Reaction mass of 1,3-dioxan-5-ol and 1,3-dioxolan-4-ylmethanol

Endpoint summary

Description of key information

No NOAEL has been indentified in available studies. However, it is clear that adverse effects in testes of treated animals are observed (in rats by oral route at 1.0 ml/kg/d; . Oral NOAEL can be stated at 0.1 ml/kg/d in the MSD study (1973) regarding effects observed in male rats testes, epididymes and seminal vesicles, corresponding to 121.85 mg/kg/d (considering a GF density of 1.2185).

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: In comparison with OECD TG 408 requirements, ophalmological examinations, functional observation, water consumption were not recorded

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

In comparison with OECD TG 408 requirements, ophalmological examinations, functional observation, water consumption were not recorded

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Own SPF-breeding
- Age at study initiation: not specified
- Weight at study initiation: 182-202 g for females; 252-295 for males
- Fasting period before study: not specified
- Housing: Separation of animals according to sex. Animals were kept in pairs in Macrolon containers of type III in a fully air-conditionned test room.
- Diet (e.g. ad libitum): ALTROMIN-R-powder ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/-1
- Humidity (%): 50-60
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 10/10

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

GF was administered daily by means of a stomach tube.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0.01; 0.10 and 1.00 ml/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before treatment, after 90 days (and 6 weeks after discuntinuation of treatment)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10+2/sex/group
- Parameters checked: count of erythrocyte, leucocyte and reticulocyte, colorimetric determination of the hemoglobin, determination of the hematocrit, differentiation of the white blood cells

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood and liver: after 90 days (and 6 weeks after discuntinuation of treatment)
- Animals fasted: No data
- How many animals: 10+2 /sex/group
- Parameters checked: enzyme activities (AP and GPT in plasma and liver), lipid content in plasma and liver, total protein and albumin content in the plasma, urea and hemoglobin in plasma, sulfhydryl groups in the liver

URINALYSIS: Yes
- Time schedule for collection of urine: before begin of the test, at 6 weeks and at the end of the test.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: protein, glucose, urobilinogen, biliburin and blood. The pH-value was determined and the sediment was microscopically examined. The leucin amino peptidase activity was determined in the urine.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Liver function test with phenacetin
Renal function test with phenol red

Sacrifice and pathology:

Animals were sacrified by exsanguination. Following organs were taken from animals and fixed in 10% formol after determination of their weight: liver, spleen, kidney, aorta, lung, stomach, intestine, pituitary gland, adrenals, thyroid of all animals; ovary and uterus of females; testes, epididymes and seminal vehicles of males.

Statistics:

Statisics were performed with indication of statistical significances as against control and as against initial value

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No conspicuous changes were observed in comparison to the controls. 5 deaths were observed: 1 control animal died on the 124th day of the test; 4 animals died in the highest dose group (1 female after 3 days, 1 female after 77 days, 1 male after 5 days and 1 male after 74 days).

BODY WEIGHT AND WEIGHT GAIN
The female animals of the 0.01 ml/kg and 0.1 ml/kg dose groups showed a low delayed bw gain whereas the group 1.0 ml/kg showed a clearly delayed gain in weight in comparison with the control group.

Relative increase in female weight in % of control at the end of the test was:
0.01 ml/kg group: 89.74
0.1 ml/kg group: 94.87
1.0 ml/kg group: 66.67

The course of weight of the male animals was equal to the control group until the 6th week. Animals from the highest, the middle and the lowest dosage group showed a delayed gain of weight from the 6th, the 7th and the 11th week, respectively.

Relative increase in male weight in % of control at the end of the test was:
0.01 ml/kg group: 80.87
0.1 ml/kg group: 68.31
1.0 ml/kg group: 57.36

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The relation between gain in weight and intake of feed is clearly demonstrated with a decrease of feed efficiency depending on the dose.

HAEMATOLOGY
Significant differences against the control were observed among
- females from 0.1 ml/kg dose group with discontinuation of GF application (95%) for erythrocytes 10E6/mm3
- females from 0.01 and 1.0 ml/kg dose group with 3 months continuous application of GF (95%) for hemoglobin g%
- females from 0.1 ml/kg dose group with 3 months continuous application of GF (99%) for hemoglobin g%
- males from 1.0 ml/kg dose group with 3 months continuous application of GF (95%) for hemoglobin g%
- females from 0.1 ml/kg dose group with 3 months continuous application and discontinuation of GF application (95%) for mean corpuscular hemoglobin
- males from 0.1 ml/kg dose group with 3 months continuous application of GF (95%) for mean corpuscular hemoglobin
- females from 0.01 ml/kg dose group with 3 months continuous application of GF (99%) for hematocrit %
- females from 0.1 ml/kg dose group with 3 months continuous application of GF (95%) for hematocrit %
- females from 1.0 ml/kg dose group with 3 months continuous application of GF (99.9%) for hematocrit %
- females from 1.0 ml/kg dose group with 3 months continuous application of GF (95%) for mean corpuscular hemoglobin concentration
- males from 1.0 ml/kg dose group with 3 months continuous application of GF (99%) for mean corpuscular hemoglobin concentration
- males and females from 0.1 ml/kg dose group with 3 months continuous application of GF (95%) for mean corpuscular volume

CLINICAL CHEMISTRY
The determination of AP and GPT in the plasma did not show any increase of the enzyme activity. In the liver the AP-values of the treated males showed an upward tendency, the mean values yet for the 0.1 ml/kg group were significantly changed in comparison to the control. These higher AP-values were observed even after discontinuation of the GF supply. The GPT values in the liver showed to be increased in comparison to the control, yet after discontinuation of the supply, no decrease was observed.
Total cholesterol and lipoid phosphor showed no significances depending on the dose, so that an influence of GF can be excluded. No influence of GF on total protein and albumin fractions in the plasma was detected. The blood urea is not influenced by GF. No hemoglobin in the plasma was determined. An increase of the non-protein bound SH-groups in the liver is observed and considered as an adaptation of the liver to application of GF.
The liver and kidney functions were not disturbed by GF

URINALYSIS
No pathologic findings were made.

ORGAN WEIGHTS
Among all weighed organs, testes and epididymes of the animals from the highest dose group showed lower weights.

GROSS PATHOLOGY and HISTOPATHOLOGY
A) Observations in control group:
The studies revealed the following findings deviating from the perfect structure:
- Liver: few small round cell nodules in 2 animals and a greater charge with fat of the lobular periphery in 1 animal
- Spleen: phagocytes with blood pigment in moderate quantity in 8 and in great quantity in 2 animals
- Kidney: calcareous inclusions in the uriniferous tubules in 7 animals with sporadic small round cell infiltrates in 1 of them
- Heart: cell nodules and degenerated muscular fibres in 6 animals
- Lung: area void of air and desquamative catarrh in 1 animal
- Suprarenal glands: richness in lipids in all 3 layers in 5 animals
- Thyroid gland: follicles of fluctuating size poorer in colloid with partly desquamed epithelium in all anaimals

B) Observation after continuous application of GF:
Observations in dose I group (0.01 ml/kg):
The studies revealed the following findings deviating from the perfect structure:
- Spleen: phagocytes charged with blood pigment in moderate quantity in 5 and in great quantity in 3 animals
- Kidney: calcareous inclusions in the uriniferous tubules in 6 animals with round cell nodules in 1 of them
- Heart: degenerated myocardial fibres in 6 animals with round cell nodules in the papillary muscles in 3 of them
- Suprarenal glands: follicles poorer in colloid with partly desquamed epithelium in all animals in fluctuating extend
- Uteri: inflammatory manifestations in 2 animals
- Testes slight inhibition of spermatogenesis in 1 animal
The same findings in spleen, kidneys, heart, suprarenal glands and thyroid gland were also made with the control group

Observations in dose II group (0.1 ml/kg):
The studies revealed the following findings deviating from the perfect structure:
- Liver: small round cell nodules in 1 animal and liver cells charged with fat in great number in 2 animals
- Kidney: calcareous inclusions in the tubules in 8 animals
- Heart: round cell nodules in the papillary muscles in 4 animals and myocardial fibres with degenerative changes in 2 others animals
- Suprarenal glands: especially lipid-rich cortica in 5 animals and a lipid distribution deviating from the control in 4 animals
- Thyroid gland: follicles poorer in colloid partly with desquamation of the epithelium in 16 animals and a higher epithelium in 4 of them
- Uteri: inflammatory endometrium in 3 animals
- Testes: partly an inhibition of spermiogenesis in 2 animals with single atrophic seminiferous tubules in one of them
The same findings were also made in liver, kidneys and heart of the control group, with some deviations also in suprarenal and thyroid glands

Observations in dose III group (1.0 ml/kg):
The studies revealed the following findings deviating from the perfect structure:
- Liver: single round cell nodules in 1 animal
- Spleen: phagocytes charged with blood pigment in bigger quantity in 5 and in big quantity in another 5 animals
- Kidney: calcareous inclusions in the tubules in 6 animals
- Heart: round cell nodules in 4 animals
- Lungs: atelectases in 3 animals
- Hypophysis: enlarged cells in the anterior lobe of 4 animals
- Thyroid gland: follicles poorer in colloid in different extent in 11 animals
- Epididymes: deviations in the pattern of the lipid distribution in 3 animals; changes in 6 animals
- Uterus: inflammatory manifestations in 1 animal
- Testes: changes in all animals, from slight inhibition of spermiogenesis to total atrophy
- Seminal vehicles: considerable changes in 4 animals
Of the above findings those in testes, epididymes and seminal vesicle are clearly to be attributed to the test item effect. The findings in hypophyses and suprarenal glands deviating from the controls appeared collectively only in a small part of the animals. In the spleen the number of phagocytes containing hemosiderin corresponded in 5 animals to about the highest quantity found in controls and in 5 other animals it was even higher.
The other findings deviating from perfect condition were also made in the organs of the control group.

Observations in the 4 animals dead in course of the study in dose III group (1.0 ml/kg):
Apart from the changes caused or influenced by the autolysis the following finding deviating from the perfect structure were made in the studied organs:
- Kidney: calcareous inclusions in 1 animal
- Heart: degenerated muscle fibres in all 3 animals
- Lungs: massive edema in all 3 animals
- Hypophysis: vessels rich in blood in all 3 animals
- Suprarenal glands: poor in lipid in all 3 animals
- Thyroid gland: follicles poor in colloid
The direct cause of death was apparently the pulmonary edema

C) Observation after discontinuation of the application of GF (4 animals/dose):
Observations in dose I group (0.01 ml/kg):
In the examined organs, the following findings deviating from the perfect structure of the organ were made:
- Kidney: calcareous inclusions in the uriniferous tubules in 1 animal
- Heart: round cell nodules in the papillary muscles in 1 animal
- Thyroid glands: partly follicles poorer in colloid in all animals
- Uterus: inflammatory manifestations in 1 animal

Observations in dose II group (0.1 ml/kg):
In the examined organs, the following findings deviating from the perfect structure of the organ were made:
- Kidneys: calcareous inclusions in the uriniferous tubules in 1 animal
- Thyroid glands: partly follicles poorer in colloid in all animals
- Testis: atrophic seminiferous tubules in low number in 1 animal
Only the findings in the testis differ from the control group

Observations in dose III group (1.0 ml/kg):
In the examined organs, the following findings deviating from the perfect structure of the organ were made:
- Kidneys: Ca-containing inclusions in 2 animals
- Lung: atelectases in 1 animal
- Thyroid gland: follicles poorer in colloid in fluctuating extent in all animals
- Testis: tubules with disturbed spermiogenesis to complete atrophy in both animals
- Epididymes: degenerated cells from the seminal epithelium in the ductus epididymidis in both animals
The findings in testes and epididymes have to be considered as deviations from the control

Dose descriptor:

NOAEL

Sex:

male

Basis for effect level:

other: Whereas no NOAEL have been determined in this study, adverse effects have been recoded in male testes in the hight dose group (1.0 ml/kg) with changes in all animals (8/8), from slight inhibition of spermiogenesis to total atrophy

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Conclusions:

From all these observations, the author stated that findings in testes, epididymes and seminal vesicles are clearly to be attributed to the test item effect among male animals treated with GF at 1.0 ml/kg dose during 3 months.

Executive summary:

Toxicity test after oral application of the test item glycerol formal was performed with rats for a period of 3 months. The test item was applied daily as aquous solution in a dosage of 0.01 ml/kg (dose I); 0.10 ml/kg (dose II); 1.00 ml/kg (dose III) to groups of 12 males and 12 females each. Control group received aqua at a dose of 2 ml/kg (12 male and 12 females) instead of glycerol formal. After 3 months of application, 10 males and 10 females of each group were sacrificed and subjected to the respective tests. The other animals were sacrificed only 6 weeks after discontinuation of the supply of test item, so as to make it possible to observe a restitution of the changes appearing by the application of glycerol formal.

The following controls ans examinations were made during the test:

- Daily observation of the general behaviour and control of weight.

- Weekly determination of the feed consumption.

- Hematological studies (ery-, leuco- and reticulocyte count, hemoglobin, hematocrit, differential blood picture) before begin of the application, after 3 monhs of application and 6 weeks after discontinuation of the treatment.

Medico-chemical tests (alkaline phosphatase, GPT, triglycerides, total cholesterol, lipoid phosphor in plasma ans liver; total protein in the plasma, separation of blood properties, urea in the plasma, sulfhydryl groups in the liver) after 3 months of application of glycerol formal and 6 weeks after discountinuation of the application. Hemoglobin in the plasma was monthly determined.

- Urinalyses before application, after 3 months and 6 weeks after discontinuation of application.

- Renal and liver function tests before application as well as after 3 months of application.

- Histo-pathological tests after 3 months and 6 weeks after discontinuation of the treatment.

The general behaviour did not show any difference in comparison to the controls. During the test 1 control animal and 4 animals from the 1.0 ml/kg dosage group died.

The course of weight of the male animals of the 1.0 ml/kg group showed a clearly delayed weight gain from the 6th week onwards. Also the other groups showed - though only to a low extent - a delayed increase in weight. Intake ans efficiency of feed were diminished with dependence on the dose in comparison to the control values.

In the hematologic studies, the greater decrease in the number of retyculocytes and the number of leucocytes showed an effect exerted at the highest dose group. All other hematologic findings were within normal limits which 6 weeks after dicontinuation of feeding GF were also reached again by the findings declared as deviation (number of reticulocytes and leucocytes).

The medico-chemical tests revealed no influence on the AP and on the GPT in the plasma. In the liver the AP-values of the female and male animals and the GPT-values of male and female animals were increased in comparison to the control values. The determination of the lipid content (triglycerides, total cholesterol and lipoid phosphor) in plasma and liver did not reveal any indication referring to the application of GF. Also total protein, albumin fractions, urea and hemoglobin in the plasma did not show any influence by GF. The non-protein bound SH-groups in the liver of the treated animals were increased in comparison to the control.

The urinalysis brought no pathological finding whatever.

Also the liver function tests (excretion of phenacetin) and the renal function tests (excretion of phenol red, leucin amino peptidase activity in the urine) showed no reaction to the administration of GF.

Apart from the male gonads ad the higher hemosiderin content of the spleens in the highest dose group, the treated animals did not show any difference in comparison to the control animals from the pathologico-anatomical point of view. The weight of testes and epididymes as well as their relation to the body weight were lower, the histological study showing an inhibition of the spermiogenesis in the highest dose (1.0 ml/kg).

From all these observations, the author stated that findings in testes, epididymes and seminal vesicles are clearly to be attributed to the test item effect among male animals treated with GF at 1.0 ml/kg dose during 3 months.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

121.85 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good quality as 2 oral route tests are available in addition to 3 other tests performed with less relevant routes of administartion.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

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

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Five repeated dose toxicity studies are available. Two of these tests were performed by oral route whereas less appropriate routes of administration were selected in the remaining tests.

Appropriate route of exposure:

90-day oral toxicity study in previously "in-utero" exposed rat (concentrations of 1; 5 and 25 mg/kg/d):

no treatment-related effects were observed at tested doses that are relatively low in comparison with limit concentration normally used in OECD TG 408 test.

90-day oral toxicity study in rat (concentrations of 0.01; 0.1 and 1.0 ml/kg/d):

Among the changes observed in treated rats, the author stated that findings in testes (changes in all male animals (8/8), from slight inhibition of spermiogenesis to total atrophy), epididymes and seminal vesicles are clearly to be attributed to the test item effect in 1.0 ml/kg test group.

Less appropriate routes of exposure:

28-day intravenous toxicity study in rabbit (concentrations of 0.024 and 0.24 ml/kg/d):

None of the observed effects were stated as grave according to the author.

90-day subcutaneous toxicity study in rat (concentrations of 0.24; 0.48 and 1.20 ml/kg/d; this last concentration was tested at 2 dilutions corresponding to the same final GF content):

5/10 animals from the 0.48 ml/kg dose group presented disturbance of spermiogenesis with interstitial edema in testis whereas 10/10 animals from the 1.20 ml/kg dose group showed atrophy of the seminiferous tubules connected with an interstitial edema in testis and changes in testes of all males from the second 1.20 ml/kg dose group with a total atrophy of the seminal tubes in 8/10.

90-day intramuscular study in dog (concentrations of 0.024 and 0.24 ml/kg/d; this last concentration was tested at 2 dilutions corresponding to the same final GF content):

Because of the low number of animals used in each test group, the following observations are less reliable than the ones recorded in other tests. According to the author, the observations made in the testes of treated dogs are noteworthy in connection with GF (with a negligible depression of spermiogenesis and also changes in the content of the tubules of the epididymes in 2/2 animals from dose group II1 and partly atrophy of the seminiferous tubules and changes of the epididymes in 1/2 animal in dose group II2).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Among all the available repeated dose toxicity tests, this study is the only one in which a relevant route of administration and dose levels closed to the ones required in OECD TG were used.

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: testes

Justification for classification or non-classification

Whereas clear adverse effects have been recorded in testis of animals in the different repeated dose toxicity tests, these effects have to be considered as adverse effects on sexual function and fertility that covers alterations to the male reproductive system as mentionned in CLP Regulation.

The test item is not classified according to STOT RE.