
Fetal examinations:
- External examinations: Yes: all live fetuses
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
SAS software
Indices:
% Resorptions or fetal deaths/litter
% Litters with resorptions or deaths
% Litters with 100% prenatal mortality
No. live fetuses/litter
Average female body weight/litter
Average male body weight/litter
% Malformed fetusesllitter (all, extemal, visceral, or skeletal)
% Litters with malformations (all, extemal, visceral, or skeletal)
% Fetuses with any variations/litter
% Litters with any variations
% Fetuses with skeletal variations
Historical control data:
None
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Notable changes included clinical signs of piloerection and sedation at 500 and 1,000 mg/kg body weight/day.
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Compared to control, 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 .
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Changes included lower maternal relative food intake (statistically significant values of 15.3% at 1,000 mg/kg body weight/day).
Food efficiency:
not examined
Description (incidence and severity):
Gavage
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Maternal relative water intake in the high dose group was reduced on gd 6-9, but was significantly elevated in the high dose group throughout the treatment and gestation periods. In the mid dose group, relative maternal water consumption was elevated during every time period from gd 9-20 with the exception of gd 18-19.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Lower gravid uterine weight (statistically significant values of 8.54% and 10.5% at 500 and 1,000 mg/kg body weight/day, respectively). Maternal absolute liver weight showed a decreasing trend, but relative liver weight was significantly increased at all dose levels.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No treatment-related group mean differences
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No treatment-related group mean differences
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Confinned pregnancy rates were 92-100% per group.
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment-related group mean differences for the following reproductive parameters: number of corpora lutea per dam, number of implantation sites per dam, average litter size, and percentage of male foetuses per litter
Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The only developmental effect noted was a 7 to 9% decrease in body weight in high-dose foetuses when compared with control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The only developmental effect noted was a 7 to 9% decrease in body weight in high-dose foetuses when compared with control group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Live litter size in treated groups was 94-98% of the control mean.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No statistically significant differences
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
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 with 1 (1/184), 4 (1/180), 3 (3/184), and 14 (14/179) fetuses in the 0, 250, 500, and 1000 mglkg/day dose groups exhibiting unossified sternebrae. I
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
ncidental occurrences of visceral malformations were noted in one high-dose foetus (1/179); however, these did not occur in a dose-related manner and were therefore considered not to be relate to the test article; these variations included enlarged lateral ventricle, enlarged nasal sinus, agenesis of the innominate artery, and distended ureter.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
Description (incidence and severity):
1 (1/184), 4 (1/180), 3 (3/184), and 14 (14/179) fetuses in the 0, 250, 500, and 1000 mglkg/day dose groups exhibiting unossified sternebrae
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
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.
Executive summary:

In an embryo-foetal developmental study with E-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. 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 with 1 (1/184), 4 (1/180), 3 (3/184), and 14 (14/179) fetuses in the 0, 250, 500, and 1000 mglkg/day dose groups exhibiting unossified sternebrae. Incidental occurrences of visceral malformations were noted in one high-dose foetus (1/179); 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 the dose level with the maternal toxicity.  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 conclusion drawn by 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.

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

A key study was identified on the registered substance (NTP, 1999, Rel.1). In this key embryo-foetal developmental study with Trans-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 non-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 Trans-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 the dose level with maternal toxicity.  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 conclusion drawn by 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. However, this finding is likely secondary to maternal toxicity and not indicative of a teratogenic effect.

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 classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Reproductive toxicity was only reported at concentrations greater than maternal toxicity for Trans-Isoeugenol. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008 and to the GHS.