Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 31 March to 28 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 31 March to 28 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The deviations were considered not to have affected the outcome or the achievement of the study objectives.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI (Han)
Details on species / strain selection:
The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. Background data and control data for the strain are available at the Test Facility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Age at study initiation: Males: Approximately 10 weeks. Females: Approximately 12 weeks.
- Weight at study initiation: Males: 298 to 365 g. Females: 186 to 231 g.
- Fasting period before study: no
- Housing: One air-conditioned room in a barrier protected unit (building K1).
Caging:Animals were caged as follows:
Number of animals per cage:
Pre-mating: 5 males and 5 females
Mating: 1 male and 1 female (housed together)
Gestation: 5 males and 1 female
Lactation: 1 female + litter
Group housed males and females and individual housed females, including females during mating and with litters, were housed in plastic cages (550 x 376 x 215 mm) meeting European directive 201
0/63/EU requirements.
- Diet (e.g. ad libitum): Rat powdered complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
- Water (e.g. ad libitum): Softened and filtered (0.2 μm) mains drinking water was available ad libitum (via an automatic watering system). Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
- Bedding: Cellulose bedding (Serlab, Montataire, France) or dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood, analysed at least twice a year for chemical and bacterial
contaminants.
- Enrichment: Animals received a small amount (handful) of shredded paper (SDS/Dietex) and the isolated animals had free access to a wooden gnaw block (Aspen Bricks, Le comptoire des sciures).
Furthermore, tissues were also provided as enrichment for females towards the end of gestation.
- Contaminants: No known contaminants were present in the diet, water or bedding at levels which might have interfered with achieving the objectives of the study.
- Acclimation period: 7 days between animal arrival and start of pre-test estrous cycle smears (females) or start of treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): > 35%
- Air changes (per hr): At least 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark (except when required for technical acts).

IN-LIFE DATES: From: 13 January 2017 To: 28 May 2017
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
supplier: SIGMA, batch number: MKCB2122V, expiry date: 23 January 2019
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was prepared as a suspension in the vehicle at concentrations of 50, 100 and 200 mg/mL according to Standard Operating Procedures of the Test Facility. No correction factor was taken into account for dose calculations.
Frequency of preparation: At least weekly (up to eight days).
Storage of formulations: Refrigerated (between +2 and +8 °C).

DIET PREPARATION
not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable
- Concentration in vehicle: 0; 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5mL/kg bw/d
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item in the vehicle: 24h at ambient temperature or room temperature (between +15 and + 25 °C), 8 days refrigerated (between +2 and +8°C) or 3 weeks frozen (between -25 and -15 °C) (based on stability study no. AB21611).
Analysis of preparations: Quadruple accurately weighed (4 x 1g) samples were taken from each formulation, including the vehicle according to the table below, used on the first day of treatment of the main study phase only. The first duplicate samples (2 x 1g) were stored refrigerated (between +2 and +8 °C). The second set of formulation samples (2 x 1g) were stored frozen (between -25 and -15 °C).
One set of samples (2 x 1g) was analysed at the Test Facility using a validated method. The transfer and validation of the analytical method was the subject of a separate study plan (study no.AB21611).
The second set of formulation samples (2 x 1g) kept at the Test Facility will be discarded following the issue of the final study report without any further notice to the Study Monitor/Study Sponsor.
Duration of treatment / exposure:
For both sexes: 14 days before mating, throughout the mating period and up to the day before necropsy (i.e. 31 days for males).
For females: During gestation (the first day of gestation was designated as G0) and at least 13 days after parturition up to and including the day before necropsy (the first day of birth is designated as
L0).
Frequency of treatment:
daily
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Rationale for the dose selection: Dose levels were selected based on the results of the DRF phase. For the Dose Range Finding phase, ethylvanillin was administered to WI (Han) female rats, (3 per dose), by oral route at dose levels of 500 and 1000 mg/kg/day for 10 days.
Under these experimental conditions of the study, the doses of 500 and 1000 mg/kg/day were not associated with any systemic effects.
Positive control:
not required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All adults were observed twice daily at the beginning and at the end of each working day (including weekends and public holidays). Any animal found dead were necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed daily during the study. To detect any clinical signs or reactions to treatment, the animals were observed before and at least twice after dosing (based on information provided from the DRF phase). A full clinical examination was performed on each weighing day. Towards the end of the gestation, females were examined daily for signs of parturition.
Besides, a hypersalivation examination was performed weekly, from Day 10, just after treatment and approximately between 1 hour and 3 hours after treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: Male and females were weighed during pretest (Day -5 for males, Day -18 for females), on the first day of dosing (prior to the first dose) and weekly thereafter during
pre-mating and mating periods.
Mated females were weighed:
- on Days 0, 6, 9, 12, 15, 18 and 20 of gestation
- on Days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption of males was recorded weekly during the pre-mating period.
Food consumption of females was recorded for the following periods:
- weekly during the pre-mating period
- gestation: Days 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 20
- lactation: Days 1 to 4, 4 to 7 and 7 to 13.

WATER CONSUMPTION: No
Sacrifice and pathology:
SACRIFICE
All adult males and females were weighed before necropsy (except found dead or killed moribund) and killed by carbon dioxide inhalation followed by exsanguination and necropsied according to the
following schedule:
- Males: after completion of the mating period on study Day 32 (31 days of dose administration)
- Females:
on Day 14 of lactation.
on Day 26 post-coitum for mated females that failed to produce a viable litter (total litter resorption).
On Day 24 post-coitum for female that was unable to deliver
within 24 hours of litter loss for females with total litter death.
as soon as possible on the day of death for found dead females.

GROSS NECROPSY
All animals (including any found dead) were submitted to necropsy procedures including an examination of following:
- external surface
- all orifices
- cranial cavity
- thoracic and abdominal cavities and organs and their contents
- the carcass.
Special attention was paid to the organs of the reproductive system.
For females necropsied before parturition, the ovaries and uterus were removed and examined including examination of the placentae. The following data were recorded:
- pregnancy status
- number of corpora lutea
- number of intrauterine implantations
- number of live embryos
- number of intrauterine deaths (resorption sites).
The uterus will be placed in ammonium sulphide solution in order to stain any previously undetected implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs listed in the following table were weighed at scheduled necropsy for all animals. Organs from any animals found dead or moribund were not weighed.
Paired organs were weighed together and if a difference in size is observed, the abnormally-sized organ(s) were weighed separately. The organs were weighed after dissection of fat and other contiguou
s tissues.
Organ weights were expressed as absolute and relative to body weight values.
Organs/tissues listed in the organ processing table were sampled for all animals (see organ processing table).
Fixatives: all tissue samples were fixed and preserved in 10 % neutral formalin with the followingexceptions:
– testes and epididymides, were fixed in modified Davidson's fluid.
The same sampling and trimming procedures were used for all applicable tissues in all applicable dose groups. Blocks were prepared only for tissues which will be evaluated histopathologically.
Histopathological examinations were performed as follows:
-for all organs/tissues from all adult animals found dead or killed moribund during the study
-for all macroscopic lesions from all dose group animals
-for all organs/tissues from all adult males and females of groups 1 (control) and 4 (high dose) (forfemales, only those with live pups)
-for the reproductive organs (see “ * ” in the organ processing table) and thyroid of females which failed to deliver and females with total litter loss.
Any exceptions are recorded in the individual animal data.
Statistics:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon
- the kurtosis of the data
- the probability of the Bartlett's test for homogeneity of the variances and
- an assessment of whether the size of the groups were approximately equal or not.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Five out of 10 females given 1000 mg/kg/day showed transient test item-related clinical signs during the pre-mating period. These consisted of decreased activity and/or abnormal breathing associated or not with partly closed eye(s), piloerection, cold body and/or recumbency. A transient decreased activity was observed also for 4/10 males at this dose level. These clinical signs were observed mainly during the first week of the premating period. These clinical signs were considered as adverse.

Noisy and/or laboured breathing was observed occasionally for 3 and 2 males in each of the 250 and 1000 mg/kg/day group. This isolated finding was considered as incidental in the absence of similar findings at 500 mg/kg/day.
During gestation, occasional piloerection was noted in two females given 1000 mg/kg/day. This was considered as unrelated to test item in the absence of such findings in other females.
On Day 22 of gestation, female no. 174 in the 1000 mg/kg/day group had a cold body, showed decreased activity, recumbency, partly-closed eyes, piloerection and was pale. This female had total litter death thereafter. These signs observed in one female only were considered due to the pregnancy/parturition status rather than to the test item.
There were no test item-related maternal clinical signs during the lactation period.
Hypersalivation associated or not with abnormal foraging and/or pedalling was observed in both sexes in all groups, including controls. There was, however, a higher incidence and persistance of these signs in all treated groups when compared with controls. This sign may be related to irritancy/palatability of the test item.
One female (no. 131) given 250 mg/kg/day had bilateral exophtalmos from mating. In isolation, this was considered incidental.
Clinical signs such as localized hairloss, erythema, scabs or scratches were incidental.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female no.139 given 250 mg/kg/day was found dead just after dosing on Day 18 of treatment. At the necropsy, the lungs were dark, correlating histologically with moderate alveolar haemorrhage. This death was considered likely related to the administration procedure and unrelated to test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
Mean body weight gain was slightly lower in the 1000 mg/kg/day group during the dosing period when compared with the control group (-6%). The final mean body weight was, however, only 1% lower than controls.
There was a slightly lower mean body weight gain during the first week or third weeks of dosing for males given 500 and 1000 mg/kg/day, respectively, when compared with the 250 mg/kg/day and control groups. During the last week of dosing, the mean body weight gain was higher in all treated groups than in the control group.
There was no test-item related effect on body weight gain for males in the 250 mg/kg/day group during premating and mating periods.

Females:
The mean body weight gain was lower in all treated groups (from 0 to 2.2g) than in the control group (9.3g) during the first week of the premating period. In addition, 2/10, 6/10 and 5/10 females lost weight during this period (from 0.8 to 8.9g) in the 250, 500 and 1000 mg/kg/day groups, respectively,
compared with none in the control group. Thereafter, the mean body weight gain in all treated groups was comparable or superior to the control group. The mean body weight on Day 15 was 5% lower in the 1000 mg/kg/day group than in the control group.
The overall mean body weight gain from G0 to G20 was similar in all groups, although there was a transient slightly lower mean body weight gain between gestation Days 6 and 12 in the 500 and 1000 mg/kg/day groups than in the control group. The mean body weight was approximately 5 to 7% lower in the 1000 mg/kg/day throughout the gestation period, due to a lower initial mean body weight.
The mean body weight gain was not affected by test item during lactation. The mean body weight was approximately 5 to 8% lower in the 1000 mg/kg/day throughout the lactation period, due to a lower initial mean body weight.
Since the effects observed on body weight and body weight changes are slight and transient, there are not considered as adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
Mean food consumption for males was comparable or higher than controls in all treated groups during the premating period.
Females:
There was a slightly lower food consumption for females in the 1000 mg/kg/day group (- 11%) when compared with other groups on the first week of the premating period.
During gestation and lactation, food consumption was 4 to 6% lower in the 500 and 1000 mg/kg/day groups than in controls. Since the decrease is slight, the effect is not considered as adverse.
There was no effect of test-item on mean food consumption for the 250 mg/kg/day group during the premating, gestation and lactation periods.
Food efficiency:
not specified
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, non-treatment-related
Description (incidence and severity):
When compared with controls, there were no treatment-related changes in organ weights. Occasional organ weight differences in treated animals when compared to the control group were considered to be incidental or only to reflect normal individual variation.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no observations that were considered to be associated with administration of the test item. One male (no.139) given 500 mg/kg/day had unilateral small testis and epididymis histologically correlated with marked unilateral tubular hypoplasia/atrophy with epididymal aspermia. Both changes were considered to be incidental since not observed in the other treated group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related microscopic findings in animals given 1000 mg/kg/day.
The nature or incidence of histological findings in the organs examined in both sexes did not indicate any relationship to treatment. All changes were considered to be incidental and part of the normal background changes encountered in reproductive/developmental studies in rodents.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Premature Decedents
Female no.139 given 250 mg/kg/day was found dead just after dosing on day 18 of treatment. At the necropsy, the lungs were dark, correlating histologically with moderate alveolar haemorrhage. This death was considered likely related to the administration (i.e. misgavage) procedure and unrelated to test item.
Details on results:
All analyzed samples for formulations prepared at nominal concentrations of 50, 100 and 200 mg/mL were in agreement with acceptance criteria and no test item was present in the vehicle sample.
Furthermore, the test item prepared as a suspension/dispersion in the vehicle was homogenous.
There was no test item-related mortality in any group.
Transient test item-related clinical signs in 5/10 females given 1000 mg/kg/day during the pre-mating period consisted of decreased activity and/or abnormal breathing associated or not with partly closed eye(s), piloerection, cold body and/or recumbency. A transient decreased activity was observed also for 4/10 males at this dose level. These effects are considered as adverse.
The mean body weight gain was lower than in controls in both males and females during the premating period in the 1000 mg/kg/day group. Some females in all treated groups lost weight. The final mean body weight of males and females in the high dose group was not, however, overtly affected (-1% for males on Day 29 and – 5% for females on Day 15). There were no test item-related body weight changes thereafter nor adverse changes in body weight gain at 250 and 500 mg/kg/day. Consistent with effects on body weight gains, there was a slightly lower food consumption in the 1000 mg/kg/day female group than in other groups.
There were no test item-related effects on mating performance of the males and females or on fertility in any group.
There were 9, 9, 10 and 9 females that successfully completed delivery in the control, 250, 500 and 1000 mg/kg/day groups, respectively. There was no evidence of any test item-related parental histological findings.
Statistically significantly lower Total T4 levels in F0-males of the 500 mg/kg/day and 1000 mg/kg/day groups when compared to Total T4 levels in control F0-males were observed, but considered to be slight in nature. In addition, the thyroid and parathyroid glands did not show histopathological or weight changes in any group.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
body weight and weight gain
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
other: Clinical signs
Organ:
other: Clinical signs
Treatment related:
yes
Dose response relationship:
yes

Table: Summary of salient mean body weight changes of males (mean values, in grams – percentage of difference with the control between brackets)

Intervals (days)

Group 1

Control

Group 2

250 mg/kg/day

Group 3

500 mg/kg/day

Group 4

1000 mg/kg/day

1-15 (Premating)

48.59

-

45.31

(-7 %)

40.47

(-17 %)

1-29

71.80

-

-

67.67

(-6%)

Conclusions:
Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 500 mg/kg/day was derived.
Executive summary:

The test item, 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin), was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 250, 500 and 1000 mg/kg/day. A control group of 10 rats/sex was given the vehicle (Propylene glycol). Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated during 2 weeks prior to mating, during mating, during post-coitum, and up to the day before necropsy (i.e. on day 13 of lactation for females delivered, on day 26 of gestation for female with total litter resorption and on day 1 of lactation for

female with total litter death). The following observations and examinations were performed: mortality / morbidity, clinical signs, body weight, food consumption, estrous cycle determination, clinical pathology,

measurement of thyroid hormone T4, macroscopy at termination, organ weights and histopathology on a selection of tissues.

All analyzed samples for formulations prepared at nominal concentrations of 50, 100 and 200 mg/mL were in agreement with acceptance criteria and no test item was present in the vehicle sample.

Furthermore, the test item prepared as a suspension in the vehicle was homogenous. There was no test item-related mortality in any group. Transient test item-related clinical signs in 5/10 females given 1000 mg/kg/day during the pre-mating period consisted of decreased activity and/or abnormal breathing associated or not with partly closed eye(s), piloerection, cold body and/or recumbency. A transient decreased activity was observed also for 4/10 males at this dose level. The mean body weight gain was lower than in controls in both males and females during the pre-mating period in the 1000 mg/kg/day

group. Some females in all treated groups lost weight. The final mean body weight of males and females in the high dose group was not, however, overtly affected (-1 % for males on Day 29 and -5 % for females on Day 15). There were no test item-related body weight changes thereafter nor adverse changes in body weight gain at 250 and 500 mg/kg/day. Consistent with effects on body weight gains, there was a slightly lower food consumption in the 1000 mg/kg/day female group than in other groups.

In conclusion, 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin) given by oral gavage in male and female Wistar Han rats at dose levels of 250, 500 and 1000 mg/kg/day revealed transient parental toxicity (considered as adverse) at 1000 mg/kg/day. Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 500 mg/kg was derived.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The deviations were considered not to have affected the outcome or the achievement of the study objectives.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
This study provided initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, parturition and early postnatal development.
The oral route was selected since it is a route of administration which is recommended by the Regulatory Authorities for this type of study.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI (Han)
Details on species / strain selection:
The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. Background data and control data for the strain are available at the Test Facility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Age at study initiation: Males: Approximately 10 weeks. Females: Approximately 12 weeks.
- Weight at study initiation: Males: 298 to 365 g. Females: 186 to 231 g.
- Fasting period before study: no
- Housing: One air-conditioned room in a barrier protected unit (building K1).
Caging:Animals were caged as follows:
Number of animals per cage:
Pre-mating: 5 males and 5 females
Mating: 1 male and 1 female (housed together)
Gestation: 5 males and 1 female
Lactation: 1 female + litter
Group housed males and females and individual housed females, including females during mating and with litters, were housed in plastic cages (550 x 376 x 215 mm) meeting European directive 2010/63/EU requirements.
- Diet (e.g. ad libitum): Rat powdered complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
- Water (e.g. ad libitum): Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via an automatic watering system). Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
- Bedding: Cellulose bedding (Serlab, Montataire, France) or dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood, analysed at least twice a year for chemical and bacterial contaminants.
- Enrichment: Animals received a small amount (handful) of shredded paper (SDS/Dietex) and the isolated animals had free access to a wooden gnaw block (Aspen Bricks, Le comptoire des sciures).
Furthermore, tissues were also provided as enrichment for females towards the end of gestation.
- Contaminants: No known contaminants were present in the diet, water or bedding at levels which might have interfered with achieving the objectives of the study.
- Acclimation period: 7 days between animal arrival and start of pre-test estrous cycle smears (females) or start of treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): > 35%
- Air changes (per hr): At least 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark (except when required for technical acts).

IN-LIFE DATES: From: 13 January 2017 To: 28 May 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
(supplier: SIGMA, batch number: MKCB2122V, expiry date: 23 January 2019)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was prepared as a suspension in the vehicle at concentrations of 50, 100 and 200 mg/mL according to Standard Operating Procedures of the Test Facility. No correction factor was taken into account for dose calculations.
Frequency of preparation: At least weekly (up to eight days).
Storage of formulations: Refrigerated (between +2 and +8 °C).

DIET PREPARATION
not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable
- Concentration in vehicle: 0; 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5mL/kg bw/d
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until mating occurs or 14 days have elapsed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not applicable
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item in the vehicle: 24h at ambient temperature or room temperature (between +15 and + 25 °C), 8 days refrigerated (between +2 and +8°C) or 3 weeks frozen (between -25 and -15 °C) (based on stability study no. AB21611).
Analysis of preparations: Quadruple accurately weighed (4 x 1g) samples were taken from each formulation, including the vehicle according to the table below, used on the first day of treatment of the main study phase only. The first duplicate samples (2 x 1g) were stored refrigerated (between +2 and +8 °C). The second set of formulation samples (2 x 1g) were stored frozen (between -25 and -15 °C).
One set of samples (2 x 1g) was analysed at the Test Facility using a validated method. The transfer and validation of the analytical method was the subject of a separate study plan (study no. AB21611).
The second set of formulation samples (2 x 1g) kept at the Test Facility will be discarded following the issue of the final study report without any further notice to the Study Monitor/Study Sponsor.


Duration of treatment / exposure:
For both sexes: 14 days before mating, throughout the mating period and up to the day before necropsy (i.e. 31 days for males).
For females: During gestation (the first day of gestation was designated as G0) and at least 13 days after parturition up to and including the day before necropsy (the first day of birth is designated as L0).

Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 10 weeks for males, 12 weeks for females
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Rationale for the dose selection: Dose levels were selected based on the results of the DRF phase. For the Dose Range Finding phase, ethylvanillin was administered to WI (Han) female rats, (3 per dose), by oral route at dose levels of 500 and 1000 mg/kg/day for 10 days. Under these experimental conditions of the study, the doses of 500 and 1000 mg/kg/day were not associated with any systemic effects.
Positive control:
not required

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All adults were observed twice daily at the beginning and at the end of each working day (including weekends and public holidays). Any animal found dead were necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed daily during the study. To detect any clinical signs or reactions to treatment, the animals were observed before and at least twice after dosing (based on information provided from the DRF phase). A full clinical examination was performed on each weighing day. Towards the end of the gestation, females were examined daily for signs of parturition.
Besides, a hypersalivation examination was performed weekly, from Day 10, just after treatment and approximately between 1 hour and 3 hours after treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: Male and females were weighed during pretest (Day -5 for males, Day -18 for females), on the first day of dosing (prior to the first dose) and weekly thereafter during pre-mating and mating periods.
Mated females were weighed:
- on Days 0, 6, 9, 12, 15, 18 and 20 of gestation
- on Days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption of males was recorded weekly during the pre-mating period.
Food consumption of females was recorded for the following periods:
- weekly during the pre-mating period
- gestation: Days 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 20
- lactation: Days 1 to 4, 4 to 7 and 7 to 13.


WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily and used to determine the cycle stage beginning 14 days prior to treatment, the first 14 days of treatment and during mating until evidence of copulation is observed. Vaginal smears continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal smear was taken to determine the stage of the estrous cycle and allow correlation with histopathology of female reproductive organs.
Sperm parameters (parental animals):
No data
Litter observations:
STANDARDISATION OF LITTERS
Not applicable

PARAMETERS EXAMINED
For each F1 litter the following data were recorded:
– number of pups born (live and dead);
– external abnormalities of the pups;
– number, weight and sex of pups alive on PND 1, 4, 7 and 13.
– anogenital distance (AGD) was measured on PND 1. The AGD was normalized to the cube root of body weight.
– all males in each litter were examined for the number of areola/nipples on PND 13.
On Day 4 post-partum, the size of each litter was adjusted (culling) to 8 pups, by eliminating extra pups by random selection to yield 4 males and 4 females per litter where possible.
Blood samples were collected from two surplus pups where possible, pooled and used for determination of serum T4 levels).
No pups were eliminated when litter size dropped below the culling target (8 pups/litter). When there is only one pup available above the culling target, only one pup was eliminated and used for blood collection for possible serum T4 assessments.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities. No tissues were preserved.
Postmortem examinations (parental animals):
SACRIFICE
All adult males and females were weighed before necropsy (except found dead or killed moribund) and killed by carbon dioxide inhalation followed by exsanguination and necropsied according to the following schedule:
- Males: after completion of the mating period on study Day 32 (31 days of dose administration)
- Females:
on Day 14 of lactation.
on Day 26 post-coitum for mated females that failed to produce a viable litter (total litter resorption).
On Day 24 post-coitum for female that was unable to deliver
within 24 hours of litter loss for females with total litter death.
as soon as possible on the day of death for found dead females.


GROSS NECROPSY
All animals (including any found dead) were submitted to necropsy procedures including an examination of following:
- external surface
- all orifices
- cranial cavity
- thoracic and abdominal cavities and organs and their contents
- the carcass.
Special attention was paid to the organs of the reproductive system.
For females necropsied before parturition, the ovaries and uterus were removed and examined including examination of the placentae. The following data were recorded:
- pregnancy status
- number of corpora lutea
- number of intrauterine implantations
- number of live embryos
- number of intrauterine deaths (resorption sites).
The uterus will be placed in ammonium sulphide solution in order to stain any previously undetected implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs listed in the following table were weighed at scheduled necropsy for all animals. Organs from any animals found dead or moribund were not weighed.
Paired organs were weighed together and if a difference in size is observed, the abnormally-sized organ(s) were weighed separately. The organs were weighed after dissection of fat and other contiguous tissues.
Organ weights were expressed as absolute and relative to body weight values.
Organs/tissues listed in the organ processing table were sampled for all animals (see organ processing table).
Fixatives: all tissue samples were fixed and preserved in 10 % neutral formalin with the following exceptions:
– testes and epididymides, were fixed in modified Davidson's fluid.
The same sampling and trimming procedures were used for all applicable tissues in all applicable dose groups. Blocks were prepared only for tissues which will be evaluated histopathologically.
Histopathological examinations were performed as follows:
-for all organs/tissues from all adult animals found dead or killed moribund during the study
-for all macroscopic lesions from all dose group animals
-for all organs/tissues from all adult males and females of groups 1 (control) and 4 (high dose) (for females, only those with live pups)
-for the reproductive organs (see “ * ” in the organ processing table) and thyroid of females which failed to deliver and females with total litter loss.

Any exceptions are recorded in the individual animal data.



Postmortem examinations (offspring):
SACRIFICE
Pups (extra pups on PND 4, any moribund pups and surviving pups on PND 13) will be killed by intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
Pups (including any found dead or killed moribund) will be necropsied. Any external abnormalities observed will be recorded but not preserved. For any pups found dead or killed moribund, the stomach will be examined for the presence of milk and defects or cause of death will be evaluated, if possible.
Each pup will be sexed and examined for external defects with special attention being paid to the external reproductive organs.


HISTOPATHOLOGY / ORGAN WEIGTHS
For pups, the thyroid glands of one male and one female of each litter were fixed and preserved.
Statistics:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon
- the kurtosis of the data
- the probability of the Bartlett's test for homogeneity of the variances and
- an assessment of whether the size of the groups were approximately equal or not.
Reproductive indices:
Pre-coital interval (in days): sum of days until successful insemination
_________________________________
number of inseminated females

Irregularity index: Mean standard deviation of length of the oestrous cycle
__________________________________________________
√ (length of the oestrous cycle)

Days in estrus: number of estrus days x 100
____________________
number of smears

Copulation index (%): number of inseminated females x 100
____________________________
number of paired animals

Fertility index (%) number of pregnant females x 100
________________________
number of inseminated females

Pre-birth loss (%): number of implantations - number of offspring born x 100
_____________________________________________
number of implantations


Offspring viability indices:

Live birth index (in %): number of pups born alive x 100
______________________
number of pups born

Viability index (in %): number of pups alive on PND 4 (before culling) x 100
______________________________________
number of pups alive at birth

Lactation index (%): number of pups alive on PND 13 x 100
_______________________________
number of pups alive on PND 4 (after culling)


Sex ratio (proportion of male) (in %): number of males x 100
____________________
number of pups

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Five out of 10 females given 1000 mg/kg/day showed transient test item-related clinical signs during the pre-mating period. These consisted of decreased activity and/or abnormal breathing associated or not with partly closed eye(s), piloerection, cold body and/or recumbency. A transient decreased activity was observed also for 4/10 males at this dose level. These clinical signs were observed mainly during the first week of the premating period. These clinical signs were considered as adverse.

Noisy and/or laboured breathing was observed occasionally for 3 and 2 males in each of the 250 and 1000 mg/kg/day group. This isolated finding was considered as incidental in the absence of similar findings at 500 mg/kg/day.

During gestation, occasional piloerection was noted in two females given 1000 mg/kg/day. This was considered as unrelated to test item in the absence of such findings in other females.

On Day 22 of gestation, female no. 174 in the 1000 mg/kg/day group had a cold body, showed decreased activity, recumbency, partly-closed eyes, piloerection and was pale. This female had total litter death thereafter. These signs observed in one female only were considered due to the pregnancy/parturition status rather than to the test item.

There were no test item-related maternal clinical signs during the lactation period.

Hypersalivation associated or not with abnormal foraging and/or pedalling was observed in both sexes in all groups, including controls. There was, however, a higher incidence and persistance of these signs in all treated groups when compared with controls. This sign may be related to irritancy/palatability of the test item.

One female (no. 131) given 250 mg/kg/day had bilateral exophtalmos from mating. In isolation, this was considered incidental.

Clinical signs such as localized hairloss, erythema, scabs or scratches were incidental.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female no.139 given 250 mg/kg/day was found dead just after dosing on Day 18 of treatment. At the necropsy, the lungs were dark, correlating histologically with moderate alveolar haemorrhage. This death was considered likely related to the administration procedure and unrelated to test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
Mean body weight gain was slightly lower in the 1000 mg/kg/day group during the dosing period when compared with the control group (-6%). The final mean body weight was, however, only 1% lower than controls.
There was a slightly lower mean body weight gain during the first week or third weeks of dosing for males given 500 and 1000 mg/kg/day, respectively, when compared with the 250 mg/kg/day and control groups. During the last week of dosing, the mean body weight gain was higher in all treated groups than in the control group.

There was no test-item related effect on body weight gain for males in the 250 mg/kg/day group during premating and mating periods.

Females:
The mean body weight gain was lower in all treated groups (from 0 to 2.2g) than in the control group (9.3g) during the first week of the premating period. In addition, 2/10, 6/10 and 5/10 females lost weight during this period (from 0.8 to 8.9g) in the 250, 500 and 1000 mg/kg/day groups, respectively, compared with none in the control group. Thereafter, the mean body weight gain in all treated groups was comparable or superior to the control group. The mean body weight on Day 15 was 5% lower in the 1000 mg/kg/day group than in the control group.

The overall mean body weight gain from G0 to G20 was similar in all groups, although there was a transient slightly lower mean body weight gain between gestation Days 6 and 12 in the 500 and 1000 mg/kg/day groups than in the control group. The mean body weight was approximately 5 to 7% lower in the 1000 mg/kg/day throughout the gestation period, due to a lower initial mean body weight.
The mean body weight gain was not affected by test item during lactation. The mean body weight was approximately 5 to 8% lower in the 1000 mg/kg/day throughout the lactation period, due to a lower initial mean body weight.

Since the effects observed on body weight and body weight changes are slight and transient, there are not considered as adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
Mean food consumption for males was comparable or higher than controls in all treated groups during the premating period.

Females:
There was a slightly lower food consumption for females in the 1000 mg/kg/day group (- 11%) when compared with other groups on the first week of the premating period.
During gestation and lactation, food consumption was 4 to 6% lower in the 500 and 1000 mg/kg/day groups than in controls. Since the decrease is slight, the effect is not considered as adverse.

There was no effect of test-item on mean food consumption for the 250 mg/kg/day group during the premating, gestation and lactation periods.
Food efficiency:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related microscopic findings in animals given 1000 mg/kg/day.
The nature or incidence of histological findings in the organs examined in both sexes did not indicate any relationship to treatment. All changes were considered to be incidental and part of the normal background changes encountered in reproductive/developmental studies in rodents.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Premature Decedents
Female no.139 given 250 mg/kg/day was found dead just after dosing on day 18 of treatment. At the necropsy, the lungs were dark, correlating histologically with moderate alveolar haemorrhage. This death was considered likely related to the administration (i.e. misgavage) procedure and unrelated to test item.

Thyroid hormone analysis
There was no adverse effect of treatment on the total T4 levels for F0-males.
Statistically significantly lower Total T4 levels in F0-males of the 500 mg/kg and 1000 mg/kg groups when compared to Total T4 levels in control F0-males were observed, however, the thyroid and parathyroid glands did not show histopathological or weight changes in any group.
No differences in Total T4 levels were noted among the different groups of PND 13 pups.
Based on these results, analysis of T4 level has not been performed in females and in PND4 pups and TSH was also deemed not necessary for PND13 pups or F0 animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment.
An irregular cycle was noted for female no. 177 before mating. This finding was not correlated to pregnancy status and was therefore considered incidental.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
All pairs of rats mated in the control, 250, 500 and 1000 mg/kg/day groups. All mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal oestrus cycle).
Mating performance was therefore comparable in all groups.

Details on results (P0)

All analyzed samples for formulations prepared at nominal concentrations of 50, 100 and 200 mg/mL were in agreement with acceptance criteria and no test item was present in the vehicle sample.
Furthermore, the test item prepared as a suspension/dispersion in the vehicle was homogenous.
There was no test item-related mortality in any group.
Transient test item-related clinical signs in 5/10 females given 1000 mg/kg/day during the pre-mating period consisted of decreased activity and/or abnormal breathing associated or not with partly closed eye(s), piloerection, cold body and/or recumbency. A transient decreased activity was observed also for 4/10 males at this dose level.
The mean body weight gain was lower than in controls in both males and females during the pre-mating period in the 1000 mg/kg/day group. Some females in all treated groups lost weight. The final mean body weight of males and females in the high dose group was not, however, overtly affected (-1% for males on Day 29 and – 5% for females on Day 15). There were no test item-related body weight changes thereafter nor adverse changes in body weight gain at 250 and 500 mg/kg/day. Consistent with effects on body weight gains, there was a slightly lower food consumption in the 1000 mg/kg/day female group than in other groups.
There were no test item-related effects on mating performance of the males and females or on fertility in any group.
There were 9, 9, 10 and 9 females that successfully completed delivery in the control, 250, 500 and 1000 mg/kg/day groups, respectively. There was no evidence of any test item-related parental histological findings.
Statistically significantly lower Total T4 levels in F0-males of the 500 mg/kg/day and 1000 mg/kg/day groups when compared to Total T4 levels in control F0-males were observed, but considered to be slight in nature. In addition, the thyroid and parathyroid glands did not show histopathological or weight changes in any group.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
Reproductive performance
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no reproduction toxicity.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no pup observations that suggested any association with maternal treatment. One pup in each of the control and 1000 mg/kg/day groups appeared cold to touch.
Incidental clinical signs included incomplete hair growth and partial or complete absence of tail.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of dead, missing or cannibalised pups was higher in the 250 (14) and 1000 (8) mg/kg/day on post-natal Day 0 than in the 500 (2) and control (0) groups. The live birth index was therefore lower at 250 (86.9 %) and 1000 (91.9 %) mg/kg/day than in the control (100.0 %) and 500 (98.2 %) mg/kg/day groups. However, the lower values was due to female no. 133 given 250 mg/kg/day and female no. 174 given 1000 mg/kg/day which had 14 and 7 dead pups at birth, respectively, and finally lost their entire litter on PND 1. This findings were isolated and in the absence of a dose-related effect, this was considered incidental.
The number of live offspring on Day 4 before culling compared to the number of offspring alive at birth was higher in the 1000 mg/kg/day group. Two, three and eight pups at 250, 500 and 1000 mg/kg/day, respectively, were found dead or missing versus none in the control group between post-natal Days 1 and 4. The viability index was therefore lower at 1000 mg/kg/day (91.2 %) than in the control (100.0 %), 250 (97.8 %) and 500 (97.2 %) mg/kg/day groups. However, the low value at 1000 mg/kg/day was due to a single female (no. 174) that lost the entire litter (8 pups) on day 1 of lactation. This was therefore considered incidental.
The number of live offspring on Day 13 compared to the number of live offspring on Day 4 after culling was not affected by treatment. The lactation index was 100 % in all groups.
There was no test item-related effect on sex ratio.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight of pups was not affected by the treatment.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Parturition
There were 9, 9, 10 and 9 females that successfully completed delivery in the control, 250, 500 and 1000 mg/kg/day groups, respectively. There was one female in the control group (no. 117) with total litter resorption, one female in each of the 250 and 1000 mg/kg/day groups (nos. 133 and 174, respectively) with total litter death and one in the 1000 mg/kg/day group that was unable to deliver and consequently euthanized for ethical reason (no. 175). In the absence of a clear dose-related trend, these findings were considered as unrelated to the test-item. The mean duration of gestation was comparable in all groups (approximately 22 days).

Post implantation data
The total number of offspring born compared to the total number of uterine implantations was not affected by treatment. The percentage pre-birth loss was lower in all treated groups compared to controls. The percentage pre-birth loss in the control group (13.0 %) can be considered equal to the upper limit of historical control data range (1.2 - 12.9 %).

Pup viability and litter sizes
The mean number of pups delivered per litter was slightly lower in the 500 and 1000 mg/kg/day groups (11.0 each) than in the control (11.7) and 250 (11.9) mg/kg/day groups. The difference was minor and all values remained above the historical control mean value (10.9). This difference was therefore not considered as toxicologically relevant.

Number of Areola / Nipples (Male Pups)
For none of the examined male pups, nipples were observed at post-natal Day 13.

Pup Anogenital Distance
Anogenital distance (normalized for body weight) in male and female pups was not affected by treatment.

Details on results (F1)

There were no test item-related effects on mating performance of the males and females or on fertility in any group. There were 9, 9, 10 and 9 females that successfully completed delivery in the control, 250, 500 and 1000 mg/kg/day groups, respectively. There were no test item-related effects on the number of implantation, pre-birth loss, litter size, pup viability or body weight.
There were no test item-related effects on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Anogenital distance (normalized for body weight) in male and female pups was not affected by test item whatever the dose level.
No differences in Total T4 levels were noted among the different groups of PND 13 pups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table: Summary of salient mean body weight changes of males (mean values, in grams – percentage of difference with the control between brackets)

Intervals (days)

Group 1

Control

Group 2

250 mg/kg/day

Group 3

500 mg/kg/day

Group 4

1000 mg/kg/day

1-15 (Premating)

48.59

-

45.31

(-7 %)

40.47

(-17 %)

1-29

71.80

-

-

67.67

(-6%)

Applicant's summary and conclusion

Conclusions:
Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 500 mg/kg/day and a reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day were derived.
Executive summary:

The test item, 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin), was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 250, 500 and 1000 mg/kg/day. A control group of 10 rats/sex was given the vehicle (Propylene glycol). Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated during 2 weeks prior to mating, during mating, during post-coitum, and up to the day before necropsy (i.e. on day 13 of lactation for females delivered, on day 26 of gestation for female with total litter resorption and on day 1 of lactation for female with total litter death). The following observations and examinations were performed: mortality / morbidity, clinical signs, body weight, food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4, macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating and fertility index, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

All analyzed samples for formulations prepared at nominal concentrations of 50, 100 and 200 mg/mL were in agreement with acceptance criteria and no test item was present in the vehicle sample. Furthermore, the test item prepared as a suspension in the vehicle was homogenous. There was no test item-related mortality in any group. Transient test item-related clinical signs in 5/10 females given 1000 mg/kg/day during the pre-mating period consisted of decreased activity and/or abnormal breathing associated or not with partly closed eye(s), piloerection, cold body and/or recumbency. A transient decreased activity was observed also for 4/10 males at this dose level. The mean body weight gain was lower than in controls in both males and females during the pre-mating period in the 1000 mg/kg/day group. Some females in all treated groups lost weight. The final mean body weight of males and females in the high dose group was not, however, overtly affected (-1 % for males on Day 29 and -5 % for females on Day 15). There were no test item-related body weight changes thereafter nor adverse changes in body weight gain at 250 and 500 mg/kg/day. Consistent with effects on body weight gains, there was a slightly lower food consumption in the 1000 mg/kg/day female group than in other groups. There were no test item-related effects on mating performance of the males and females or on fertility in any group. There were 9, 9, 10 and 9 females that successfully completed delivery in the control, 250, 500 and 1000 mg/kg/day groups, respectively. There were no test item-related effects on the number of implantation, pre-birth loss, litter size, pup viability or body weight. There were no test item-related effects on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13. Anogenital distance (normalized for body weight) in male and female pups was not affected by test item whatever the dose level. There was no evidence of any test item-related parental histological findings. Statistically significantly lower Total T4 levels in F0-males of the 500 mg/kg/day and 1000 mg/kg/day groups when compared to Total T4 levels in control F0-males were observed, but considered to be slight in nature. In addition, the thyroid and parathyroid glands did not show histopathological or weight changes in any group. No differences in Total T4 levels were noted among the different groups of PND 13 pups.

In conclusion, 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin) given by oral gavage in male and female Wistar Han rats at dose levels of 250, 500 and 1000 mg/kg/day revealed transient parental toxicity (considered as adverse) at 1000 mg/kg/day, but no reproduction and developmental toxicity for treatment up to 1000 mg/kg/day. Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 500 mg/kg and a reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg were derived.