coconut oil, reaction products with glycerol esters of 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

To reduce the number of animals, it was decided to combine a 90-day toxicity study (Part A) with a one-generation study (Part B) performed simultaneously with the same test item and dose levels at NOTOX. This was done by using the male animals exposed for ten weeks during the 90-day toxicity study for mating with the female animals exposed for two weeks at the same dose level during the one-generation study.
The treatment period for the males and females of the 90-day toxicity study was at least 13 weeks continuously. The treatment period for the females of the one-generation study was at least 8 weeks continuously.
For the 90-day toxicity study 48 male rats and 40 female rats were used; for the one-generation study 96 female rats were used.
On request of the UK authorities, the following was added:
Semen analysis (sperm motility, count and morphology)
Staging of spermatogenesis (detailed histopathology of the testes)
Oestrus cycle monitoring

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-400-6

EC Name:

-

Cas Number:

179986-09-5

Molecular formula:

UVCB substance

IUPAC Name:

methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate; propane-1,2,3-triol; tetradecanoic acid

Details on test material:

- Physical state: colourless paste
- Analytical purity: not indicated by the sponsor; treated as 100% pure
- Storage condition of test material: at room temperature protected from light

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: males 175-177 g, females 133-138 g
- Housing: Males were housed in groups of 5 males/cage in Macrolon cages. The additional males were housed in groups of 2 males/cage in Macrolon cages. Females were housed in groups of 5 females/cage in Macrolon cages.
- Diet (ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF® SpeziaIdiaten GmbH, Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
daily, 2 hours prior to dosing

VEHICLE
- Concentration in vehicle: 5, 10, 20 mg/ml
- Amount of vehicle (if gavage): 2 ml/kg bw
- Lot/batch no. (if required): 17644FC6

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Quantitative analysis was based on the analytical method validated for the test substance in NOTOX project 487737.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
14, 56, 222 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 males, 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were based on the results of a 28-day toxicity study (RCC Project 636456).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at begin and end of the study
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: white blood cells, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the treatment
- Dose groups that were examined: all animals
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

On request of the UK authorities, the following was added:
- Semen analysis (sperm motility, count and morphology)
- Staging of spermatogenesis (detailed histopathology of the testes)
- Oestrus cycle monitoring

Statistics:

The following statistical methods were used to analyse the data:
- lf the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one ranktest) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The T-test was applied for testis and epididymidis concentrations.
- The percentage of motile spermatozoa, progressive motile spermatozoa and sperm with normal morphology was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference. Ifthe results ofthe ANOVA were significant (p<0.05), the Wilcoxon test was applied to the data to compare the treated groups to the control group.
AII tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
No statistical analysis was performed on histopathology findings.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity noted during the observation period, except salivation and incidental findings (e.g. alopecia, wounds, broken tails).

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no clinical signs of toxicity noted during the observation period, except salivation and incidental findings (e.g. alopecia, wounds, broken tails).

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of treated animals remained in the same range as controls over the study period.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment related effects were noted at ophthalmoscopic examination up to 222 mg/kg body weight/day.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Slightly reduced values for prothrombin time were noted for males and females treated at 56 and 222 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 222 mg/kg, slight increased values for total protein, albumin and total bilirubin were observed for males and females. Additionally at this dose, females showed a decreased glucose level.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the treated animals when compared to control animals. A variation in motor activity did not indicate a relation with treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

at 56 and 222 mg/kg bw/day (see deteails)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 222 and 56 mg/kg bw hepatic changes (enlarged and discolorated livers) in males and females were observed (see details).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment related changes were present in the liver (periacinar hepatocytic hypertrophy) and thyroid (follicular epithelial hypertrophy) noted in males and females treated at 56 and 222 mg/kg bw (see details).

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Thyroid follicular adenoma was noted for one male of the 222 mg/kg bw dose group.

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no clinical signs of toxicity noted during the observation period.
Salivation was noted in all males treated at 56 and 222 mg/kg and to a lesser extent in males treated at 14 mg/kg. Salivation was noted at least once in one female treated at 14 mg/kg, in all females treated at 56 mg/kg and was repeatedly noted in all females treated at 222 mg/kg. Salivation was considered to be a physiological response to the taste of the formulation rather than a sign of systemic toxicity, considering the nature, minor severity of the effect and its time of occurrence (i.e. after dosing). Therefore, this was considered not toxicologically relevant. Incidental findings that were noted included alopecia, scabs and wound at several body parts, broken tail apex, broken upper incisors, and rales. These findings are commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption (absolute and relative) of treated animals remained in the same range as controls over the study period.

OPHTHALMOSCOPIC EXAMINATION
No treatment related effects were noted at ophthalmoscopic examination up to 222 mg/kg bw.

HAEMATOLOGY
Slightly reduced values for prothrombin time were noted for males and females treated at 56 and 222 mg/kg body weight/day.
AII other statistical significant changes were not considered to be due to treatment as no dose response relationship was noted, the values were within normal limits, the control values were at the upper/lower end of the normal range and/or no corroborative findings were noted.
Individual increases of neutrophil counts with concurrently reduced lymphocyte counts were noted for one high dose male and two low dose females.
This shift in type of white blood cells was considered to be a secondary non-specific response to stress and to be of no toxicological significance. At scheduled necropsy, all three animals showed reddish or red-brown discolouration of the thymus.

CLINICAL CHEMISTRY
At 222 mg/kg, slight increased values for total protein, albumin and total bilirubin were observed for males (5, 3 and 20%) and females (8, 5, 37%). Additionally at this dose, females showed a decreased glucose level (about 20 %). AII other statistical significant changes were not considered to be due to treatment as no dose response relationship was noted, the values were within normal limits, the control values were at the upper/lower end of the normal range and/or no corroborative findings were noted.

ORGAN WEIGHTS
Organ weight changes were noted at 56 and 222 mg/kg for both sexes. These changes consisted at 222 mg/kg bw of increased liver weights for males (about 40%) and females (about 46%) and increased thyroids weights both sexes (80% for males, 46 % for females).
Mid and high dose males showed increased kidneys and prostate weights (absolute and relative). As these values were within normal limits and no microscopic correlate was observed, these changes were considered not toxicologically significant.
AII other statistical significant changes were not considered treatment-related as no dose response relationship was noted.

GROSS PATHOLOGY
At 222 mg/kg, hepatic changes were noted. These consisted of accentuated lobular pattern (one male and one female), enlarged livers (seven males), black-brown or red-brown discoloration (five males and nine females) and thickened and dark-red discoloured papillary process (one female). To a lesser extent hepatic abnormalities were seen in animals treated at 56 mg/kg/day (one male showed an enlarged liver and two females showed black-brown discolouration).
One control male showed the left testes and epididymidis reduced in size. During in-life this was noted as undescended left testicle.Incidental findings included bent tail apex, nodules on and enlargement of the preputial glands, alopecia and scab formation of the skin, reduced seminal vesicles size, discoloration of the adrenals and the lymph node (mandibular), reduced size of papillary process of the liver, foci on the lungs, and discoloration and foci on the thymus. These findings are occasionally seen among rats used in these types of study and in the absence of correlated microscopic findings they were not considered changes of toxicological significance.
Some females from each dose group were found with watery fluid in the uterus; this is related to a stage in the oestrous cycle and constitutes a normal finding.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment related changes were present in the liver (periacinar hepatocytic hypertrophy) and thyroid (follicular epithelial hypertrophy) noted in males and females treated at 56 and 222 mg/kg bw.
Periacinar hepatocytic hypertrophy was seen in six males and six females treated at 56 mg/kg and in all animals treated at 222 mg/kg. Thyroid follicular epithelial hypertrophy was observed for eight males/two females of the 56 mg/kg bw dose group and nine males/five females of the 222 mg/kg bw dose group.
The hypertrophic changes in liver and thyroid were both treatment and dosage related changes.
The hepatic enlargement and accentuated lobular pattern recorded at necropsy are gross correlates of periacinar hepatocytic hypertrophy. There was no microscopic evidence of pigmentation to correlate with gross discolouration.
Other findings were usual occurrences in laboratory rats and none were related to treatment.

HISTOPATHOLOGY: NEOPLASTIC
Thyroid follicular adenoma was noted for one male of the 222 mg/kg bw dose group.

OTHER FINDINGS
Sperm examination: At all dose levels, decreased sperm motility and progressive motility was noted (at 222 mg/kg bw: 20 resp. 40%). The concurrent control was right at the historical control mean. The values of the treated groups were at the lower end of the historical control range.
The number of testicular and epididymidal sperm cells and the percentage of normal sperm cells was similar for the 222 mg/kg bw dose group and control group.
Left testis weight was statistically significantly increased in the animals of the 222 mg/kg bw dose group (about 12%). These values were considered to be within normal limits and as this effect was not observed for the total testes weight it was not considered toxicologically relevant.
In the testes sections which were stained PAS/haematoxylin, in addition to sections stained H&E, no variation was present in the staging of spermatogenesis between the 222 mg/kg bw treated and the control group.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: hepatic and thyroidal changes

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Summary tables are given in the attachments

Tables 1 Absolute organ weights males

Table 2 Relative organ weights males

Table 3 Absolute organ weights females

Table 4 Relative organ weights females

Table 5: Macroscopic findings summary

Table 6: Incidence of histopathology findings for liver, kidney and thyroid, males

Table 7: Incidence of histopathology findings for liver and thyroid, females