4-trans-propenylveratrole

Endpoint summary

Description of key information

Repeated dose toxicity oral: NOAEL = 264 mg/kg bw/day in female rats;  NOAEL = 275 mg/kg bw/day in male rats (similar to OECD 407, K, rel. 2). No adverse effect reported at the highest dose level tested in this study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study conducted similarly to OECD guideline 407 with some deviations: sensory activity, grip strength and motor activity not assessed)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

(sensory activity, grip strength and motor activity not assessed.)

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Not reported

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Olac Ltd, Bicester, Oxon, UK
- Age at receipt: 3 weeks
- Weight at study initiation: Males: 118-124 g; females: 101-111 g
- Housing: Animals were caged in groups of four of the same sex and treatment, in plastic and stainless steel grid-floored cages.
- Diet: Basic diet (Rat and Mouse No.1 Maintenance diet; Special Diet Services, Witham, Essex), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 45-70 %

Route of administration:

oral: feed

Details on route of administration:

No details available

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Diets containing test material (cis- and trans-isomers) at concentrations calculated to provide intakes of 0 (control), 30, 100 and 300 mg/kg bw/day (nominal in diet).
- Storage temperature of food: Stored at 2-6 °C in closed metal tins sealed with adhesive tape.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Diets were analysed for test material (sum of cis- and trans-isomers) by GC after preparation and accepted for use if the concentration of the test material was within 10 % of the desired value.
- Diets were extracted into acetone and analysed by GC after adding the internal standard, isopropylbenzyl carbinol.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Target dose

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Target dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Target dose

No. of animals per sex per dose:

16

Control animals:

yes, plain diet

Details on study design:

No data

Positive control:

None

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During entire study period.

BODY WEIGHT: Yes
- Time schedule for examinations: Rats were weighed 3 days before the start of treatment, on the first day of treatment, and then twice weekly throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from the caudal vein of each animal on the day before autopsy.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes; animals were fasted overnight.
- How many animals:
Haematology: 16 animals/sex/dose (except for red blood cells, mean cell volume and haematocrit: 15 males in control and 30 mg/kg bw/day group; white blood cells: 15 males in 30 mg/kg bw/day dose group); haemoglobin: 15 males and females in 30 and 300 mg/kg bw/day dose group, respectively; platelets: 9 males and 5 females in all dose groups; reticulocytes: 15 males in control group; mean prothrombin time and activated partial thromboplastin time: 7 males in control and 30 mg/kg bw/day dose group, 9 males in 100 mg/kg bw/day dose group, 8 males in 300 mg/kg bw/day dose group and 6* females in all dose groups; * 5 females in control group for mean activated partial thromboplastin time). Reticulocytes and differential WBC count were made only in control and highest dose (300 mg/kg bw/day) group.
Clinical chemistry: 16 animals/sex/dose (except alanine aminotransferase and alkaline phosphatase and albumin: 15 males in control group; glucose: 15 males in control, 15 females in control and 300 mg/kg bw/day dose group and 14 females in 30 and 100 mg/kg bw/day dose groups; urea: 15 males in control, 15 females in control and 100 mg/kg bw/day dose groups and 14 females in 30 mg/kg bw/day dose group; total protein: 15 males in control group, 15 females in control and 100 mg/kg bw/day dose group; globulin, triglycerides, cholesterol, chloride, bilirubin, creatinine and sodium: 5 males and females* at all dose groups, * 4 females in control and 30 mg/kg bw/day dose group for bilirubin).
- Parameters examined:
Haematology: Red blood cells (RBC), mean cell volume (MCV), haematocrit (HCT), white blood cells (WBC), haemoglobin (Hb), platelets, reticulocytes, differential WBC count, mean prothrombin time (PT), mean activated partial thromboplastin time (APTT).
Clinical chemistry: Aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase, glucose, urea, total protein, albumin, globulin, triglycerides, cholesterol, chloride, bilirubin, creatinine and sodium.

URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of treatment urine was collected from each animal over a 6 h period.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters examined: Appearance, pH, semi-quantitative measurements of blood, bilirubin, ketones, glucose, protein and urobilinogen.
The renal concentrating and diluting abilities were assessed by measuring the volume and refractive index (as a measure of urine concentration) of urine produced in a 6 h period without water, a 4 h period commencing after 16 h without water and a 2 h period immediately after an oral water load of 25 mL/kg bw. A urinary cell count was made using the latter sample.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- At the end of the 28-day treatment period, equal numbers of animals from each group were killed on a daily basis over the next 4 days. The animals were killed by exsanguination from the dorsal aorta under barbiturate anaesthesia following an overnight fast (with water available). All macroscopic abnormalities were noted.

HISTOPATHOLOGY: Yes
- All macroscopic abnormalities were noted and the brain, heart, caecum (with and without its contents), kidneys, adrenals, gonads, spleen and liver were weighed. Samples of these organs together with samples of the skin, eye, Harderian gland, salivary glands, trachea, oesophagus, pancreas, rectum, colon, urinary bladder, urethra, spinal cord, skeletal muscle, lymph nodes, aorta, vena cava, thymus, pituitary, lung, prostate, epididymis, seminal vesicles, uterus, vagina, mammary glands, tongue, stomach, duodenum and ileum, and any other tissue that appeared abnormal, were preserved in 10 % buffered formalin.
- Paraffin-wax sections of these tissues from the control animals and those given the highest dietary level of test material (300 mg/kg bw/day (nominal)) were stained with haematoxylin and eosin for histopathological investigations.

Other examinations:

Organ weight:
- Brain, heart, caecum (with and without its contents), kidneys, adrenals, gonads, spleen and liver were weighed.

Statistics:

- The continuous variable data from the control and test groups were compared using analysis of variance followed by the least significant difference test.
- Incidence data from the histopathological examination and the semi quantitative urine analysis were analysed using Fisher's exact test (Fisher, 1934).
- In all cases, a level of probability less than 0.05 was taken to indicate statistical significance.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

not considered as adverse

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

not considered as adverse

Urinalysis findings:

no effects observed

Description (incidence and severity):

not considered as adverse

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

not considered as adverse

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

not considered as adverse

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
- No treatment-related effects were observed on the condition or behaviour of the rats during the study.

BODY WEIGHT AND WEIGHT GAIN:
- Mean body weights for the three groups of treated males and for female given either 30 or 300 mg/kg bw/day (nominal) did not differ significantly from the control values during the entire study period.
- Females treated with 100 mg/kg bw/day (nominal) showed significantly (p< 0.05) increased mean body weight as compared to control before start of the study, on the first day of treatment and on Day 3, but the difference was not apparent for the remainder of the study period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Occasional statistically significant differences in food intakes between treated groups and controls were observed during the study, but none showed any consistent relationship to dose or duration of treatment.
- Calculation of the average daily intake of test material showed that the dose levels achieved were close to the proposed values.

HAEMATOLOGY:
- Haematological examination revealed no differences between control and treated male rats, except for a significantly higher lymphocyte count in male rats at 300 mg /kg bw/day (nominal). The white blood cell count was also raised in this group. Minor differences in haematological measurements were seen between control and treated female rats in the two highest dose groups. The red blood cell count and haematocrit value were significantly lower in the 100 mg /kg bw/day group (nominal) than in controls, while the mean cell volume and white blood cell and reticulocyte counts were significantly higher in the 300 mg /kg bw/day group (nominal) than in controls. The lymphocyte count was also raised in the latter treatment group, but not significantly.
- Clotting measurements for all the treated animals (prothrombin and activated partial thromboplastin times) were not significantly different from control values.

CLINICAL CHEMISTRY:
- There were no significant differences between control and treated male rats in the results of serum chemistry measurements.
- Females in all three treated groups had significantly lower alkaline phosphatase activities than the control group, though the decrease was not linearly related to the dose.
- Significantly increased alanine aminotransferase activity compared with the control group was observed in females treated with 300 mg/kg bw/day (nominal).

URINALYSIS:
- 6 h urine samples from males treated with 300 mg/kg bw/day (nominal) and female treated with 30 mg/kg bw/day (nominal) had a higher refractive index than that of the control group.
- A significantly higher refractive index compared with the control group was observed in the 2 h urine samples collected from females in all the three treatment groups but these differences were not seen in 2 h urines from males.
- Protein was found in the majority of 6 h urine samples from males in both the control and treated groups.
- A high incidence of ketones in the urine of male rats treated with 300 mg/kg bw/day (nominal) compared with the control group was observed (p < 0.05).

ORGAN WEIGHTS:
- Livers of males and females treated with 300 mg/kg bw/day (nominal) were heavier than the controls; these differences were significant for the absolute and relative liver weights of male rats and the relative liver weights of female rats.
- Other significant differences for organ weights (absolute or relative) between control and treated animals occurred infrequently and were restricted, for the organ concerned, to one sex and dose group.

GROSS PATHOLOGY:
- No treatment related macroscopic changes were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Histopathological examination of tissues from rats in this study revealed few abnormalities.
- A small number of animals from both the control and 300 mg/kg bw/day group (nominal) showed minor lesions of the lung, trachea or nasal mucosa, indicative of a mild respiratory infection.
- Occasional focal, degenerative and inflammatory lesions were observed in eye, nerve, skeletal muscle, stomach, liver, kidney, Harderian gland and prostate. In most cases the lesions were slight or mild, and were restricted to only one or two animals in either the control or 300 mg/kg bw/day group (nominal).
- The liver of a female animal treated with 300 mg/kg bw/day (nominal) showed multifocal parenchymal necrosis, which was extensive in places.
- A small number (less than one-third) of male and female rats treated with 300 mg/kg bw/day (nominal) exhibited a focal interstitial nephritis compared with only one out of 16 rats in each of the control groups. The observed lesions were light in all cases.
- Inflammation of the Harderian gland was found in 5/16 females treated with 300 mg/kg bw/day (nominal) compared with one out of 16 control animals. This lesion was accompanied in the treated females by acinar degeneration, the incidence of which was statistically significant.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

None

Conclusions:

Based on the results of the study, the oral no observed adverse effect level (NOAEL) was considered to be equivalent to 275 mg/kg bw/day in male rats and 264 mg/kg bw/day in female rats).

Executive summary:

In a repeated dose toxicity study, test material was administered in the diet for a minimum of 28 days to the groups of Sprague-Dawley rats (16/sex/dose) at nominal target dose levels of 0 (plain diet), 30, 100 and 300 mg/kg bw/day (corresponding to actual dose level of 0, 30, 89 and 275 mg/kg bw/day for males and 0, 27, 91 and 264 mg/kg bw/day for females). Clinical signs, food intakes and body weights were monitored during the study. Urine analysis, haematology and blood chemistry were performed. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed. 

 

No mortality or clinical signs were observed. During the treatment period both male and female animals gained approximately the same amount of weight as did the control animals. The observed minor differences in food intakes between control and treated animals were not considered to be treatment related.

Slightly higher lymphocyte and white blood cell counts compared with controls were seen in both sexes treated with 300 mg/kg bw/day. These changes, in isolation, are not considered to be an adverse effect of treatment. Other changes in leucocytes parameters were minor and were not observed in both sexes. Small differences in a few erythrocyte parameters, seen between control females and females treated with either 100 or 300 mg/kg bw/day, were not dose-related. It is considered unlikely that the isolated differences seen for these haematological measurements between treated and control animals were related to treatment.

Difference in some of the refractive index measurements of the 2 -hr and 6 -hr urine samples from treated rats compared with controls were not dose-related or observed in both sexes. Although all treated female groups had a higher mean refractive index than controls for the 2 -hr urine samples, it is more likely that the control value is unusually low than these difference represent an effect of treatment. Other isolated differences in urinary components between control and treated animals are not thought to be related to treatment.

 

Changes in serum chemical measurements for alkaline phosphatase and alanine aminotransferase activities seen for treated female rats were not found for treated male rats. The differences between control and treated animals for alkaline phosphatase activities in the three dose groups may be due to a relatively high level of enzyme activity in the female control group [226.2 + 29.5 IU compared with 140 + 59.8 IU, a value reported for the alkaline phosphatase activity for young (~< 6 months) Sprague-Dawley rats.

Both male and female rats treated with 300 mg/kg bw/day had livers heavier (10-15%) than those of controls when expressed relative to body weight. Isolated differences in some other organ weights were seen in a few treated groups, but these are not considered to be related to the administration of methyl isoeugenol.

In female rats treated with 300 mg/kg bw/day, the elevated alanine aminotransferase activity coupled with the slightly enlarged livers might indicate a hepatotoxic effect. Apart from multifocal parenchymal necrosis seen in the liver of one female rat in this group, there was little histopathological evidence, however, to support this view. The difference between control and treated animals for alanine aminotransferase activity was observed at the highest dose only and, although statistically significant, was only 30% of control animals. Less than twofold magnitude is commonly considered to be reversible (Hall et al, 2012) and due to a hypertrophic adaptive response to increased metabolic load.

 

Rats given 300 mg/kg bw/day showed very few other histopathological abnormalities. Most of the observed lesions were of minimal or mild severity and showed a similar incidence and frequency in the control group, though a higher incidence of inflammatory lesions was seen in the kidneys and Harderian glands of treated animals.

Kidney lesions are frequently found in rats as they age and are not attributed in the present study to treatment with methyl isoeugenol.

Inflammatory and degenerative changes in the rat Harderian gland are commonly associated with sialodachryoadenitis infection.

Sialodachryoadenitis is primarily an upper respiratory tract infection with variable effects in the salivary and Harderian glands. It is frequently epizootic and subclinical in rat colonies, but may become more prevalent in high dose groups due to additional stress factors (Otto et al, 2006). Its occurrence in the present study is not considered to be a direct effect of treatment.

It is concluded that methyl isoeugenol in the diet of rats for 28 days at a dose of 300 mg/kg bw/day may result in slight liver enlargement and a slight increase in white blood cell counts. Neither of these effects can be considered to be adverse in the absence of any related functional or structural observations, thus treatment with methyl isoeugenol at the doses used in this study is without adverse effects.

 

As a summary, the administration of methyl isoeugenol in the diet did not adversely affect the growth or general health of the animals or their food intakes. Although high dose animals of both sexes had increased lymphocyte and total white blood cell counts, these are not considered, in isolation, to be an adverse effect of treatment. None of the minor variations observed in the serum chemical analyses or urine analyses is considered to be indicative of a treatment-related toxic effect. An increase in liver weight, adjusted for body weight, was seen in male and female rats receiving 300 mg/kg bw/day. Few histopathological abnormalities were observed. Although the incidence of kidney and Harderian gland lesions was higher for high dose animals compared with the controls, the lesions are of a type that occurs spontaneously and are thus not considered to be attributable to treatment with methyl isoeugenol. While the increased liver weight and white blood cell counts of rats given 300 mg/kg bw/day may represent effects of treatment, it is not considered that there is any reason to regard these as adverse effects.

 

Based on the results of the study, the oral no observed adverse effect level (NOAEL) was considered to be equivalent to 275 mg/kg bw/day in male rats and 264 mg/kg bw/day in female rats).

References:

[Hall et al, 2012] Liver Hypertrophy: A Review of Adaptive (Adverse and Non-adverse) Changes—Conclusions from the 3rd International ESTP Expert Workshop. Toxicologic Pathology, 40: 971-994, 2012.

[Otto et al, 2006] The laboratory rat, 2nd edition. Chapter 16 Medical Management and Diagnostic Approaches. 2006, 1980 Elsevier Inc.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

264 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is of high quality although its GLP status was not reported (Klimisch score = 2).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A 28-day repeated dose toxicity study was available (Purchase, 1992, Rel.2). This study is considered as the key study for repeated dose toxicity endpoint. In this 28-day study, the substance was administered in the diet to rats at equivalent nominal dose levels of 0 (plain diet), 30, 100 and 300 mg/kg bw/day (corresponding to actual dose level of 0, 30, 89 and 275 mg/kg bw/day for males and 0, 27, 91 and 264 mg/kg bw/day for females). The study was conducted similarly to OECD TG No. 407. However, neurotoxicity evaluations were not conducted since they were not required at the time of the study completion.

As a summary, the administration of the substance in the diet did not adversely affect the growth or general health of the animals or their food intakes. Although animals of both sexes in the high dose groups had increased lymphocyte and total white blood cell counts, these are not considered, in isolation, to be an adverse effect of treatment. None of the minor variations observed in the serum chemical analyses or urine analyses is considered to be indicative of a treatment-related toxic effect. An increase in liver weight, adjusted for body weight, was seen in male and female rats receiving 300 mg/kg bw/day. Few histopathological abnormalities were observed. Although the incidence of kidney and Harderian gland lesions was higher for animals in the high dose groups compared with the controls, the lesions are of a type that occurs spontaneously and are thus not considered to be attributable to treatment with the substance. While the increased liver weight and white blood cell counts of rats given 300 mg/kg bw/day may represent effects of treatment, it is not considered that there is any reason to regard these as adverse effects.

Based on the results of the study, the oral no observed adverse effect level (NOAEL) was considered to be equivalent to 275 mg/kg bw/day in male rats and 264 mg/kg bw/day in female rats).

This result is supported by the Reproduction/Developmental Toxicity Screening Test (Envigo, 2015, OECD TG No. 421, GLP, Rel.1) in which the NOAEL for males and females was 4500 ppm (equivalent to daily intakes between 295 and 702 mg/kg bw/day, depending on the age and weight of the animals) on the basis that the animals of the 15000 ppm dose group suffered adverse effects in food consumption and weight gain, which were not fully alleviated in the recovery period. [Cf. Iuclid Section 7.8 for further details on this study]

Additionally, the results of a 90-day repeated dose toxicity study were available (Osborne, 1981). Groups of 24 male and 24 female Crl : CD(SD)BR albino rats were fed diets containing the substance at an equivalent dose of 6 mg/kg bw/day for 13 weeks. All animals were observed twice daily throughout the study for mortality and signs of toxicity. Evaluations of haematological and blood chemistry parameters and urine analyses were performed on randomly selected rats (12 of each sex per dose) at weeks 6 and 12. No toxicologically significant variations in any of the parameters examined were reported between treated animals and controls. Weekly measurements of body weights and food consumption revealed no significant differences between test animals and controls. At necropsy, gross and histopathological examinations revealed no lesions that could be associated with administration of the substance. Statistically significant variations in organ weights of treated animals compared with the controls were limited to reduced absolute thyroid weights in males and females, and reduced relative weights of both lobes in females and the right lobe in males; however, these differences were not associated with any morphological changes and consequently, were considered by the authors not to be toxicologically significant. It has to be highlighted that the dose levels used in this study were related to intended use conditions of the substance and are therefore not adequate for the purpose of hazard characterization. Higher dose levels should have been tested to comply with OECD TG No. 408. As such the study summary for this particular study is not included in the dossier.

References:

(Osborne, 1981), as cited in WHO FOOD ADDITIVES SERIES: 52 - Hydroxypropylbenzenes (addendum). IPCS INCHEM

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

Based on the available data, no additional classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure.