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

Endpoint summary

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP lab following OECD guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

Animals
Number: six nulliparous and non-pregnant female rats were received at CiToxLAB France on 09 and 14 August 2012.
Strain and Sanitary status: Sprague-Dawley rat, Rj Han: SD, Indemn of Organism Pathogen Specific Han (IOPS Han).
Breeder: Janvier, Le Genest-Saint-Isle, France.
Age/Weight: on the day of treatment, the females were approximately 8 weeks old and had a mean body weight of 215 g (range: 208 g to 220 g).
Receipt: on arrival, the animals were given a clinical examination to ensure that they were in good condition.
Acclimation: the animals were acclimated to the study conditions for a period of 5 days before treatment.
Allocation to groups: the day before treatment, the required number of animals (three females per treatment step) were allocated to the groups according to a computerized randomization procedure.
Identification: the day before treatment, the animals were individually identified by ear notches (unique CiToxLAB France identity number).

Environmental conditions
From arrival at CiToxLAB France, the animals were housed in a barriered rodent unit.
The animal room conditions were set as follows:
. temperature : 22 ± 2°C,
. relative humidity : 50 ± 20%,
. light/dark cycle : 12h/12h,
. ventilation : approximately 12 cycles/hour of filtered, non-recycled air.
The corresponding instrumentation and equipment are checked and calibrated at regular intervals. The temperature and relative humidity are recorded continuously (recording devices equipped with alarm systems).

Housing
The animals were housed by three from the same group in polycarbonate cages with stainless steel lids (Tecniplast 2154, 940 cm²) containing autoclaved sawdust (SICSA, Alfortville, France). Rat Nylabone was given as enrichment.

Food and water
All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 2626975 (SSNIFF Spezialdiäten GmbH, Soest, Germany), and tap water (filtered with a 0.22 μm filter).
During periods of fasting, food was removed, but the animals were not deprived of water.

Contaminant analyses
The batches of diet and sawdust were analyzed by the suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants that could have interfered with or prejudiced the outcome of the study were found in the diet, drinking water or sawdust.

Route of administration:

oral: drinking water

Vehicle:

water

Remarks:

The vehicle was drinking water treated by reverse osmosis using ELIX 5 (Millipore SA, Saint-Quentin en Yvelines, France).

Details on oral exposure:

Vehicle
The vehicle used in this study was selected from the results of solubility assays performed by the CiToxLAB France Pharmacy.
In the absence of recommendation from the Sponsor, the solubility assay first started at the concentration of 200 mg/mL, and the first choice vehicle was drinking water treated by reverse osmosis. A solution was obtained at the concentration of 200 mg/mL in this vehicle.

Dose formulation preparation
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle.
Dose formulations preparations were prepared by the CiToxLAB France Pharmacy extemporaneously on the day of each administration.
The dose formulations were stored at room temperature and delivered to the study room in brown flasks.

Administration
The dose formulations were administered once by gavage, using a plastic syringe fitted with a plastic gavage tube.
The quantity of dose formulation administered to each animal was adjusted according to the body weight recorded on the day of dose administration. A constant dosage-volume of 10 mL/kg was used. The dose formulations were kept at room temperature and were stirred continuously throughout the dosing procedure.

Doses:

Rationale for dose-level selection
The starting dose-level was selected in agreement with the Sponsor, based on the following rationale:
. based on available test item toxicity data, no morbidity or mortality was expected to occur at the dose-level of 2000 mg/kg. This was therefore chosen as the starting dose-level.
After treatment at the starting dose-level, the next dose-levels were administered in a sequential manner, under the same conditions to different animals, based on the dose-levels indicated in the flow charts of OECD Guideline No. 423, 17th December 2001, which are equivalent to those of Commission Regulation (EC) No. 440/2008, B.1tris (30 May 2008).

No. of animals per sex per dose:

Three female animals were used at each dose-level. The time intervals between the treatment of each group were 7 days and were determined by the onset, duration and severity of any signs of toxicity. The next group was treated according to the results obtained for the previously treated group.

Control animals:

no

Details on study design:

Duration
The dose formulations were administered once followed by an observation period of 14 days. For each animal, the day of dose administration was recorded as day 1 of its observation period.

CLINICAL EXAMINATIONS

Morbidity and mortality
Each animal was checked for mortality and morbidity, frequently during the hours following administration, then at least once a day until the end of the observation period, including weekends and public holidays.

Clinical signs
Each animal was observed after treatment as follows:
. at least once during the first 30 minutes,
. periodically during the first 4 hours,
. then once a day, at approximately same time, for the recording of clinical signs.
Any clinical signs observed were recorded individually for each animal, along with the times of onset and recovery.

Body weight
The body weight of each animal was recorded the day of group allocation then on the day of treatment and on days 8 and 15.
The body weight gain of the test item-treated animals was compared to that of CiToxLAB France historical control data generated from animals of the same strain and age treated with drinking water treated by reverse osmosis under similar experimental conditions.

PATHOLOGY

Euthanasia
On completion of the observation period, animals were deeply anesthetized by an intraperitoneal injection of pentobarbital sodium and euthanized by exsanguination.

Macroscopic post-mortem examination
A macroscopic post-mortem examination was performed on all animals. After opening the thoracic and abdominal cavities, a macroscopic examination of the main organs (digestive tract, heart, kidneys, liver, lungs, pancreas, spleen and any other organs with obvious abnormalities) was performed. All gross observations were recorded individually for each animal.

Preservation of tissues
For all animals, the macroscopic lesions were preserved in 10% buffered formalin. Histological specimens (tissues in fixative) were destroyed at the finalization of the study report.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No unscheduled deaths occurred during the study.

Clinical signs:

other: No clinical signs were observed in any animals.

Gross pathology:

There were no macroscopic post-mortem findings related to test item treatment.
The macroscopic findings observed correlated with common histological findings. Thus they were
considered to be incidental.

Table 1: Body weight (BW) changes in g

	CiToxLAB France historical control data	Group 1 (2000 mg/kg/bw)	Group 2  (2000 mg/kg/bw)
BW (Day 1)	219	213	216
BW (Day 8)	264	244	255
BW (Day 15)	284	263	271
BW change (Days 1 -8)	+44	+31	+39
BW change (Days 8 -15)	+20	+19	+15
BW change (Days 1 -15)	+64	+50	+54

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions of this study, the oral LD50 of the test item, Glycerol formal, was higher than 2000 mg/kg in rats.
Therefore, the test item is not classified as toxic by oral route according to the criteria of CLP Regulation.

Executive summary:

The objective of this study was to evaluate the potential acute toxicity of the test item, Glycerol formal, following a single oral administration (gavage) to rats.

Methods

The test item, Glycerol formal (batch No. 1206191600R), was administered once by oral route (gavage) to two groups of three fasted female Sprague-Dawley rats under a dosage-volume of 10 mL/kg. The test item was prepared in drinking water treated by reverse osmosis. Based on available test item toxicity data, the starting dose-level of 2000 mg/kg was chosen. After the first assay, as no toxicity was observed, the results were confirmed in other females. Each animal was observed at least once a day for mortality and clinical signs for 15 days. Body weight was recorded on day 1 and then on days 8 and 15. On completion of the observation period, the animals were sacrificed and then submitted for a macroscopic post-mortem examination. Macroscopic lesions were preserved in buffered formalin then destroyed at the finalization of the study report as no microscopic examination was performed.

Results

No unscheduled deaths occurred during the study and no clinical signs were observed in any animals. When compared to CiToxLAB France historical control data, a trend to a slightly lower mean body weight gain was observed in 3/6 females during the first week of study and 2/6 females during the last week of the study. There were no macroscopic post-mortem findings related to test item treatment.

Conclusion

Under the experimental conditions of this study, the oral LD50 of the test item, Glycerol formal, was higher than 2000 mg/kg in rats. Therefore, the test item is not classified as toxic by oral route according to the criteria of CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

3 tests are available for acute oral toxicity endpoint:
- 1 key study performed according to OECD TG 423 and GLP with a DL50> 2000 mg/kg bw (Duclos, 2012)
- 2 supporting studies that followed no guidelines with LD50 of 8000 mg/kg and > 4000 mg/kg (Cheav et al., 1986 and Sanderson, 1958)

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP lab following OECD guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
Strain and Sanitary status: Sprague-Dawley rat, Rj Han: SD, Indemn of Organism Pathogen Specific Han (IOPS Han).
Breeder: Janvier, Le Genest-Saint-Isle, France.
Age/Weight: on the day of treatment, the animals were approximately 8 weeks old. The males had a mean body weight of 376 g (range: 370 g to 387 g) and the females had a mean body weight of 228 g (range: 218 g to 242 g).
Receipt: on arrival, the animals were given a clinical examination to ensure that they were in good condition.
Acclimation: the animals were acclimated to the study conditions for a period of 7 days (females) or 10 days (males) before treatment.
Allocation to study: the day before treatment, the required number of animals was allocated by sex to the groups according to a computerized randomization procedure. The skin of each animal (previously clipped) was examined in order to use only animals with healthy intact skin.
Identification: the day before treatment, the animals were individually identified by ear notches (unique CiToxLAB France identity number).

Environmental conditions
From arrival at CiToxLAB France, the animals were housed in a barriered rodent unit.
The animal room conditions were set as follows:
. temperature : 22 ± 2°C (see § Study plan adherence),
. relative humidity : 50 ± 20%,
. light/dark cycle : 12h/12h,
. ventilation : approximately 12 cycles/hour of filtered, non-recycled air.
The corresponding instrumentation and equipment are checked and calibrated at regular intervals. The temperature and relative humidity are recorded continuously and the records checked daily and filed.

Housing
The animals were housed by five from the same sex and group in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust (SICSA, Alfortville, France). Nylabone was given as enrichment.

Food and water
All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 2626975 (SSNIFF Spezialdiäten GmbH, Soest, Germany), and tap water (filtered with a 0.22 μm filter).

Contaminant analyses
The batches of diet and sawdust were analyzed by the Suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants that could have interfered with or prejudiced the outcome of the study were found in the diet, drinking water or sawdust.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

Preparation of the animals
On the day before treatment, an area representing approximately 10% of the total body surface area (i.e. approximately 7 cm x 5 cm for males and 6 cm x 5 cm for females (1)) was clipped on the back of each animal using an electric clipper. Care was taken to avoid abrading the skin. Only animals with healthy intact skin were used for the study.

(1) The total body surface is calculated according to Meeh's formula: S = k.p2/3 where S = body surface (cm2), p = body weight (g), k = 7.47 for rat [Diack, S L. (1930). The determination of the surface area of the white rat. J. Nutr. 3(3): 289-296].

Duration of exposure:

The dose formulations were applied once followed by an observation period of 14 days. For each animal, the day of treatment was recorded as day 1 of its observation period.

Doses:

A limit test was carried out by application of 2000 mg/kg to five females in the first instance.
As no mortality occurs, the limit test was completed using five males. As no mortality also occurs in males, the study was considered as complete.

No. of animals per sex per dose:

5

Control animals:

not required

Details on study design:

Application
The test item was applied as a film (as thin and uniform as possible) to the clipped area of skin. The application site was covered with a hydrophilic gauze pad.
The quantity of test item applied to each animal was adjusted according to the body weight recorded on the day of dose application.
The gauze pad was held in place with an aerated hypoallergic dressing for 24 hours to avoid ingestion of the test item by the animals.
No residual test item was noted after removal of the dressing.

CLINICAL EXAMINATIONS
Morbidity and mortality
Each animal was checked for mortality and morbidity, frequently during the hours following treatment, then once a day until the end of the observation period, including weekends and public holidays.

Clinical signs
Each animal was observed after treatment as follows:
. at least once during the first 30 minutes,
. periodically during the first 4 hours,
. then once a day, at approximately same time, for the recording of clinical signs.
Any clinical signs observed were recorded individually for each animal, along with the times of onset and recovery.
From day 2, any local reactions at the treatment site were recorded.

Body weight
The body weight of each animal was recorded the day of group allocation then on the day of treatment and on days 8 and 15.
The body weight gain of the test item-treated animals was compared to that of CiToxLAB France historical control data generated from animals of the same strain and age treated with drinking water treated by reverse osmosis under similar experimental conditions.

Statistics:

not necessary as a limit test was performed.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No unscheduled deaths occurred during the study.

Clinical signs:

other: No clinical signs indicative of systemic toxicity were observed in any animals. A well-defined erythema was noted at the application site on day 2 and then a very slight erythema associated with a very slight dryness was observed from day 3 to day 6 in 1/

Gross pathology:

There were no macroscopic changes at necropsy.

Table 1 - body weight evolution

	CiToxLAB France historical control data (FEMALE)	Females exposed at 2000 mg/kg	CiToxLAB France historical control data (MALE)	Males exposed at 2000 mg/kg
BW (day 1)	228	228	332	376
BW (day 8)	264	246	377	418
BW (day 15)	283	265	422	419
BW change (days 1 - 8)	+36	+18	+45	+41
BW change (days 8 - 15)	+18	+19	+45	+2
BW change (days 8 - 15)	+55	+37	+90	+43

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

higher than 2000 mg/kg in rats.
Therefore, the test item should not be classified as toxic by dermal route according to the criteria of CLP
Regulation.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following a single dermala pplication to rats.

Methods

The test item, Glycerol formal, was applied in its original form to the skin of five female then five male Sprague-Dawley rats at the dose-level of 2000 mg/kg. The application site was covered by a semi-occlusive dressing for 24 hours.

Each animal was observed at least once a day for mortality and clinical signs for 15 days. From day 2, any local reactions at the treatment site were also noted. Body weight was recorded on day 1 and then on days 8 and 15.

On completion of the observation period, the animals were sacrificed and then submitted for a macroscopic post-mortem examination. No tissues were preserved.

Results

No unscheduled deaths occurred during the study and no clinical signs indicative of systemic toxicity were observed in any animals.

A well-defined erythema was noted at the application site in 1/5 females on day 2 then a very slight erythema associated with a very slight dryness was observed from day 3 to day 6.

No cutaneous reactions were observed in any males during the study period.

When compared to CiToxLAB France historical control data, lower mean body weight and mean body weight gain were recorded in both sexes during the study.

No macroscopic changes were observed at necropsy.

Conclusion

Under the experimental conditions of this study, the dermal LD50 of the test item, Glycerol formal, was higher than 2000 mg/kg in rats. Therefore, the test item should not be classified as toxic by dermal route according to the criteria of CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Good quality as the test was performed according to OECD TG 402 and GLP.

Additional information

Oral route

Under the experimental conditions of an OECD 423 and GLP study, the oral LD50 of the test item, Glycerol formal, was higher than 2000 mg/kg in rats (Duclos, 2012). Therefore, the test item is not classified as toxic by oral route according to the criteria of CLP Regulation.

Dermal route

Under the experimental conditions of an OECD 402 and GLP study, the dermal LD50 of the test item, Glycerol formal, was

higher than 2000 mg/kg in rats (Duclos, 2012). Therefore, the test item should not be classified as toxic by dermal route according to the criteria of CLP Regulation.

Other route

Intraperitoneal route

Sanderson (1959) stated that the toxicity of glycerol formal appeared to be low after intraperitoneal injection to rats with a DL50 estimated as approximately 3000 mg/kg. Intraperitoneal injection of 1000 mg/kg was given to two male guinea pigs and to six male albino mice causing no apparent toxic effectsor macroscopic pathology (DL50>1000 mg/kg).

Subcuaneous route

Rats treated with 1000 mg/kg subcutaneously were completely unaffected, and showed no evidence of local irritation at the injection site. DL50 is >1000 mg/kg (Sanderson, 1959).

Justification for selection of acute toxicity – oral endpoint

This study was selected as the most reliable one (OECD TG 423 and GLP) and is supported by 2 studies that followed no guidelines.

Justification for selection of acute toxicity – inhalation endpoint

In accordance with column 2 of REACH Annex VIII relative to Acute toxicity (Point 8.5), in addition to the oral route (8.5.1), for substances other than gases, the information mentioned under 8.5.2 (acute toxicity by inhalation) to 8.5.3 (acute toxicity by derma route) shall be provided for at least one other route. The choice for the second route will depend on the nature of the substance and the likely route of human exposure. If there is only one route of exposure, information for only that route need be provided.

Testing by dermal route is appropriate regarding to the test substance exposure and physicochemical and toxicological properties.

Testing by inhalation is not appropriate.

Justification for selection of acute toxicity – dermal endpoint

This study was selected as the only one available.

Justification for classification or non-classification

With a LD50 > limit dose in acute oral and dermal toxicity test, glycerol formal is not classified.