Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

An OECD 421 study has been conducted in 2017 on the registered substance. In this study, 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 but adverse parental toxicity at 1000 mg/kg/day. However, no reproduction and developmental toxicity were observed in this OECD 421 study up to the dose level of 1000 mg/kg/day (highest tested dose).

Based on this study 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.

Link to relevant study records
Reference
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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
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. QA statement)
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.
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 or test system 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
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
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
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
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: 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.
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.
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.
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.
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.
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).
Reproductive effects observed:
not specified

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 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
One reliability 1 study according to the OECD 421 guideline is available.

Effects on developmental toxicity

Description of key information

An OECD 414 study has been performed in rats in 2017. In this study, oral (gavage) administration of 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin) to the pregnant Wistar rat from implantation through to the day before caesarean section at 250, 500 or 1000 mg/kg/day was associated with transient and non-adverse slightly lower body weight gain and food consumption at 1000 mg/kg/day. Transient but adverse clinical changes at the start of treatment in the 1000 mg/kg/day group included irregular and/or rapid breathing, partly closed eye(s), decreased activity, dark eyes, recumbency and /or subdued behaviour. In view of the nature of the clinical signs, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was established as 500 mg/kg/day in the female rat.

Post-implantation data were not affected by test item in any group. A slight increase of the foetal and litter incidences of malformations was observed at 1000 mg/kg/day compared to the control. However, all these malformations were already noted in the more recent historical control data of the laboratory with an incidence comparable or lower to the maximal incidence per study in the HCD. Therefore a test item-related effect was doubtful and the malformations were more likely attributed to a maternal mediated effect .

Based on these findings, the NOAEL for embryo-fetal development was considered to be 500 mg/kg/day. However this effect is more likely attributed to a maternal mediated effect.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 January 2017 to 28 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI (Han)
Details on test animals or test system and environmental conditions:
- Number: 88 virgin mated females. The females were mated at the supplier with a documented day of mating. They were received at the Test Facility on Day 0 of gestation (G 0).
- Strain and sanitary status: Wistar rats: Crl: WI (Han). All animals received a clinical inspection for ill-health on arrival.
- Breeder: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Age/Weight:
Age at mating: 10 to 13 weeks.
Body weight range at mating: 189 to 238 g
- Housing: One air-conditioned room in a barrier protected unit (building K1), Caging: Females were individually housed in plastic cages (dimensions 375 x 215 x 180 mm) meeting European directive 2010/63/EU requirements.
- Food and water: Rat powdered/pelleted commercial complete rodent diet ad libitum (Diet reference SAFE 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. The rats had free access to the food throughout the study. Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via bottles). Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
- Acclimation: no information
- Allocation to group: Performed by random assignment after arrival (computer-generated random numbers).
- Identification: Animals were identified using transponder implants (Biolog-id).

ENVIRONMENTAL CONDITIONS
- temperature: 22 + 3 °C (target range),
- relative humidity: > 35 % (target),
- light/dark cycle: 12 hours light (artificial)/12 hours dark,
- ventilation: At least 10 air changes per hour.

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
DOSE FORMULATION PREPARATION
Preparation: 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: Weekly.
Storage of formulations (including vehicle): Refrigerated (between +2 and +8 °C).
Stability of the test item in the vehicle: Formulations, protected from light and in the range 5-200 mg/mL, were demonstrated to be stable for 24 hours at room temperature (between +15 and +25 °C), for 8 days refrigerated (between +2 and +8 °C) and for 3 weeks under frozen conditions (between -15 and 25 °C) (Test Facility study no. AB21611).



VEHICLE
The vehicle was propylene glycol..
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
The females were mated at the supplier with a documented day of mating. They were received at the Test Facility on Day 0 of gestation (G 0).

Duration of treatment / exposure:
The dose formulations were administered daily from day 6 to day 20 p.c., inclusive. Formulations were maintained under continuous stirring for at least 15 minutes before and throughout the dosing procedure.
Frequency of treatment:
Daily
Duration of test:
One month
Dose / conc.:
250 mg/kg bw/day
Remarks:
dose volume : 5 ml/kg/day
Dose / conc.:
500 mg/kg bw/day
Remarks:
dose volume : 5 ml/kg/day
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
dose volume : 5 ml/kg/day
No. of animals per sex per dose:
22 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose-level selection
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of the DRF phase.




Maternal examinations:
MORBIDITY AND MORTALITY:
All animals were observed at least twice daily.

CLINICAL SIGNS:
All animals were observed daily for clinical signs during the study.
During the treatment period, the animals were observed before and at least twice after dosing to detect any abnormalities in appearance, behaviour or other signs of reaction to treatment. A full clinical examination was performed on each weighing day.

BODY WEIGHT:
All animals were weighed individually on Days 0, 6, 9, 12, 15, 18 and 21 of gestation .

FOOD CONSUMPTION:
Individual food consumption was measured for the periods (Days) 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 21 during gestation .

POST-MORTEM EXAMINATIONS: Yes
All females were weighed before necropsy and killed by carbon dioxide inhalation followed by exsanguination on Day 21 of gestation, on the day after the last treatment. They were dissected and examined for macroscopic pathological changes.
Any abnormal organs/tissues were sampled and preserved in 10 % neutral formalin. No further examinations were performed.

Ovaries and uterine content:
The ovaries and uterus of each female were removed and examined.
The placentae were also examined. The following data were recorded:
- pregnancy status
- number of corpora lutea
- number of implantations
- number and distribution of live foetuses
- number and distribution of intrauterine implantations, classified as follows:
- live foetuses
- dead foetuses
- early resorptions
- late resorptions
- gravid uterus weight
- individual foetal weights
- foetal sex.

Resorptions were classified on the basis of the presence (late) or absence (early) of foetal or decidual tissue in addition to placental tissue.
The uterus of all females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites.




Fetal examinations:
Each foetus was examined for external defects. All foetuses were killed by an oral administration of sodium pentobarbitone (CEVA Santé Animale).
Approximately one half of each litter was submitted to fresh visceral examination of the body (abdominal and thoracic cavities) and then processed for skeletal examination. The ossified skeleton was stained with Alizarin red following maceration of the soft tissues in aqueous potassium hydroxide solution. The stained specimens were preserved in glycerol.
The remaining foetuses were preserved in Harrisson's fluid for subsequent examination of the head only by the modified Wilson-Barrow technique. The remaining carcass was retained fixed but not examined further.
Soft tissue and skeletal examinations were performed using a binocular microscope.
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 normality of the data distribution tested by the Shapiro-Wilk's test
- the homogeneity of the variances across groups tested by the Bartlett's test.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
The number of resorptions, number of dead foetuses and all litter-based percentages were analysed using non-parametric methods, i.e. Kruskal-Wallis test followed by non-parametric Dunnett’s test if the Kruskal-Wallis is significant.
Selected incidence data were analysed using a chi2 test for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were 10 out 22 females given 1000 mg/kg/day that showed transient but adverse clinical signs including irregular and/or rapid breathing associated or not with partly closed eye(s), wheezing, decreased activity or dark eyes. Among these animals, six displayed recumbency and /or subdued behaviour. These clinical signs were observed mainly shortly after the first or second dosing (G 6 and G 7).
There was a higher incidence and persistence of hypersalivation associated or not with abnormal foraging and/or pedalling in all treated groups (21/22, 22/22 and 22/22 females in the 250, 500 and 1000 mg/kg/day groups, respectively) when compared with the control group (6/22 females). Hypersalivation, with associated observations of abnormal foraging, pedalling and/or Straub tail, is commonly observed in rodents given oral formulations with poor palatability and is, therefore, considered of no toxicological significance.
There were no other treatment-related clinical signs in any group.
Wheezing was noted for control female no. 101 on G 17. This isolated finding in the control group was considered incidental. Other incidental clinical signs included hairloss and scab(s).
Mortality:
no mortality observed
Description (incidence):
There is no unscheduled deaths in any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Although the mean body weight gain was slightly lower from G 6 to G 9 for females given 1000 mg/kg/day (9.7 g) than for other groups (13.0, 13.0 and 11.1 g in the control, 250 and 500 mg/kg/day groups, respectively), the mean final body weight was comparable in all groups. This slight decrease is considered as non-adverse.
There were no treatment-related effects on mean body weight gain during the dosing period (G 6 to G 21) in other groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a slightly lower mean food consumption between G 6 and G 12 for females given 1000 mg/kg/day (19.6 g/day) than in other groups (20.8, 21.8 and 21.4 g/day in the control, 250 and 500 mg/kg/day groups, respectively). This was no longer observed thereafter.
There was no treatment-related effect on food consumption during the dosing period (G 6 to G 21) in other groups.
This slight and transient decrease is not considered as adverse.

Food efficiency:
not examined
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:
no effects observed
Description (incidence and severity):
There was no treatment-related effect on gravid uterus weight.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related macroscopic findings in any group.
A cyst on the left ovary was noted for female no. 139 given 500 mg/kg/day and a dark focus on the liver of female no. 185 given 1000 mg/kg/day. These isolated findings were considered incidental.
Localized or diffuse alopecia was occasionally observed with a sporadic incidence.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
There were no unscheduled deaths in any group.
Transient but marked test item-related clinical changes were observed in 10 out of 22 females in the 1000 mg/kg/day group on G 6 or G 7 shortly after dosing. These included irregular and/or rapid breathing associated or not with partly closed eye(s), wheezing, decreased activity or dark eyes. Among these animals, six displayed recumbency and /or subdued behaviour.
Hypersalivation noted for essentially all females in the vehicle, 250, 500 and 1000 mg/kg/day groups, with or without abnormal foraging and/or pedalling were clearly associated with the vehicle (Polyethylene glycol 400) but might possibly be slightly exacerbated by the test item. Hypersalivation, with associated observations of abnormal foraging, pedalling and/or Straub tail, is commonly observed in rodents given oral formulations with poor palatability and is, therefore, considered of no toxicological significance.
There was a transient slightly lower mean body weight gain and mean daily food consumption for females given 1000 mg/kg/day compared with the concurrent control towards the initiation of treatment. The mean body weight at the end of the dosing period was, however, comparable in all groups.
There were no treatment-related macroscopic findings or effects on mean gravid uterus weight in any group.
There were 22 pregnant females at terminal caesarean in each of the control, 250, 500 and 1000 mg/kg/day groups, all of which had viable foetuses.
Number of abortions:
no effects observed
Description (incidence and severity):
There were 22 pregnant females at the terminal caesarean sections in the control and in each of the treated groups, all of which had viable foetuses.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The mean numbers of corpora lutea and implantation sites were comparable in all groups. The pre-implantation loss was slightly higher for females given 250 and 500 mg/kg/day (12.14 and 12.92%, respectively) when compared with other groups (7.03 and 7.44% for group 1 and 4, respectively). However mean values were within the historical control range and these differences were considered incidental.
There was no test item-related effect on embryo-foetal survival in any group. Mean live litter size was consequently comparable in all groups.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
There were 22 pregnant females at the terminal caesarean sections in the control and in each of the treated groups, all of which had viable foetus. There was no-test item effect on embryo-foetal survival in any group. Mean live litter size was consequently comparable in all groups.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were 22 pregnant females at the terminal caesarean sections in the control and in each of the treated groups.
Details on maternal toxic effects:
Under the experimental conditions of the study, oral (gavage) administration of 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin) to the pregnant Wistar rat from implantation through to the day before caesarean section at 250, 500 or 1000 mg/kg/day was associated with transient and non-adverse slightly lower body weight gain and food consumption at 1000 mg/kg/day. Transient but adverse clinical changes at the start of treatment in the 1000 mg/kg/day group included irregular and/or rapid breathing, partly closed eye(s), decreased activity, dark eyes, recumbency and /or subdued behaviour.
In view of the nature of the clinical signs, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was established as 500 mg/kg/day in the female rat.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment on mean foetal weight in any group.
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect of treatment on sex ratio in any group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean litter size was comparable in all groups.
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 3 foetuses from as many litters with external malformation in the 1000 mg/kg/day group compared with none in other groups.
One foetus had meningoencephalocele (foetus no.4 from dam no. 167), one had anasarca (foetus no 4 dam no. 174) and the other had anal atresia and thread-like tail (foetus no. 11 from dam no. 175).
The incidence of each external malformation was equal or lower to the maximal incidence per study in the historical control data. None of these observations were therefore clearly attributed to the test item.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 5 foetuses from as many litters with skeletal malformations in the 1000 mg/kg/day group compared with none in other groups.
Consistent with the meningoencephalocele observed externally, the foetus no.4 from dam no.167 had interparietal and parietal misshapen associated with hole in cranium. This foetus also showed exoccipital fused with right first vertebral cervical arch.
Correlated with the anal atresia and thread-like tail noted at the external examination, the foetus no.11 from dam no.175 showed multiple skeletal abnormalities including sacral arch vertebra misshapen, 2nd sacral centrum absent, absence of the 3rd and 4th sacral vertebrae and the absence of all caudal vertebrae.
Foetus no.4 from dam no.174 with anasarca also had mandible fused and palatine split and one foetus from each of dams nos. 182 (foetus no. 7) and 188 (foetus no. 3) had fused sternebra or fused mandible, respectively.
All skeletal malformations were observed in the historical control data (2014-2016) with a maximal incidence comparable or higher to that of the 1000 mg/kg/day treated group. Therefore, these skeletal malformations can not be clearly related to an effect of the test item.
The incidence of other less severe skeletal anomalies and variations did not suggest any association with the test item.

Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In addition to the foetus no.4 (dam no.174) with multiple abnormalities of the heart, likely associated with the anasarca noted externally, 2 foetuses from two litters had visceral malformations in the 1000 mg/kg/day group, compared with none in other groups.
One foetus had retroesophageal subclavian artery (foetus no. 6 from dam no. 168) and the other one had a situs inversus (foetus no.13 from dam no. 174).
In view of the lower incidence of each visceral malformation when compared with the maximal incidence per study of the historical control data (2014-2016) , none of these malformations were considered clearly related to an effect of the test item.
The incidence of other less severe visceral anomalies and variations such as absent innominate artery, transposed umbilical artery and convoluted or dilated ureters did not suggest any association with the test item.

Details on embryotoxic / teratogenic effects:
There was no test item-related effect on embryo-foetal survival, foetal weight or foetal sex ratio in any group.
In total, the number of foetuses (litters) with external, skeletal and/or visceral malformations in the 1000 mg/kg/day group were 7/240 (6/22) leading to a foetal (litter) incidence of 2.9% (27%) compared with none in other groups. All malformations were already noted in our more recent historical control data with an incidence comparable or lower to the maximal incidence per study in the historical control data (2014-2016). In addition, in view of broad spectrum of these malformations (including general, head, caudal malformations and other visceral or skeletal malformations such as situs inversus, retroesophagial subclavian artery and sternebra or mandible fused), a test item-related effect was doubtful and the malformations were more likely attributed to a maternal mediated effect in view of the marked clinical signs observed on the first days of dosing at the high dose of 1000 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: based on a slight increase of the foetal and litter incidences of malformations at 1000 mg/kg/day which was more likely attributed to the maternal clinical signs.
Abnormalities:
not specified
Developmental effects observed:
not specified

Summary of malformations – Individual descriptions:

 

Dose level (mg/kg/day)

Female identification number

Foetus identification number(s)

Malformation(s)#

0

/

/

No malformations observed

250

/

/

No malformations observed

500

/

/

No malformations observed

1000

167

4

Meningoencephalocele

Interparietal and parietal misshapen associated with hole in cranium

Exoccipital fused with right 1stvertebral cervical arch

168

6

Retroesophageal right subclavian artery (arising from descending aorta)

174

4

Anasarca

Multiple abnormalities of the heart (interventricular septum defect with aorta arising from right ventricle)

Mandible, fused

Palatine, split

13

Situs inversus (abdominal and thoracic cavities)

175

11

Anal atresia

Thread‑like tail

2ndsacral arch vertebra misshapen; 2ndsacral centrum absent; absence of the 3rdand 4thsacral vertebrae; absence of all caudal vertebrae

182

7

Sternebra, fused (3rdand 4th)

188

3

Mandible, fused

#: including external, visceral and skeletal examinations.

maximal foetal incidence per study or total incidence of external malformations in the historical control data (2014-2016) compared with study no.AB21616 (in percent):

Type of malformation

Incidence in study AB21616

Maximal incidence per study in HCD

Total incidence in HCD (mean)

Head malformation

0.4 (meningoencephalocele)

0.4 (exencephaly)

0.04

Anasarca

0.4

0.5

0.12

Anal atresia

0.4

1

0.04

maximal foetal incidence per study or total incidence of visceral malformations in the historical control data (2014-2016) compared with study no.AB21616 (in percent):

Type of

 malformation

Incidence in study AB21616

Maximal incidence per study in HCD

Total incidence in HCD (mean)

Retroesophageal subclavian artery

0.8

1.8

0.05

Situs inversus

0.8

1.4

0.23

maximal foetal incidence per study or total incidence of skeletal malformations in the historical control data (2014-2016) compared with study no.AB21616 (in percent):

Type of malformation

Incidence in study AB21616

Maximal incidence per study in HCD

Total incidence in HCD

Mandible fused

1.6

1.8

0.1

Sternebra fused

0.8

0.8

0.05

Palatine split

0.8

0.8

0.1

Conclusions:
Under the experimental conditions of the study, oral (gavage) administration of 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin) to the pregnant Wistar rat from implantation through to the day before caesarean section at 250, 500 or 1000 mg/kg/day was associated with transient and non-adverse slightly lower body weight gain and food consumption at 1000 mg/kg/day. Transient but adverse clinical changes at the start of treatment in the 1000 mg/kg/day group included irregular and/or rapid breathing, partly closed eye(s), decreased activity, dark eyes, recumbency and /or subdued behaviour.
In view of the nature of the clinical signs, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was established as 500 mg/kg/day in the female rat.
Post-implantation data were not affected by test item in any group.
A slight increase of the foetal and litter incidences of malformations was observed at 1000 mg/kg/day compared to the control. However, all these malformations were already noted in our more recent historical control data with an incidence comparable or lower to the maximal incidence per study in the HCD. Therefore a test item-related effect was doubtful and the malformations were more likely attributed to a maternal mediated effect .
Based on these findings, the NOAEL for embryo-fetal development was considered to be 500 mg/kg/day.

Executive summary:

The objectives of the study were to determine the potential of the test item, 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin), to induce developmental toxicity after maternal exposure during the critical period of organogenesis, to characterize maternal toxicity at the exposure levels tested and to determine the NOAEL (no observed-adverse effect level) for maternal toxicity and developmental toxicity.

The test item, 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin), was administered by daily gavage at dose levels of 250, 500 and 1000 mg/kg/day to groups of 22 mated female Wistar rats from days 6 to 20 of gestation inclusive. A control group received a similar volume (5 mL/kg) of the vehicle Propylene Glycol. Clinical condition, body weights and food consumption were monitored throughout the study. The females were submitted to a caesarean examination on day 21 of gestation and litter parameters were recorded. At necropsy, the females were examined macroscopically and the gravid uterus was weighed.

All foetuses were weighed, sexed and examined for external abnormalities. Half of the foetuses were submitted to fresh visceral examination of the body (abdominal and thoracic cavities) prior to processing for skeletal examination. The remaining foetuses were preserved for subsequent examination of the head only by the modified Wilson-Barrow technique.

All formulations at 50, 100 and 200 mg/mL of Test item: 3-ethoxy-4-hydroxybenzaldehyde in vehicle (Propylene Glycol) including the vehicle, used on the first day of treatment were in agreement with acceptance criteria. No significant amount of test item was detected in the vehicle sample. There were no unscheduled deaths in any group. Transient but marked test item-related clinical changes were observed in 10 out of 22 females in the 1000 mg/kg/day group on G 6 or G 7 shortly after dosing. These included irregular and/or rapid breathing associated or not with partly closed eye(s), wheezing, decreased activity or dark eyes. Among these animals, six displayed recumbency and /or subdued behaviour. Hypersalivation noted for essentially all females in the vehicle, 250, 500 and 1000 mg/kg/day groups, with or without abnormal foraging and/or pedalling were clearly associated with the vehicle (Polyethylene glycol 400) but might possibly be slightly exacerbated by the test item. Hypersalivation, with associated observations of abnormal foraging, pedalling and/or Straub tail, is commonly observed in rodents given oral formulations with poor palatability and is, therefore, considered of no toxicological significance.

There was a transient slightly lower mean body weight gain and mean daily food consumption for females given 1000 mg/kg/day compared with the concurrent control towards the initiation of treatment. The mean body weight at the end of the dosing period was, however, comparable in all groups. There were no treatment-related macroscopic findings or effects on mean gravid uterus weight in any group.

There were 22 pregnant females at terminal caesarean in each of the control, 250, 500 and 1000 mg/kg/day groups, all of which had viable foetuses. Post-implantation data were not affected by test item in any group. There was no test item-related effect on embryo-foetal survival, foetal weight or foetal sex ratio in any group. In total, the number of foetuses (litters) with external, skeletal and/or visceral malformations in the 1000 mg/kg/day group were 7/240 (6/22) leading to a foetal (litter) incidence of 2.9 % (27 %) compared with none in other groups. All malformations were already noted in our more recent historical control data with an incidence comparable or lower to the maximal incidence per study in the HCD. In addition, in view of broad spectrum of these malformations (including general, head, caudal malformations and other visceral or skeletal malformations such as situs inversus, retroesophagial subclavian artery and sternebra or mandible fused), a test item-related effect was doubtful and the malformations were more likely attributed to a maternal mediated effect in view of the marked clinical signs observed on the first days of dosing at the high dose of 1000 mg/kg/day.

In conclusion, in view of the nature of the clinical signs, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was established as 500 mg/kg/day in the female rat. Some malformations were observed at 1000 mg/kg/day compared with none in the control group. However, all these malformations were already noted in our more recent historical control data with an incidence comparable or lower to the maximal incidence per study in the HCD. Therefore a test item-related effect was doubtful and the malformations were more likely attributed to a maternal mediated effect. Based on these findings, the NOAEL for embryo-fetal development was considered to be 500 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One reliability 1 study according to the OECD 414 guideline is available.
Additional information

In an available OECD 414 performed on rats, a slight increase of the foetal and litter incidences of malformations was observed at 1000 mg/kg/day compared to the control. However, all these malformations were already noted in the more recent historical control data of the laboratory. The incidence of each malformation observed in this study was equal or lower to the maximal incidence per study in the historical control data. In addition, in view of broad spectrum of these malformations, a test item-related effect was doubtful and the malformations were more likely attributed to a maternal mediated effect in view of the marked clinical signs observed on the first days of dosing at the high dose of 1000 mg/kg/day. Therefore in order to protect the pups from these malformations, parents must be protected against general toxicity. DNELs have been calculated based on general toxicity observed at 1000 mg/kg/day also in the OECD 421 study.

Toxicity to reproduction: other studies

Description of key information

No other studies are available.

Justification for classification or non-classification

No effect on reproduction was observed in the OECD 421 study performed in rats in 2017 up to the highest treated dose (1000 mg/kg/day).

In an available OECD 414 performed on rats, a slight increase of the foetal and litter incidences of malformations was observed at 1000 mg/kg/day compared to the control. However, all these malformations were already noted in the more recent historical control data of the

laboratory. The incidence of each malformation observed in this study was equal or lower to the maximal incidence per study in the historical control data. In addition, in view of broad spectrum of these malformations, a test item-related effect was doubtful and the malformations were more likely attributed to a maternal mediated effect in view of the marked clinical signs observed on the first days of dosing at the high dose of 1000 mg/kg/day. Therefore since the developmental effect observed is link to the maternal toxicity, no classification is required for the developmental toxicity.

Regarding the available studies, and according to classification criteria of EC regulation 1272/2008, Ethylvanillin should not be classified for reprotoxicity.

Additional information