3-methyl-1,5-pentanediyl diacrylate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September -October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Version / remarks:

September, 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1,5-pentanediyl diacrylate

EC Number:

264-727-7

EC Name:

3-methyl-1,5-pentanediyl diacrylate

Cas Number:

64194-22-5

Molecular formula:

C12H18O4

IUPAC Name:

3-methyl-5-(prop-2-enoyloxy)pentyl prop-2-enoate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 9 weeks
- Weight at study initiation (mean): Group 1: males: 271.33 g; females: 174.43 g. Group 2: males: 275.80 g; females: 175.27 g
- Housing: except for the exposure period, animals were housed three animals to a cage, separated by sex, in Macrolon cages. Wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany) were used as bedding material and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a piece of gnaw wood (ABEDD, Vienna, Austria) were provided as environmental enrichment. During exposure, animals were housed individually in the inhalation exposure units. Animals were immediately returned to their home cages after exposure.
- Diet: cereal-based (closed formula) rodent diet (VRF1) from a commercial supplier (SDS Special Diets Services, Witham, England); available ad libitum (except during exposure)
- Water: domestic mains tap-water suitable for human consumption; available ad libitum (except during exposure)
- Acclimation period: Experiment/Group 1 (5 g/m3): 3 weeks; Experiment/Group 2 (1 g/m3): 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.79 - <= 3.23 µm

Geometric standard deviation (GSD):

>= 2.17 - <= 2.38

Remark on MMAD/GSD:

For group 1, the MMAD was 3.04 and 3.23 µm with a GSD of 2.23 and 2.28, respectively.
For group 2, the MMAD was 2.98 and 2.79 µm with a GSD of 2.17 and 2.38, respectively.

Details on inhalation exposure:

EXPOSURE EQUIPMENT
The animals were exposed to the test atmosphere in a nose-only inhalation chamber manufactured by Versteeg Verspaningstechniek B.V. (Maarsbergen, the Netherlands) as a modification of the chamber manufactured by ADG Developments Ltd. (Codicote, Hitchin, Herts, SG4 8UB, United Kingdom). The inhalation chamber consisted of a cylindrical stainless steel column, surrounded by a transparent cylinder. The column had a volume of 47.6 liters and consisted of a top assembly with the entrance of the unit, two mixing sections, a rodent tube section and at the bottom the base assembly with the exhaust port. The rodent tube section had 20 ports for animal exposure. Several empty ports were used for test atmosphere sampling (for analysis of the actual concentration and particle size) and measurement of oxygen, carbon dioxide, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Male and female rats were placed in alternating order. Unused ports were closed. Only the nose of the rats protruded into the interior of the column. Habituation to the restraint in the animal holders was not performed because, based on previous experience, habituation does not help to reduce possible stress. In the experience of the laboratory, the animal’s body does not exactly fit in the animal holder which always results in some leakage from high to low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder, which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animals’ thorax to the nose was avoided. The unit was illuminated externally by normal laboratory fluorescent tube lighting. The total air flow through the unit was at least 1 litre/min for each rat. The air temperature and relative humidity in the unit were maintained at 22 ± 3°C and between 30 and 70%, respectively.

GENERATION OF THE TEST ATMOSPHERE
The inhalation equipment is designed to expose the rats to a continuous supply of fresh test atmosphere. To generate the test atmosphere, the test material was nebulized using an airdriven atomizer (Schlick type 970/S, Coburg, Germany), which was placed at the top inlet of the exposure chamber. From there, the aerosol was directed downward and led to the noses of the animals. At the bottom of the unit, the test atmosphere was exhausted. The animals were placed in the exposure unit after stabilization of the test atmosphere (T95/time to attain chamber equilibration was about 2.5 - 3 minutes). The period between the start of the generation of the test atmosphere and the start of exposure of the animals was 17 minutes for group 1 and 20 minutes for group 2. Test atmosphere generation was done in an illuminated laboratory at room temperature.

MONITORING OF EXPOSURE CONDITIONS
- Actual concentration: During preliminary experiments, the evaporation of test material from gravimetric filters (added to the filter in several droplets) was investigated and was found to be extremely low (around 1% in 24 hours). Based on the available data on molecular weight and vapor pressure, the test material concentration in the test atmosphere present as vapor could theoretically reach a maximum of 0.01 g/m3. Therefore, the contribution of vapor to the test atmosphere was minimal. The actual concentration of the test material in the test atmosphere was determined six times during exposure of group 1 and eight times during exposure of group 2 by means of gravimetric analysis. Representative test atmosphere samples were obtained by passing approximately 5 L (group 1) or 10 L (group 2) test atmosphere at 5 L/min through fibre glass filters (Sartorius 13400-47, Ø 47 mm). Filters were weighed before sampling, loaded with a sample of test atmosphere, and were then weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter by the volume of the sample taken.
- Time to attain chamber equilibration (T95): The time to reach 95% of the steady state concentration (T95) was calculated as: 3V/F. This calculation follows from the formula C = C8 * (1 – e-(F*T/V)), describing the increase in concentration C in a perfectly stirred chamber with volume V [L] and flow F [L/min], where T [min] is the time and C8 is the steady state concentration.
- Nominal concentration and generation efficiency: The nominal concentration was determined by dividing the total amount of test material used (by weight) by the total volume of air passed through the inhalation chamber. The generation efficiency was calculated from the actual concentration (determined by gravimetric analysis) and the nominal concentration (efficiency = actual concentration as percentage of nominal concentration).
- Particle size measurement: For each of the two exposure sessions, three particle size distribution measurements were carried out (one during preliminary generation and two during the exposure) using a cascade impactor. The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation of the distribution of particle sizes (GSD) was calculated (Lee, 1972).
- Total air flow, temperature, relative humidity, oxygen and carbon dioxide concentration: The chamber air flow, temperature and relative humidity of the test atmosphere were recorded eight times during exposure. The flow of dry compressed air that was used to nebulize the test substance was measured using a Mass View Meter; the bypass flow of humidified air was measured using a Mass Flow Controller. The temperature and relative humidity were measured using an RH/T device (TESTO type 0636 9735 probe with 635-1 read-out unit, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany). The oxygen (Oxygen analyser type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70 probe with MI70 read-out unit, Vaisala, Helsinki, Finland) concentrations were measured once.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

EXPERIMENT 1
- Target concentration: 5 g/m3
- Actual concentration: 5.14 (± 0.10) g/m3
- Nominal concentration: 10.4 g/m3

EXPERIMENT 2
- Target concentration: 1 g/m3
- Actual concentration: 1.05 (± 0.11) g/m3
- Nominal concentration: 1.8 g/m3

No. of animals per sex per dose:

- Experiment 1: 3 males, 3 females
- Experiment 2: 3 males, 3 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for clinical signs just before exposure, four times during exposure (about once per hour), and twice after exposure. During the observation period, each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. The body weight of each animal was recorded once during the acclimatization period (on day -1 for group 1 and on day -3 for group 2) and on days 0 (just before exposure) and 1 for both groups; on days 3, 4, 5, 6, 7 and on day 14 prior to necropsy for group 2 only. Due to the high mortality in group 1, body weight could only be determined for this group until day 1 post-exposure and for group 2 additional body weight measurements were made on days 4, 5 and 6 post-exposure.
- Necropsy of survivors performed: yes, surviving animals were sacrificed for necropsy 14 days after exposure by exsanguination from the abdominal aorta under sodium pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital). At necropsy, abdominal and thoracic organs were examined in situ. Animals from group 1 that were in moribund condition on the day after exposure were sacrificed in the same way as described above. These two animals and the four animals from group 1 that died during the first night after exposure, were subjected to post-mortem examination as soon after death as possible.

Statistics:

no

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

>= 1 050 - <= 5 140 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other:

Remarks:

corresponding to 1.05 and 5.14 mg/l

Mortality:

Experiment 1:
- All animals survived the exposure. During the night after the exposure, two males and two females died. The health situation of the remaining two animals had deteriorated compared to the day before, so they were humanely sacrificed.

Experiment 2:
- All animals survived the exposure.

Clinical signs:

other: See 'Any other information on results incl. tables'

Body weight:

Experiment 1:
- Because of the death of four out of six animals before the body weight determination on day 1, the post-exposure body weight change could only be determined for one male and one female animal. The surviving animals lost around 10-11% body weight. Due to the humane sacrifice of both of these animals on day 1, no further body weight data are available.

Experiment 2:
- On day 1 post-exposure, the average weight loss compared to the body weight pre-exposure was 9.4% for males and 5.9% for females. The animals’ weight continued to diminish until day 3 or 4, where it reached its lowest value; the average weight loss on day 3 was 17.5% for males and 11.0% for females, compared to the pre-exposure values. On day 4, all females regained weight while for male animals, the average weight showed a minimal increase. From day 5 onwards, all animals regained weight. Females recovered from the pre-exposure weight loss on day 6, while for males it took more than a week to reach a body weight that was equal to or higher than the pre-exposure weight. Although a relatively small and transient decrease in weight after exposure is commonly observed in acute inhalation studies (because of the restraint during exposure), it is likely that the exposure to 3-Methyl-1,5-pentanediyl diacrylate had an effect on the body weight loss observed in group 2 for male animals especially.

Gross pathology:

Experiment 1:
- In the male and female that survived the night after exposure, subcutaneous edema and gastrointestinal distension were found. The latter finding was likely caused by the gasping for air, which causes air to enter the gastrointestinal tract. Subcutaneous edema and gastrointestinal distension were also observed upon macroscopic examination of the four animals that were found dead. In addition, two of these animals showed nasal discharge.

Experiment 2:
- No major pathological lesions, indicative of marked toxicity of the test material, were observed. The only finding was a single red spot on the cranial lung lobe of one male animal. This is an isolated finding and should thus be considered as background pathology. In general, animals appeared to have recovered from the exposure to 1.05 g/m3 test material.

Other findings:

- Time to attain chamber equilibration: based on a chamber volume of 47.6 L and airflows of 50.3 and 50.2 L/min for groups 1 and 2 respectively, the time to reach 95% of the steady state concentration (T95) was about 2.5-3 minutes for both groups.
- Total air flow, temperature, relative humidity, oxygen and carbon dioxide concentration: the average chamber airflow was 50.3 L/min for group 1 and 50.2 L/min for group 2. The mean temperature in the exposure chamber during exposure was 21.2 ± 0.1°C (range 21.1– 21.3°C) and the relative humidity was 37.7 ± 3.4% (range 34.2 – 44.2%) for group 1; for group 2, measurements indicated values of 21.3 ± 0.1°C (range 21.1– 21.5°C) for the temperature and 38.2 ± 2.9% (range 34.9 – 43.1%) for relative humidity. The oxygen concentration, measured once during each exposure period, was 20.7% (v/v) for both groups, while the CO2 concentration was 0.103% (v/v) for group 1 and 0.122% (v/v) for group 2.

Any other information on results incl. tables

CLINICAL SIGNS

- Experiment 1:

All animals showed breathing abnormalities on all observation time points during exposure. When animals were first observed around one hour after the start of exposure, moderately decreased breathing rate and moderate shallow breathing were noted in all animals. In five out of six animals, the decrease in breathing frequency became severe as the exposure continued, while for two animals, the observed shallow breathing became severe. Also, towards the end of the exposure, five out of six animals were moderately restless. The last animal showed a slight restlessness only. Shortly after exposure, breathing abnormalities persisted (grunting, decreased rate, dyspnoea, sniffing), and in addition a large range of other clinical signs was observed. The most noticeable of these was erythema on the face and feet in all animals. But also, low body temperature (6/6), muscle weakness (4/6), hunched posture (6/6), piloerection (6/6), eye blepharospasm (4/6) and tremors (2/6) were observed. When animals were observed again at the end of the day, they no longer felt cold and they no longer showed a hunched posture. However, abdominal distension was observed in two males and one female, while hypoactive behavior, swollen nose and nasal discharge were observed in four, three and five animals, respectively.

- Experiment 2:

During exposure, all animals showed slight to moderate breathing abnormalities characterized by a decreased breathing rate and shallow breathing. Also, all animals were slightly lethargic during the last observation time point. Shortly after exposure, dyspnoea and piloerection observed in all animals. Erythema was observed on the paws and around the noses of five out of six animals. One animal was grunting and another displayed a hunched posture. At the end of the day, similar clinical signs were observed. During the post-exposure period, hemorrhagic nasal discharge was observed until day 6 in five out of six animals. Also, for all animals piloerection, sniffing and dyspnoea were observed. These signs were no longer seen from day 4 (piloerection), day 7 (sniffing), or day 10 (dyspnoea). Finally, one male and one female were found to breathe through their mouths on days 3 to 4, one male and one female displayed a hunched posture on days 1 to 4 and one male had nasal encrustations on day 7.

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The 4-hour LC50 was determined to be between 1.05 and 5.14 g/m3.

Executive summary:

A GLP-compliant acute inhalation toxicity study was performed according to OECD guideline 436, where 3 male and 3 female Wistar rats were exposed nose-only to a target concentration of aerosols of 5 g/m3 for 4 hours. Two male and 2 female animals died during the first night after exposure and the 2 remaining animals were humanely sacrificed because of their very poor condition. Therefore, a second group (three males, three females) was exposed to a lower target concentration of 1 g/m3. The animals were kept for an observation period of 14 days. To detect adverse effects, clinical observations were made and body weights were determined before, during and after exposure. Furthermore, animals were examined for gross pathological changes at the end of the observation period. The mean actual concentration was determined to be 5.14 (±0.10) in the first and 1.05 (± 0.11) g/m3 in the second experiment.

In the first experiment (5.14 g/m3) animals showed moderate to severe breathing abnormalities during exposure. Directly after exposure, animals showed a number of clinical signs, including erythema of the skin on the paws and face, low body temperature, muscle weakness, hunched posture, piloerection, erythema of the skin, blepharospasm and tremors. At the end of the day, they no longer felt cold and they no longer showed a hunched posture. However, abdominal distension was observed in two males and one female, while hypoactive behavior, swollen nose and nasal discharge were observed in four, three and five animals, respectively. During the night, two males and two females died. Due to the poor condition of the surviving animals (clinical signs observed included severe breathing impairment, hunched posture, ataxia, blepharospasm, hypoactive behavior), they were humanely sacrificed. Upon macroscopic examination, subcutaneous edema and gastrointestinal distension were noted in all six animals. Also, in two animals nasal discharge was noted.

In the second experment (1.05 g/m3), all animals survived the exposure. During exposure, all animals showed slight to moderate breathing abnormalities, and all were slightly lethargic at the last observation time point. Shortly after exposure, dyspnoea and piloerection were observed in all animals. Erythema was observed on the paws and around the noses of five out of six animals. Also, one animal was grunting and another displayed a hunched posture. At the end of the day, similar signs were observed. During the post-exposure period, all animals showed piloerection, sniffing and dyspnoea while for five out of six, hemorrhagic nasal discharge was observed. These signs were resolved on day 4, 7, 10 and 7, respectively. Finally, one male and one female were found to breathe through their mouths on days 3 to 4, one male and one female displayed a hunched posture on days 1 to 4 and one male had nasal encrustations on day 7. After exposure, weight loss was observed in all animals. On day 3, the lowest values were observed; males lost 17.5% and females lost 11.0% compared to the pre-exposure values. After day 3, animals regained weight. Females took six days to recover from the weight loss, while males took more than a week to regain their pre-exposure body weight. No exposure-related pathology was observed in animals from group 2.

The LC50 in rats after 4 hours of inhalation exposure was determined to be between 1.05 and 5.14 g/m3. The test material should be classified into GHS category 4.