1-(2,6-bis(4-tolyl)-1,3-dioxano(5,4-d)-1,3-dioxan-4-yl)ethane-1,2-diol

Endpoint summary

Description of key information

Three studies have been assessed for repeated dose toxicity: 90 day oral feeding study (key study), 1-generation reproduction study and 28-day study. See discussion for details. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 March to 16 August 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

other: September 1982 pre-Annex V

Deviations:

not specified

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sankyo Lab Service.
- Age at study initiation: 6 weeks old.
- Weight at study initiation: Average of 185 g for males and 150 g for females.
- Housing: In groups of 3 to 4 and kept in plastic cages (Aircon P from Clea Japan, Inc.) in a breeding room. The floor of the cages were covered with heat sterilised wood shavings (from the Japan Charles River Co), which were changed every 2 - 3 weeks.
- Diet (e.g. ad libitum): MF Rat Feed (Oriental Yeast Company), the test substance was mixed with the diet at the density of 1.25, 2.5 and 5% per weight. Ad libitum.
- Acclimation period: 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1°C.
- Humidity (%): 60 ± 10%.
- Air changes (per hr): 12 changes per hour.

IN-LIFE DATES: Day 0 (the day of dosing) up to Day 91.

Route of administration:

oral: feed

Vehicle:

other: diet

Details on oral exposure:

(Feeding study)
DIET PREPARATION
- Rate of preparation of diet (frequency): The necessary rat feed for the experiment was prepared all at once, placed in a vinyl bag and stored in a lidded polyethylene tontainer.
- Mixing appropriate amounts with (Type of food): The test substance was mixed with MF Rat Feed (Oriental Yeast Company) at the density of 1.25, 2.5 and 5% per weight.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable.

Duration of treatment / exposure:

90 days.

Frequency of treatment:

7 days/week.

Remarks:

Doses / Concentrations:
719.8 mg/kg/day (male), 758.8 mg/kg/day (female). Density of substance in food of 1.25%.
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
1518.8 mg/kg/day (male), 1598.2 mg/kg/day (female). Density of substance in food of 2.5%.
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
2925.5 mg/kg/day (male), 3055.9 mg/kg/day (female). Density of substance in food of 5%.
Basis:
nominal in diet

No. of animals per sex per dose:

10 males and 10 females per dose.

Control animals:

yes, plain diet

Details on study design:

Substance Administration:
Unlimited feed, mixed to 1.25%, 2.50% and 5.0% levels of substance and feed with no substance added were given to 4 groups of rats, each with 10 males and 10 females for a period of 90 days. The amount of substance intake was measured once a week by measuring the total feed intake and calculating on an individual basis.

Positive control:

Not applicable.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observed every day, twice a day (morning and afternoon) throughout the study.
- Cage side observations checked: General appearance and behaviour, any signs of abnormalities.

BODY WEIGHT: Yes
- Time schedule for examinations:Once a week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
Determined by the mount of food remaining, adjusted per individual per day.

FOOD EFFICIENCY: No.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily.
Determined by the mount of water remaining, adjusted per individual per day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the 90-day period.
- Anaesthetic used for blood collection: Yes (ether).
- Animals fasted: No data.
- How many animals: All the animals.
- Parameters checked: red corpuscle count, platelet count, hematocrit value, hemoglobin, white corpuscle count and the composition of white corpuscles.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the 90-day period.
- Animals fasted: No data.
- How many animals: All the animals.
- Parameters checked: Glutamic acid-oxalcyl acetic acid-transaminase (GOT), Glutamic acid-pyruvic acid-transaminase (GPT), alkaline phosphatase (Al-p), total protein (TP), blood sugar (Glu), total cholesterol (Cho), nitrogen (BUN), sodium (Na), potassium (K), chlorine (Cl) and calcium (Ca).

URINALYSIS: Yes
- Time schedule for collection of urine: before the start of the administration period, at the 1-month stage, and at the end of the study.
- Metabolism cages used for collection of urine: No data.
- Animals fasted: No data.
- Parameters checked: pH, protein, glucose, ketone bodies, urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: No.

Sacrifice and pathology:

GROSS PATHOLOGY: At the end of the 90-day period, all of the animals were terminated and subject to necropsy.
Organs were weighed immediately after macroscopic examination.


HISTOPATHOLOGY: Yes
The organs were hardended in 10% neutral formalin, covered with parafin, sliced and dyed with hematoxylin-eosin before being examined under an electron microscope. The following were examined:
cerebrum, cerebellum, pituitary gland, thyroid gland, heart, lungs, thymus, liver, kidney, adrenal gland, spleen, testis, prostate gland, bladder, pancreas, ovary, uterus, peritoneum, lymph node, stomach, duodenum, rectum and bone marrow.

Other examinations:

Not reported.

Statistics:

Average values were compared done using t-values.

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):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

COMPOUND INTAKE
Rats consumed a normal amount, demonstrating no aversion to the feed mixed with substance. The average daily intake over the 90 day-period is as below;

Density of analyte Male Female
1.25% 719.8 mg/kg/day 758.8 mg/kg/day
2.50% 1518.8 mg/kg/day 1598.2 mg/kg/day
5.00% 2925.5 mg/kg/day 3055.9 mg/kg/day

For all of the groups the daily substance intake was relatively high at the beginning of the administration period and decreased over time, due to the relationship between weight gain and feed intake. Substance intake for all of the groups decreased by about one-half from the beginning to the end of the administration period as below;

Density of analyte Sex Beginning of period End of period
1.25% Male 1082 mg/kg/day 594 mg/kg/day
1.25% Feale 1003 mg/kg/day 646 mg/kg/day
2.50% Male 2225 mg/kg/day 1160 mg/kg/day
2.50% Female 2104 mg/kg/day 1353 mg/kg/day
5.00% Male 4529 mg/kg/day 2323 mg/kg/day
5.00% Female 4206 mg/kg/day 2509 mg/kg/day

CLINICAL SIGNS AND MORTALITY
There were no deaths in any of the groups. There were no abnormalities observed in any of the groups for behaviour, appearance, coat lustre or excretion.

BODY WEIGHT AND WEIGHT GAIN
All of the female groups had normal rates of growth, with no significant difference relative to the control group.
Among males, the 5.00% group showed an inhibited rate of weight gain around the fifth week, and a statistically significant decrease in the weight gain rate from the eighth week to the end of the experiment. The other groups showed normal weight gain trends throughout, with no difference compard to the control group.

FOOD CONSUMPTION
Daily average feed intake is shown in Table 3, attached. All of the groups consumed normal levels of feed, showing no signs of aversion toward the feed mixed with analyte. In fact, all groups, both male and female, consumed more feed than the control group.

FOOD EFFICIENCY
Not examined.

WATER CONSUMPTION
All of the groups,both male and female, consumed normal levels of water with no significant difference compared to the control group.

OPHTHALMOSCOPIC EXAMINATION
Not examined.

URINALYSIS
The pH, protein and urobilinogen levels of the urine were all within the range of normal values for all groups. There were no differences compared to the readings obtained before substance administration began.
Test for glucose, ketone bodies and occult blood were all negative.

HAEMATOLOGY
All test results fell within the normal range, with no differences observed between the control group and the substance groups.

CLINICAL CHEMISTRY
Statistically significant differences compared to the control group were observed in GOT, alkaline phosphatase (Al-p), total cholesterol (Cho) and calcium (Ca). Statistically significant low GOT values were observed for males in both the 1.25% and 5.00% groups. Statistically significant low Al-p values were observed for males in the 5.00% group; low, though not statistically significant Al-p values for males in the 1.25% and 2.50% groups and females in the 1.25% and 2.50% groups were also observed. However, females of the 5.00% group showed a high Al-p value.
There were no abnormalities in any of the other test items.

NEUROBEHAVIOUR
Not examined.

ORGAN WEIGHTS
None of the organs of the animals were significantly different in weight relative to the control group. However, the carcasses if the 5.00% male group had a statistically significant lowe weight.

GROSS PATHOLOGY
Macroscopic inspection at the time of dissection revealed no abnormalities in any of the rats in both the substance groups and the control groups.

HISTOPATHOLOGY:
Abnormalities were observed in the liver and kidneys.

In the liver, 14 cases of cell infiltration in Glisson's capsule was observed, including in the control group (1/10 in control, 2/10 at 1.25%, 2/10 at 2.5% and 0/10 at 5% in males and 1/10 in control, 3/10 at 1.25%, 1/10 at 2.5% and 4/10 at 5% in females). However, all the cases were extremely minor.

In the kidneys, 16 cases of calcinosis in corticomedullary junction was observed, including in the control group (4/10 in control, 6/10 at 1.25%, 5/10 at 2.5% and 1/10 at 5% in females).

Nephritis was observed in one female in the control group. There were no other abnormalitis observed in any of the other organs.

Dose descriptor:

LOAEL

Effect level:

700 - 750 other: mg/kg/day (daily average consumption in diet)

Sex:

male/female

Basis for effect level:

other: Recommended value by the former Competent Authority: an over cautious LOAEL of 700 - 750 mg/kg could be suggested based on the lowest dose tested. See overall remarks.

Critical effects observed:

not specified

Conclusions:

After unlimited supply of rat feed with Gel ALL-MD content varying from 0.01% to 1.25%, 2.50% , and 5.00%, was fed to rats over a 90-day period, the amount of Gel ALL-MD intake was approximately 750 mg/kg/day for the 1.25% group, 1500 mg/kg/day for the 2.50% group, and 3000 mg/kg/day for the 5.00% group. None of the animals that consumed the analyte demonstrated any toxic effects. The 5.00% male group did show an inhibited weight gain rate after the fifth week and a statistically significant lowering of weight gain rate from the eight week to the end of the 90-day period. As this is within the normal range of values for male SLc-Wister rats, a correlation between inhibited weight gain and dosage level was not established.

No other abnormalities attributable to Gel ALL-MD intake were observed in the urinalyses, blood test, blood serum analysis or examination of the internal organs.

Executive summary:

Three groups of SLc-Wister rats (10 males and 10 females each) were provided unlimited amounts of hard feed containing GEL ALL-MD levels of 1.25%, 2.50% and 5.00% by weight over a 90-day period. A fourth control group was given hard feed with no GEL ALL-MD added.

 The rats consumed normal amounts of feed, demonstrating absolutely no aversion to feed mixed with GEL ALL-MD.

 The daily average consumption of GEL ALL-MD over the 90-day period is shown in the following table.

Density of analyte	Male	Female
1.25%	719.8 mg/kg/day	758.8 mg/kg/day
2.50%	1518.8 mg/kg/day	1598.2 mg/kg/day
5.00%	2925.5 mg/kg/day	3055.9 mg/kg/day

 

There were no cases of death in any of the test groups during the test period, and no abnormal conditions were observed in behaviour, appearance, coat luster or excretion,.

 Males in the 5.00% group showed weight gain rate after the fifth week, and statistically significant decrease in weight gain rate from the eight week to the end of the experiment.

At the end of the 90-day period, microscopic and macroscopic examinations of blood, serum, urine and the internal organs were carried out; no abnormal effects attribute to GEL ALL-MD intake were observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LOAEL

720 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The LOAEL is a precautionary result based on the findings of the 90-day study.

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

Three studies have been assessed for repeated dose toxicity via the oral route; a 90 day oral feeding study, a 1-generation reproduction study and a 28-day repeat dose study.

The 90 day oral feeding study has been assessed as the key study for repeated dose toxicity as it has a longer exposure period than the 28-day study and also represents the most precautionary assessment of repeated dose toxicity.

The 1-generation study has conducted to investigate the potential adverse effects on the reproductive cycle and subsequent offspring development to rats. It has some evaluation of general systemic toxicity but not have enough evidence on its own to allow for a proper assessment of repeated dose systemic toxicity alone.

 

Therefore, the assessment of repeated dose systemic toxicity is based on the 90 day study as the key study and supported by findings from the one generation study and 28-day study.

Key Study: 90 day feeding study (Fujihir & Takahashi, 1982):

Three groups of SLc-Wister rats (10 males and 10 females each) were provided unlimited amounts of hard feed containing GEL ALL-MD levels of 1.25%, 2.50% and 5.00% by weight over a 90-day period. A fourth control group was given hard feed with no GEL ALL-MD added.

 The rats consumed normal amounts of feed, demonstrating absolutely no aversion to feed mixed with GEL ALL-MD.

 The daily average consumption of GEL ALL-MD over the 90-day period is shown in the following table.

Density of analyte	Male	Female
1.25%	719.8 mg/kg/day	758.8 mg/kg/day
2.50%	1518.8 mg/kg/day	1598.2 mg/kg/day
5.00%	2925.5 mg/kg/day	3055.9 mg/kg/day

 

There were no cases of death in any of the test groups during the test period, and no abnormal conditions were observed in behaviour, appearance, coat luster or excretion,.

Males in the 5.00% group showed weight gain rate after the fifth week, and statistically significant decrease in weight gain rate from the eight week to the end of the experiment. As this is within the normal range of values for male SLc-Wister rats, a correlation between inhibited weight gain and dosage level was not established.

At the end of the 90-day period, microscopic and macroscopic examinations of blood, serum, urine and the internal organs were carried out; no abnormal effects attribute to GEL ALL-MD intake were observed.

 

However, during histopathological investigations abnormalities were observed in both the liver and kidneys, however the study does not describe the nature of these abnormalities except to describe the incidence; 14 cases of liver abnormalities were observed (including in the control group) and 16 cases of kidney abnormality (4 in the control group). All changes were considered minor. A single case of nephritis in a female control was specifically mentioned in the report which may indicate that the severity of the observations in the test animals may not appear to be a finding related to administration of the substance.

Due to the limited reporting in the study, particularly with regard to the effects on the liver and kidneys, an over precautions LOAEL of 700 - 750 mg/kg could be suggested based on the lowest dose tested.

However, as the effects were all considered minor and also occurred in control animals, they could also be assessed to be of no toxicological importance and a NOAEL established at the top dose.

Supporting data:

28 -day repeated dose study (oral):

The substance (in 0.5% carboxymethylcellulose) was administeredby gavage to Sprague-Dawley rats (5/sex/dose) at doses of
0, 11, 110 or 1100 mg/kg/day for 28 days.

There were no deaths and no signs of toxicity. There were no effects on haematology or clinical chemistry. There were no treatment related effects on pathology or histology.

The NOEL/NOAEL was 1100 mg/kg/day.

1 -generation reproduction study:

The test material was administered by gavage to three groups, each of twenty-four male and twenty-four female Wistar Han™: HsdRccHan™: WIST strain rats, at dose levels of 100, 300 and 1000 mg/kg/day. A control group of twenty-four males and twenty-four females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study.Oestrous cycle assessment was performed daily for three weeks prior to mating.

All males were terminated following the completion of a successful mating. All surviving females were terminated on Day 21 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

Results.

Mortality.One male treated with 1000 mg/kg/day was killed in extremis on Day 71.  One female treated with 100 mg/kg/day was killed in extremison Day 43 (during expected time of parturition).There were no further unscheduled deaths during the study.

Clinical Observations .No clinically observable signs of toxicity were detected in terminal kill animals.

Bodyweight. No adverse effect on bodyweight gain was detected for males throughout the treatment period or for females throughout maturation, gestation or lactation.

Food Consumption. No adverse effect on food consumption was detected.

Water Consumption. No intergroup differences were detected.

Necropsy. No toxicologically significant effects were detected in terminal kill animals.

Organ Weights. No treatment-related effects were detected in the organ weights measured.

Histopathology. No treatment-related microscopic abnormalities were detected.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The assessment of repeated dose systemic toxicity is based on the 90 day study as the key study and supported by findings from the one generation study and 28-day study.

Justification for classification or non-classification

Based on the assessment of effects seen in the studies, it is considered that the test substance does not meet the criteria for classification for specific target organ toxicity - repeated exposure (STOT-RE).

The most precautionary assessment is based on the 90 -day study, where a LOAEL of 700 - 750 mg/kg could be suggested based on the lowest dose tested and the abnormalities observed in the livers and kidneys.

However, as these effects were considered minor and were also observed in control animals, this LOAEL is very much a worst case assessment, and it can also be argued that a NOAEL could be established at the top dose test.

According to the criteria for classification as STOT-RE Category 2 (CLP), based on a 90 -day study, significant toxic effects should be observed within the guidance dose range 10 - 100 mg/kg bw/day. Even if the effects seen in the livers and kidneys were considered to be significant/adverse effects, the dose range of 700 - 750 mg/kg bw/day is significantly over the guidance dose range for classification.

The supporting data from the 28 -day repeat dose study and 1 -generation reproduction study also showed no adverse toxicological effects, at any dose levels, from repeated dose exposure. This supports the argument that the effects in the 90 -day study were not significant.

Therefore, it is considered that classification for STOT-RE is not required.