2,4,8,10-tetra(tert-butyl)-6-hydoroxy-12H-dibenzo[d,g] [1,3,2]dioxaphosphocin, 6-oxide

Endpoint summary

Description of key information

28d repeated oral:

Adverse effects on the intestines, mesenteric lymph node and blood coagulation were observed by repeated doses of the test item. The NOAEL (No observable adverse effect level) under the conditions of this study was considered to be 170 mg/kg/day and no adverse effects were detected in the 170 mg/kg groups.

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:

From 2017-09-08 to 2017-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Purity: > 99%
Batch No.: 101Z4

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Labratories Japan, Hino Breeding Center
- Age at study initiation: five weeks old at the onset of dosing
- Weight at study initiation: 145.6-175.2 g and 127.3-158.6 g for males and females, respectively
- Housing: hanging steel cages with wire-mesh floor
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 °C
- Humidity (%): 40-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hours light per day (light on at 7:00 and off at 19:00)

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

- VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance was suspended at a concentration of 20 w/v% in olive oil. In addition, olive oil has been commonly used in general toxicity studies and we have historical control data of this vehicle.
- Concentration in vehicle: 20 w/v%
- Lot/batch no. (if required): 701019

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

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

Remarks:

vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the dose-range finding study
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

GENERAL CLINICAL OBSERVATIONS: Yes
- Time schedule: observed two times daily (before and after dosing) during the dosing period, once daily during the recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the dosing period, once weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: day of the group allocation, days 1, 3, 8, 12, 17, 21, 26 and 28 of the dosing period, days 1, 5, 10 and 14 of the recovery period (for the recovery groups), the days carrying animals to the autopsy room (on the necropsy days)

FOOD CONSUMPTION:
- Time schedule for examinations: day of the group allocation, days 1, 3, 8, 15, 22 and 28 of the dosing period, days 1, 4, 8 and 14 of the recovery period (for the recovery groups); In addition, the food was replenished on days 8, 15 and 22 of the dosing period and day 8 of the recovery period, and the weights after the replenishment were also measured.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after the urine sampling on the next day of the last dosing for the main group and of the recovery period for the recovery group after overnight fasting (16 to 20 hours)
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, overnight fasting (16 to 20 hours) of the recovery group in the recvery period

BLOOD CHEMISTRY: Yes, Serum samples were used

URINALYSIS: Yes
- Time schedule for collection of urine: the nest morning after fasted for 15-16 hours of the last day of the dosing period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

OTHER:
pathological examinations including gross necropsy, tissue collecting and organ weight measurements, histopathological examinations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Other examinations:

SENSORlMOTOR FUNCTION EXAlMINATIONS
- Time schedule for examinations: once in week 4 (days 25 and 26) of dosing. The examinations for recovery groups in week 2 of the recovery period were not performed

Statistics:

Data regarding body weights, food consumption, grip strength and locomotor activity counts during the dosing period, and parameters of the hematological and blood chemical examinations, urine volume, urine specific gravity, organ weights and body weights on the necropsy day for the main groups were analyzed by the Bart1ett's test for homogeneity of variances. When the variances were homogeneous at a significance level of 5% in this analysis, the Dunnett’s test was performed. When the variances were not homogeneous, the nonparametric Dunnett's test was performed. The frequencies of defecation (number of stool) and urination (number of pool) during the dosing period were analyzed by the nonparametric Dunnett’s test.
Data regarding body weights and food consumption during the recovery period, and parameters of the hematological and blood chemical examinations, urine volume, urine specific gravity, organ weights and body weights on the necropsy day for the recovery groups were analyzed by the F-test for variance ratio. When there were no significant differences at a significance level of 5% in this analysis, the Student’s t-test was performed. When there were significant differences, the Aspin-Welch t-test was performed. The frequencies of defecation and urination during the recovery period were analyzed by the Mann-Whitney U-test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the general clinical observations, salivation and soft stool in males and females and diarrhea in females were observed in the 1000 mg/kg groups.
In the detailed clinical observations, a significant decrease of the frequency of urination in males was observed in the 1000 mg/kg group in pre-dosing period and a significant decrease of the frequency of defecation was observed in the 30 mg/kg group in week 4.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In either sex, no significant changes were observed in any treatment groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During dosing period, in males, a significant increase was observed in the 1000 mg/kg group on day 15, 22 and 28; in females, a significant decrease was observed in the 1000 mg/kg group on day 3 and a significant increase was observed in same group on day 28.
During recovery period, a significant increase in males was observed in the 1000 mg/kg group on day 4 and 8.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males, significant prolongations of prothrombin time (PT) and activated partial thromboplastin time (APTT) were observed in the 1000 mg/kg group. A significant increase of platelet count was observed in the 1000 mg/kg group, although it was within the range of historical control data in the testing facility.
In females, a significant decrease of eosinophils ratio was observed in the 1000 mg/kg group, although it was within the range of historical control data in the testing facility. A significant increase of white blood cell count in the 30 and 170 mg/kg groups and a significant shortening of PT in the 170 mg/kg group were observed, although they were not dose-dependent.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

BLOOD CHEMICAL EXAMINATIONS:
In males, a significant increase of triglyceride (TG) in the1000 mg/kg group and a significant decrease of total bile acids in the 170 mg/kg group and higher were observed although they were within the range of historical control data in the testing facility.
In females, a significant increase of TG was observed in the 1000 mg/kg group, although it was within the range of historical control data in the testing facility. A significant increase of aspartate aminotransferase (AST) and significant decreases of sodium and chloride were observed in the 170 mg/kg group, although they were not dose-dependent.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

In either sex, no significant changes in the urine volume or urine specific gravity were observed in any treatment groups and no abnormalities were observed in the conrol or any treatment groups in the other parameters.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In either sex, no significant changes were observed in the grip strength or locomotor activity count in any treatment groups and no abnormalities were observed in the control or any treatment groups in the reflex tests.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In males, significant decreases of relative weights of the testes in the 30 and 170 mg/kg groups and the seminal vesicle in the 30 mg/kg group were observed, although they were not dose-dependent.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In males, enlargement of the cecum was observed in all the five animals of the 1000 mg/kg group. Pelvic dilatation (unilateral) in the kidney in one animal each of the 30 and 1000 mg/kg groups and cyst of the pituitary gland in one animal of the 1000 mg/kg group were observed.
In females, enlargement of the cecum was observed in two animals out of five of the 1000 mg/kg group. Blackish region of mucosa of the glandular stomach was observed in one animal of the170 mg/kg group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In males, hypertrophy of paracortex of the mesenteric lymph node in all the five animals, mucosal hyperplasia of the rectum in three animals and mucosal hyperplasia of the colon in one animal were observed in the 1000 mg/kg group. Pelvic dilatation (unilateral) of the kidney was observed in one animal each of the 30 and 1000 mg/kg groups in which pelvic dilatation (unilateral) was observed macroscopically. Cyst formation in pars intermedia of the pituitary gland was observed in one animal of the 1000 mg/kg group in which cyst of the pituitary gland was observed macroscopically. In the control group, microgranuloma of the liver and solitary cyst in medulla of the kidney were observed in one animal each.
In females, mucosal hyperplasia of the rectum and hypertrophy of paracortex of the mesenteric lymph node in four animals out of five, mucosal hyperplasia of the colon and cecum in three animals each, and mucosal hyperplasia ofthe duodenum in one animal were observed in the 1000 mg/kg group. Mineralization in cortico-medullary junction of the kidney was observed in one animal each of the 1000 mg/kg and control groups. Focal necrosis of fundic mucosa of the glandular stomach was observed in one animal of the 170 mg/kg group in which blackish region of mucosa of the glandular stomach was observed macroscopically.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Estrous cycle stage:
In the control group, two animals out of five were in estrus, one was in metestrus, and two were in diestrus. In the 30 mg/kg group, two animals out of five were in metestrus and three were in diestrus. In the 170 mg/kg group, two animals out of five were in estrus, one was in metestrus and two were in diestrus. In the 1000 mg/kg group, one animal out of five was in proestrus, two were in estrus, and two were in diestrus.
- Sensorimotor function examinations:
During dosing period: In either sex, no significant changes were observed in the grip strength or locomotor activity count in any treatment groups and no abnormalities were observed in the control or any treatment groups in the reflex tests.
During recovery period: In either sex, since no treatment-related abnormalities were observed in week 4 of dosing period, the examinations were not performed during recovery period.

Key result

Dose descriptor:

NOAEL

Effect level:

170 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

intestine

mesenteric lymph node

Treatment related:

yes

Conclusions:

Adverse effects on the intestines, mesenteric lymph node and blood coagulation were observed by repeated doses of the test item. The NOAEL (No observable adverse effect level) under the conditions of this study was considered to be 170 mg/kg/day and no adverse effects were detected in the 170 mg/kg groups.

Executive summary:

A 28-day repeated-dose oral toxicity of the test item was performed to characterize the toxicity of the test substance in accordance with OECD Guidelines for the Testing of Chemicals, No.407.

Male and female Crl: CD (SD) rats (5 weeks old) were orally treated with the test item suspended in olive oil by gavage for 28 days daily. During the dosing period, general clinical observations, detailed clinical observations, sensorimotor function examinations, body weights and food consumption measurements were performed. On the next day of last dosing, dissection was performed after blood and urine samples collection, and urinalyses, blood and pathological examinations were performed after that. The dose levels were set at 0 (olive oil), 30, 170 and 1000 mg/kg/day, and five males and five females were used for each dose level (main group).

In addition, the recovery groups were set for the vehicle control and 1000 mg/kg/day which were reared for another 14 days without dosing to examine the reversibility of adverse effects.

 

In the general clinical observations, salivation and soft stool in males and females, and diarrhea in females were observed in the 1000 mg/kg groups.

In the food consumption measurements, a significant decrease in females on day 3 of the dosing period and a significant increase in males on day 15, 22 and 28 of the dosing period and females on day 28 of the dosing period were observed in the 1000 mg/kg groups.

In the hematological examinations, significant prolongations of prothrombin time (PT) and activated partial thromboplastin time (APTT) were observed in males of the 1000 mg/kg groups.

In the gross necropsy, enlargement of the cecum was observed in males and females of the 1000 mg/kg groups.

In the histopathological examinations, mucosal hyperplasia of the colon and rectum and hypertrophy of paracortex of the mesenteric lymph node in males and females, and mucosal hyperplasia of the duodenum and cecum in females were observed in the 1000 mg/kg groups.

No abnormalities related to the test substance dosing were seen in the detailed clinical observations, sensorimotor function examinations, body weights, urinalyses, blood chemical, examinations, organ weights or estrous cycle stage.

In the recovery groups, soft stool was observed in males and females of the 1000 mg/kg group on day 1 of the recovery period. A significant increase of food consumption was observed in males of the 1000 mg/kg group on day 4 and 8 of the recovery period. The other changes disappeared.

 

As stated above, adverse effects on the intestines, mesenteric lymph node and blood coagulation were observed by repeated doses of the test item. The NOAEL (No observable adverse effect level) under the conditions of this study was considered to be 170 mg/kg/day since soft stool, mucosal hyperplasia of the colon and rectum and hypertrophy of paracortex of the mesenteric lymph node in males and females, prolongations of PT and APTT in males, and diarrhea and mucosal hyperplasia of the duodenum and cecum in females were observed in the 1000 mg/kg groups, and no adverse effects were detected in the 170 mg/kg groups.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

170 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline study, GLP study

System:

gastrointestinal tract

Organ:

intestine

mesenteric lymph node

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

28-day repeated-dose oral test:

A 28-day repeated-dose oral toxicity of the test item was performed to characterize the toxicity of the test substance in accordance with OECD Guidelines for the Testing of Chemicals, No.407.

Male and female Crl: CD (SD) rats (5 weeks old) were orally treated with the test item suspended in olive oil by gavage for 28 days daily. During the dosing period, general clinical observations, detailed clinical observations, sensorimotor function examinations, body weights and food consumption measurements were performed. On the next day of last dosing, dissection was performed after blood and urine samples collection, and urinalyses, blood and pathological examinations were performed after that. The dose levels were set at 0 (olive oil), 30, 170 and 1000 mg/kg/day, and five males and five females were used for each dose level (main group).

In addition, the recovery groups were set for the vehicle control and 1000 mg/kg/day which were reared for another 14 days without dosing to examine the reversibility of adverse effects.

 

In the general clinical observations, salivation and soft stool in males and females, and diarrhea in females were observed in the 1000 mg/kg groups.

In the food consumption measurements, a significant decrease in females on day 3 of the dosing period and a significant increase in males on day 15, 22 and 28 of the dosing period and females on day 28 of the dosing period were observed in the 1000 mg/kg groups.

In the hematological examinations, significant prolongations of prothrombin time (PT) and activated partial thromboplastin time (APTT) were observed in males of the 1000 mg/kg groups.

In the gross necropsy, enlargement of the cecum was observed in males and females of the 1000 mg/kg groups.

In the histopathological examinations, mucosal hyperplasia of the colon and rectum and hypertrophy of paracortex of the mesenteric lymph node in males and females, and mucosal hyperplasia of the duodenum and cecum in females were observed in the 1000 mg/kg groups.

No abnormalities related to the test substance dosing were seen in the detailed clinical observations, sensorimotor function examinations, body weights, urinalyses, blood chemical, examinations, organ weights or estrous cycle stage.

In the recovery groups, soft stool was observed in males and females of the 1000 mg/kg group on day 1 of the recovery period. A significant increase of food consumption was observed in males of the 1000 mg/kg group on day 4 and 8 of the recovery period. The other changes disappeared.

 

As stated above, adverse effects on the intestines, mesenteric lymph node and blood coagulation were observed by repeated doses of the test item. The NOAEL (No observable adverse effect level) under the conditions of this study was considered to be 170 mg/kg/day since soft stool, mucosal hyperplasia of the colon and rectum and hypertrophy of paracortex of the mesenteric lymph node in males and females, prolongations of PT and APTT in males, and diarrhea and mucosal hyperplasia of the duodenum and cecum in females were observed in the 1000 mg/kg groups, and no adverse effects were detected in the 170 mg/kg groups.

Justification for classification or non-classification

28-day repeated-dose oral test:

Soft stool, mucosal hyperplasia of the colon and the recturn and hypertrophy of paracortex of the mesenteric lymph node in males and females, prolongations of PT and APTT in males, and diarrhea and mucosal hyperplasia of the duodenum and cecum in females were observed in the 1000 mg/kg groups, and no adverse effects were detected in the 170 mg/kg/day. In conclusion, there is adverse effects at 1000 mg/kg/day, NOAEL value is 170 mg/kg/day.

In accordance with Regulation (EC) No. 1272/2008 Table 3.9.1 and 3.9.3, substance should be classified as category 2 if there is adverse effects observed at 30-300 mg/kg/day by oral route (Guidance values need to be multiplied by 3 when using results from 28-d repeated toxicity study). This substance cause adverse effects at 1000 mg/kg/day, therefore this substance should not be classified for this specific target organ toxicity - repeated exposure endpoint.