Bis(2,4-di-tert-butyl-6-methylphenyl)ethyl phosphate

Endpoint summary

Description of key information

In a subchronic study with rats (C.I.T. 1994), the test substance did not cause any adverse effects up to the highest dose tested (1000 mg/kg), therefore, the NOAEL was set at 1000 mg/kg bw. In a subchronic capsule feeding study with dogs (Covance, 1999), the substance caused liver/gall bladder weight increases correlating with elevated alkaline phosphatase activity and centrilobular to diffuse hepatocellular hypertrophy. The NOEL was set at 50 mg/kg and the NOAEL at 200 mg/kg, due to the adaptive nature of the observed effects at this dose level.

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:

1998-08-25 to 1999-09-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study. According to OECD Guideline 409 and EU method B.27

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

adopted on 21-Sep-1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.27 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Inc., Cumberland, Virginia
- Housing: individually housed in an elevated, stainless-steel cage measuring 90 x 83 x 79 cm
- Diet: Certified canine diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 12 days under test conditions, following a health examination

ENVIRONMENTAL CONDITIONS
- Temperature 18 - 29°C
- Humidity: 50 +/- 20 %
- Air changes: about 10 cycles/hour
- Photoperiod: 12-hrs light, 12-hrs dark

Route of administration:

oral: capsule

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was transferred to one or more size 11 capsules which were placed in labeled plastic vials and subsequently transferred to labeled capsule boards; the boards were stored at room temperature until dosing. Capsules were prepared at least once weekly, with the per-dose amount of test material for each animal based on the most recently recorded body weight.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

92 days

Frequency of treatment:

Twice daily, 4 to 5 hours apart

Remarks:

Doses / Concentrations:
10, 50, 200, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

4 dogs

Control animals:

other: The control dogs received empty capsules at a rate equal to that of the high-dose dogs.

Details on study design:

- Dose selection rationale: Dosages were based upon a preliminary 14-day capsale range-finding toxicity study in dogs.

Positive control:

None

Observations and examinations performed and frequency:

MORTALITY / VIABILITY
All dogs were observed twice daily (a.m. and p.m.) for evidence of mortality and moribundity and once daily at the time of the afternoon mortality check for evidence of toxic or pharmacologic effect.

DETAILED CLINICAL OBSERVATIONS
A thorough weekly clinical examination was conducted at each weighing interval.

BODY WEIGHTS
Body weights were recorded at least once prior to treatment on the first day of treatment, and weekly thereafter.

FOOD CONSUMPTION
Food consumption was recorded weekly.

OPHTHALMIC EXAMINATION
Ophthalmic examinations were performed prior to treatment and prior to scheduled sacrifice by a staff veterinarian using an indirect ophthalmoscope. A mydriatic solution was used for pupil dilation.

CLINICAL PATHOLOGY
Clinical pathology tests were performed once prior to treatment (Day -6) and on Days 52 and 87.

ORGAN WEIGHTS
At the scheduled sacrifice, the following organs (when present) were weighed after careful dissection and trimming of fat and other contiguous tissue: adrenal, ovary, liver with drained gallbladder, thymus, spleen, kidney, testis with epididymides, thyroid with parathyroid, brain.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete macroscopic examination was performed on all animals including those found dead during the study. All gross observations were recorded individually.

HISTOPATHOLOGY: Yes
Tissues (as appropriate) from each animal were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically from the control and high dose group. The liver was identified as a target organ, and livers were examined from all animals to establish a no-effect level.

Statistics:

The following statistical tests were used:
- Levene's test to determine variance homogeneity. In the case of heterogeneity of variance (p < 0.05), transformations were used to stabilize the variance. Comparison tests took variance heterogeneity into consideration.
- One-way analysis of variance (ANOVA) was used to analyze body weights, body weight change, food consumption, continuous clinical pathology values, and organ weight data.
- If the ANOVA was significant, Dunnett's t-test was used for control versus treated group comparisons.

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the course of the study. In general, clinical observations noted in this study were typical of beagle dogs in toxicity studies. Although the incidence of vomitus containing capsules was generally low (less than 1 % of the total number of doses administered) for the study, Group 5 dogs demonstrated a higher incidence of vomitus containing capsules than did the control or other dose groups. When intact capsules were noted within 1 hour after dosing, they were redosed; partial capsules or those which were noted more than 1 hour after dosing could not be redosed. The increased incidence of regurgitation of one or both of the dosed capsules is likely related to the test article administration, but as the incidence was low and sporadic in comparison to the total number of doses, it is of no toxicologic importance.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of treated groups were similar to those of the control. All dogs gained weight during the course of the study, with no significant differences in total weight gain noted between control and treated dogs. Occasional significant increases (Day 22-29, Group 2-5 males) or decreases (Day 78-85, Group 4 and 5 males) in the mean body weight gain were considered small in magnitude and not biologically relevant.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Fluctuations in the food consumption data were comparable between the control and treated dogs. Total food consumption over the course of the study was similar between the treated and control dogs.

OPHTHALMOSCOPIC EXAMINATION
The unilateral (right) multifocal retinal folds of the fundus noted at Day 91 (term) in animal No. G35870 is an incidental finding occasionally seen in the laboratory beagle dog during routine toxicology testing and is unrelated to test material administration.

HAEMATOLOGY
No treatment-related changes were noted in the hematology and coagulation.

CLINICAL CHEMISTRY
There were no significant changes noted in the chemistry data at any interval, but the mean values for alkaline phosphatase activity were higher in Group 5 animals relative to control values at Days 52 and 87 (Weeks 8 and 13, respectively). The individual values for most Group 5 animals increased from Day 52 to Day 87, whereas the mean and most individual alkaline phosphatase values decreased for Group 1-4 animals, as expected in young growing animals. The progressively increasing alkaline phosphatase values noted for most Group 5 animals at Days 52 and 87 are attributed to the administration of the test item and correlate with the elevated liver weights noted in this group at terminal sacrifice. No additional test article-related findings were noted in the chemistry data.

URINALYSIS
No treatment-related changes were noted in urinalysis data at any interval.

ORGAN WEIGHTS
The mean absolute liver weight, liver-to-body weight percentage, and liver-to-brain weight ratio were significantly higher in Group 5 animals. The increased mean liver values in Group 5 animals correlated with the microscopic observation of centrilobular to diffuse hepatocellular hypertrophy, centrilobular to midzonal hepatocellular cytoplasmic clearing, and the presence of hepatocellular eosinophilic inclusions noted in these animals. The mean adrenal-to-body weight percentage, absolute thyroid weight, thyroid-to-body weight percentage, and thyroid-to-brain weight ratio in Group 4 males and the mean thyroid-to-body weight percentage in Group 5 males were significantly higher. No microscopic correlates were noted for the adrenal and thyroid changes, and they appeared to be small spurious increases.

GROSS PATHOLOGY
At necropsy, there were few grossly observed alterations, and none were suggestive of any relation to the test article.

HISTOPATHOLOGY: NON-NEOPLASTIC
The incidence and severity of centrilobular to diffuse hepatocellular hypertrophy and centrilobular to midzonal hepatocellular cytoplasmic clearing in the livers were increased in animals which received 200 and 1000 mg/kg bw/day of the test material. The incidence of hepatocellular eosinophilic inclusions in the liver was increased in animals which received 1000 mg/kg bw/day. Hepatocellular eosinophilic inclusions in the liver were seen in all males and in one female of the high dose group. These liver lesions apparently correlated with the increased liver weights and alkaline phosphatase activity.

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Changes in alkaline phosphatase, organ weight increases, liver cell hypertrophy and eosinophilic inclusions.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

dog

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

Two subchronic studies performed in rats and dogs are available. In a GLP-compliant subchronic oral toxicity study (OECD 408), a total of 120 Sprague-Dawley rats (10 males and 10 females per dose group; additional 10 males and 10 females in control and high dose groups were kept for a recovery period) were dosed by gavage with 0, 50, 200 and 1000 mg/kg body weight daily for three months (C.I.T., 1994). Doses were based on a 28 -day range-finder study (C.I.T., 1993). No relevant clinical signs and no mortalities related to administration of the test substance were observed during the treatment or recovery periods. At the end of the treatment period, a slightly higher total protein level was observed blood biochemistry analysis in females given 1000 mg/kg/day. However, this difference was considered to be of minor toxicological significance since the values were within the historical control range. A slightly higher incidence of positive reaction for proteins in the urine was noted in animals of both sexes given 250 and 1000 mg/kg/day at the end of treatment. After 4 weeks of recovery, this was observed only in males. As the positivity for proteins was minimal, this was considered to be of minor toxicological importance. At the end of treatment, a minor but statistically significant increase in testis weight was noted in high dose animals. At the end of the recovery period, a decrease in spleen weights in males and a minor increase in kidney weights in females were noted in the high dose group. In the absence of any histological change noted in these organs, these minor weight differences were considered to be of no toxicological importance. Based on the minor effects found, the NOEL was set at 50 mg/kg/day and the NOAEL at 1000 mg/kg/day under the conditions of this study.

In a second GLP compliant subchronic study (OECD 409), male and female beagle dogs received the test item at 0 (empty capsules), 10, 50, 200 and 1000 mg/kg/day for 13 consecutive weeks (Covance, 1999). No mortality and clinical signs of toxicity occurred. Body weight and food consumption were unaffected. Ophthalmoscopic examinations as well as hematology and urinalysis did not reveal any treatment-related changes. At dose levels of 200 mg/kg, minimal centrilobular to diffuse hepatocellular hypertrophy and centrilobular to midzonal hepatocellular cytoplasmic clearing were reported in the liver. In the high dose group, this finding correlated with an increase in mean and relative liver/gall bladder weights and an increase in alkaline phosphatase. In addition, hepatocellular eosinophilic inclusions were increased in all males and one female treated with 1000 mg/kg. Based on these findings, the NOEL was set at 50 mg/kg. The reported effects on the liver are mostly likely adaptive in nature and are expected to be reversed after a follow-up recovery period. At the dose level of 200 mg/kg, the liver effects were minimal and are not considered to impair liver function. Therefore, the NOAEL was set at 200 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP-compliant guideline study

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the present data, classification for repeated dose oral toxicity is not warranted under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for repeated dose oral toxicity is not warranted under Regulation (EC) No.1272/2008.