Registration Dossier

Administrative data

Description of key information

Two studies have been selected as key study, a 90 day study and an OECD 421 study.

In the 13-week study (Huntingdon et al. , 1992) performed on rats, the NOAEL is 1000 mg/kg bw/day based on spleen weight which is higher than control group (relative) related with histopathological changes. Based on this pathability study, the NOAEL is 1000 mg/kg bw/day for effects on the spleen.

In the OECD 421 study performed by gavage on 2017 (Charles River, 2017), transient but adverse clinical signs were observed in male and female rats at the dose level of 1000 mg/kg/day. A NOAEL of 500 mg/kg/day was therefore identified for these effects.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 july 1991 to 07 july 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was GLP, standardized guidelines compliant and well described.
Qualifier:
according to
Guideline:
other: US EPA Food guideline 1982
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Manston Road, Margate, Kent, England
- Age at study initiation: approximately 28 days old
- Weight at study initiation: 12 g (females) to 15 g (males)
- Fasting period before study: no data
- Housing: individually
- Diet (e.g. ad libitum): free access to SDS rat and mouse N°1 modified maintenance diet.
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: 9 days. A further period of acclimatisation of 7 days was allowed between allocation of animals and beginning of treatment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12h / 12h


IN-LIFE DATES: from 16 aug 1991 to 15-20 nov 1991
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): each week
- Mixing appropriate amounts with (Type of food): SDS rat and mouse N°1 modified maintenance diet. A premix of suitable strength was prepared each week by grinding the required quantity of Ethyl vanillin with untreated diet. Blending of the premix was achieved by mixing in a turbula mixer for a minimum period of 2 minutes.
The required concentrations were prepared by direct dilution of the prepared premix. Blending of the inclusion levels for feeding was achieved by mixing in a double-cone blender for a minimum period of 7 minutes.
The concentrations of Ethyl vanillin were changes as necessary.
- Storage temperature of food: -20°C in airtight containers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Only samples of diet prepared for week 1, 6 and 13 were analysed for inclusion levels. Following issue of the final report, the stored samples will be discarded with no further analyses performed. Chemical analysis was carried out by HRC Department of Analytical chemistry. The results of these analyses are available in addendum in the study report.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0; 500; 1000; 2000 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
20 males and 20 females
Control animals:
yes, plain diet
Details on study design:
Post-exposure period: none
- Dose selection rationale: issue from the palatability study (Huntingdon, 1992).
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at time of allocation, on the day of the start of treatment, and once a week thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal: The quantity of food consumed by each animal was recorded on a weekly basis.

FOOD EFFICIENCY: Food conversion ratios were calculated, where appropriate, from bodyweight and food consumption data as weight of food consumed per unit gain in bodyweight.


WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily monitoring by visual appraisal of the water bottles was maintained throughout the study. Water consumption was measured accurately, by weight, over daily periods during Week 12 for all rats in all groups.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of the treatment, then during Week 13, the eyes of all surviving animals in the control and high dosage level groups were examined. Prior to examination the pupils were dilated using a Tropicamide ophtalmic solution.
- Dose groups that were examined: all surviving animals in the control and high dosage level groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: twice
- Anaesthetic used for blood collection: Yes (no data on identity)
- Animals fasted: Yes
- How many animals: 20 (10 males and 10 females)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: twice
- Animals fasted: Yes
- How many animals: 20 (10 males and 10 females)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Adrenals, brain, heart, kidneys, liver, lungs, ovaries, pituitary, prostate, spleen, testes (with epididymes), thyroids, uterus.

HISTOPATHOLOGY: Yes
Adrenals, alimentary tract, aorta, brain, eyes, femur, Harderian gland, heart, kidneys, larynx and pharynx, liver, lungs, lymph nodes, mammary gland, ovaries, pancreas, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal column, spleen, sternum, testes (with epididymes), thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus, vagina.
Statistics:
The following sequence of statistical tests was used for food conumption, water consumption, bodyweight, organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different form the mode was analysed by Fisher's exact test or Mantel's test. Otherwise:
- Bartlett's test was applied to test heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
- Where no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.

Analyses of variance were followed by Student's "t" test and William's test for a dose-related response, although only the one thought most appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the "t" test and William's test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY: A control female died during the Week 6 routine blood sampling procedures, as a result of suspected ether anesthesic overdose. There were no clinical signs noted during the 13-week treatment period considered to be of toxicological importance.

BODY WEIGHT AND WEIGHT GAIN: The group mean bodyweight gain of male receiving 2000 mg/kg/day was inferior to that of controls troughout the whole study. For males receiving 500 or 1000 mg/kg/day the mean bodyweight gain was inferior to that of controls during the first 4 weeks. However, although the differences from control were statistically different they were not dosage-level in degree. It was noted that for these groups the food intake was lower than control.
During the remaining weeks of the study, the weekly mean bodyweight gains of males receiving 500 or 1000 mg/kg/day were considered to be essentially comparable with those of controls. However, this improvment in bodyweight gain was not sufficient for either the overall weight gain for period Weeks 4 to 13, or the absolute bodyweights for these male rats to regain parity with concurrent controls.
For females, overall, inferior bodyweight gain was recorded in the 2000 mg/kg/day group, while weight gain of females receiving 500 or 1000 mg/kg/day was considered to be unaffected by treatment with Ethylvanillin.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): during the first 4 weeks of the study the cumulative mean food intake by all the male treated groups was stastistically lower than that of controls.
During the subsequent period the food intake by all the treated groups of males was without any notable differences from control.
During the first week of the study, the mean intake by females receiving 2000 mg/kg/day was marginally inferior to that controls. Subsequently, during the remainder of the study, the food intake by the same rats was comparable to that of controls.
The food intake by females receiving 500 or 1000 mg/kg/day was considered to be similar to that of controls throughout the study.

FOOD EFFICIENCY: The efficiency of food utilisation was assessed by calculation of food conversion ratios. The food conversion ratios for rats of either sex treated with 2000 mg/kg/day during 13 weeks were inferior to those of their respective control. The marginally inferior food conversion ratios noted for rats of both sexes treated with 500 or 1000 mg/kg/day were considered to reflect the marginally lower bodyweight gains reported earlier for these groups.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Measurement of water intake during Week 12 of treatment revealed no notable differences between the water intake of treated rats and their respective controls.

OPHTHALMOSCOPIC EXAMINATION: no treatment-related changes were recorded during the Week 13 examination.

HAEMATOLOGY: Investigation of haematological parameters during Week 6 and 13 did not reveal any differences from control that were considered to be attributable to treatment, despite the occasional parameters that achieved levels of statistical significance.

CLINICAL CHEMISTRY: biochemical investigations showed slightly higher than control values for glutamic-pyruvic transaminase, alkaline
phosphatase, cholesterol and total plasma protein at the high dosage level, only occasionally involving the intermediate dosage level. These
differences from control may be related to treatment in view of the histopathological changes in liver. The remaining differences from the control
were considered unlikely to be of toxicological importance.

ORGAN WEIGHTS : for rats receiving 1000 or 2000 mg/kg/day, liver, spleen and kidneys weights were higher than the control. The absolute liver
weights of these animals were only marginally higher than the respective controls. Spleen and kidney weights were not associated with
histopathological change.
The remaining statistical differences from control were considered to have arisen as a result of the lower than control terminal bodyweights for the
treated male groups or were considered to reflect normal biological variability for rats of this age and strain and were therefore considered to be
of no toxicological importance.


GROSS PATHOLOGY : Fur: yellow discolouration of some or all of the fur was observed in 1/20 male and 1/20 female rats treated with
500 mg/kg/day, 6/20 male and 16/20 females rats treated with 1000 mg/kg/day, and all male and female rats treated with 2000 mg/kg/day,
compared with 0/20 male and 0/19 female control rats. This finding was also noted clinically and considered to be of no toxicological importance.
Cervical lymph nodes: enlargement was noted in a greater number of male rats treated with 1000 mg/kg/day or 2000 mg/kg/day compared with male and female control rats.
Deep cervical lymph nodes: enlargement was observed in 7/20 female rats treated with 2000 mg/kg/day compared with 0/19 female control rats.
Adipose tissue: a reduction in adipose tissue was observed in 7/20 male and 1/20 female rats treated with 2000 mg/kg/day compared with 0/20 male and 0/19 female control rats.
Spleen: enlargement was seen in 2/20 male treated with 500 mg/kg/day, 3/20 male treated with 1000 mg/kg/day and in 9/20 male and 4/20 female rats treated with 2000 mg/kg/day compared with 0/20 male and 0/19 female control rats.


HISTOPATHOLOGY: NON-NEOPLASTIC : Hepatic peribiliary inflammatory change and minor bile duct hyperplasia attributable to dietary administration of ethylvanillin were seen at dosages of 2000 and 1000 mg/kg/day. No changes were seen in the liver parenchyma and no degenerative or inflammatory changes were seen in the bile duct epithelium.
Associated reactive changes of lymphoid tissues were also seen.
No treatment-related changes were observed at the 500 mg/kg/day dosage level.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Spleen weight higher than control group (relative) related with histopathological changes.
Critical effects observed:
not specified
Conclusions:
The NOAEL is 1000 mg/kg bw/day based on spleen weight which is higher than control group (relative) related with histopathological changes.
Executive summary:

In a subchronic toxicity study (Hooks, 1992), ethylvanillin was administered to CD(SD) BR rats (males and females, 20 per sex per dose), in diet at dose levels of 0; 500; 1000; 2000 mg/kg/day for 13 weeks. Yellow discolouration of some or all of the fur of rats treated with 500 mg/kg/day, 1000 mg/kg/day, or 2000 mg/kg/day, were not considered to be of toxicological importance.

For males and females, inferior bodyweight gain was recorded in the 2000 mg/kg/day group. For males receiving 500 or 1000 mg/kg/day, the mean bodyweight gain was inferior to that of controls during the first 4 weeks and the cumulative mean food intake by all the male treated groups was stastistically lower than that of controls. The food intake by females receiving 500 or 1000 mg/kg/day was considered to be similar to that of controls throughout the study. The decrease of bodyweight gain is observed in the fourth first weeks, related with a decrease in food consumption. This variation is due to the unpathability of the substance, and was not considered to related with toxicological property of the substance.

Biochemical investigations showed slightly higher than control values for glutamic-pyruvic transaminase, alkaline phosphatase, cholesterol and total plasma protein at the high dosage level, only occasionally involving the intermediate dosage level. These differences from control may be related to treatment in view of the histopathological changes in liver. The remaining differences from the control were considered unlikely to be of toxicological importance.

Concerning observations done on the increase of liver weight (relative), it appears to be not correlated with hepathological changes, because these effects were observed in the control group too, that would be due to the strain of rat used in this study. Therefore the increase of the liver weight is not considered as adverse effect.

The NOAEL is 1000 mg/kg bw/day based on spleen weight which is higher than control group (relative) related with histopathological changes.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Good quality. This NOAEL has been identified from an OECD 421 study performed in 2017 with reliability 1. The results observed on this study are consistent with the results from the other available repeated toxicity studies and give the lower NOAEL.

Additional information

Several studies were available by oral route (feed and gavage), but only 2 of them were chosen as key study (Huntingdon, 1992 and Charles River, 2017). These 2 studies have the reliability 1 according to Klimish score.

In a subchronic toxicity study (Huntingdon, 1992), Ethylvanillin was administered to CD(SD) BR rats (males and females, 20 per sex per dose), in diet at dose levels of 0 to 2000 mg/kg bw per day for 13 weeks. The NOAEL is 1000 mg/kg bw/day based on spleen weight which is higher than control group (relative) related with histopathological changes. In a subacute toxicity study (Dose-range finding) (Huntingdon, 1992) (selected as WOE), ethylvanillin was administered to CD(SD) rats (males and females, 10 per sex per dose), in diet at dose levels of 0 to 2000 mg/kg bw/day for 2 weeks. The NOAEL is 1000 mg/kg bw/day based on bodyweight gain.

The OECD 421 study performed on rats in 2017 (Charles River, 2017) was also selected as a key study. Such study is not really design to assess repeated toxicity. However in this study transient but adverse clinical signs were observed in male and female rats at the dose level of 1000 mg/kg/day. Therefore based on this study a NOAEL of 500 mg/kg/day was identified in rats. Since this NOAEL is lower than the NOAEL identified in the 90 day study, the OECD 421 study was retained, in a conservative approach, for the derivation of the DNELs.

Justification for classification or non-classification

In the different studies available, ethylvanillin did not show adverse systemic effects up to 500 mg/kg/day, and should not be classified for repeated toxicity or STOT repeated exposure, according to the CLP 1272/2008 criteria.