mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Endpoint summary

Description of key information

Acute Toxicity: Oral Route: Cummins (1990)

Under the conditions of the study, the combined acute oral LD50 for male and female CD rats was 775 mg/kg (95 % confidence limits of 666 – 885).  The test material was considered acutely toxic via the oral route (Category 4).

Acute Toxicity: Oral Route, Supporting Study: Dange (1994)

Under the conditions of this study, the acute oral LD50 of the test material was determined to be 431 mg/kg bw in male and female Sprague-Dawley rats.

Acute Toxicity: Inhalation Route: Cracknell (1990)

Under the conditions of this study, there were no deaths following the exposure for four hours of five male and five female rats to the maximum chamber concentration that could be generated using the methods described in this report. The median lethal chamber concentration for four hours' exposure (LC50 4-hour) is therefore greater than 0.87 mg per litre of air.

Acute Toxicity: Inhalation Route: Coombs (1977)

Under the conditions of this study, the acute inhalation LC50 was > 2.21 g/m^3.

Acute Toxicity: Dermal Route: Cummins (1990)

Under the conditions of this study, the acute percutaneous median lethal dosage (LD50) of the test material was greater than 2 000 mg/kg.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 February 1990 to 29 March 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA Pesticide Assessment Guidelines

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Fresh formulations of the test material were prepared on the morning of administration and any surplus remaining after dosing was destroyed on the same day. No analyses were undertaken to determine the stability, homogeneity or achieved concentrations of the test material in the vehicle.

Species:

rat

Strain:

CD-1

Remarks:

Remote Sprague-Dawley origin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately four weeks old on arrival. The animals were approximately five weeks old at dosing.
- Weight at study initiation: Pre-fasted bodyweight was recorded on the day prior to dosing and ranged for males from 105 - 144 g and for females from 92 - 132 g.
- Fasting period before study: Approximately 18 hours before administration of the test material.
- Housing: Five animals of the same sex were accommodated in each stainless steel grid cage, unless reduced by mortality.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: An acclimatisation period of at least six days was allowed between arrival at the laboratory and administration of the test material. A daily check on the general condition of the animals was made during this time and the record was consulted before each animal was accepted for use in the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target value for temperature was 21 °C (range 18 - 25 °C)
- Humidity (%): Target value for relative humidity was 55 % R.H. (range 40 % - 70 % R.H.)
- Air changes (per hr): Approximately 15 complete air changes per hour without re-circulation.
- Photoperiod (hrs dark / hrs light): 12 hours of artificial light per day.

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % w/v methylcellulose in distilled water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: The test material was prepared at appropriate concentrations in 0.5 % w/v methylcellulose in distilled water. Dosages were calculated and expressed gravimetrically in terms of the material as received.

MAXIMUM DOSE VOLUME APPLIED
- A constant volume-dosage of 20 mL/kg bodyweight.

Doses:

450, 567, 714 and 900 mg/kg bw.

No. of animals per sex per dose:

Five per sex per dose with the exception of the 450 mg/kg group in which only 4 females were dosed, as one female animal was culled prior to dosing, and no replacement was available.

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Three separate inspections were made during the first hour after dosing and two further inspections during the remainder of Day 1. From Day 2 onwards, the animals were inspected twice daily (morning and afternoon). The type, time of onset and duration of reactions to treatment and the circumstances of any death were recorded.
The bodyweight of each animal was recorded on the day before dosing and on Days 1, 8 and 15. The test was terminated on Day 15.
- Necropsy of survivors performed: Yes. Carcases were stored in a refrigerator at approximately 4 °C until trained necropsy staff were available. All decedents were weighed and thoroughly examined at necropsy for abnormality of tissues or organs. All body cavities were opened, larger organs were sectioned and the gastro-intestinal tract was opened at intervals for examination of the mucosal surfaces. All abnormalities were described or the normal appearance of major organs was confirmed. Animals surviving to termination of the study or sacrificed during the observation period were killed by carbon dioxide inhalation and were examined at necropsy by the same procedure.
- Clinical signs including body weight: As above.
- Other examinations performed: Clinical signs, body weight.

Statistics:

Probit analysis by the method of Finney (1952) was used to determine the acute median lethal dosage, 95 % confidence interval and slope of the dose response curve of the test material for both sexes. The calculations were performed by the GLIM statistics program (Baker and Nelder, 1978) using a special macro program developed by Baker (Baker, 1980).

Preliminary study:

A preliminary study was carried out using three groups of one male and one female rat given a single oral administration of the test material at dosages of 450, 900 or 1 800 mg/kg bodyweight, at a volume-dosage of 20 mL/kg in 0.5 % w/v methylcellulose in distilled water. Both animals treated at 1 800 mg/kg died.
On the basis of this result the main study was carried out using four groups of five male and five female rats given a single oral administration of the test material within the range 450 - 900 mg/kg bodyweight.
One female animal destined to receive 450 mg/kg was in an unsatisfactory condition before dosing on Day 1 and therefore culled. There was no replacement rat available.

Sex:

male

Dose descriptor:

LD50

Effect level:

803 mg/kg bw

Based on:

test mat.

95% CL:

>= 710 - <= 896

Sex:

female

Dose descriptor:

LD50

Effect level:

756 mg/kg bw

Based on:

test mat.

95% CL:

>= 651 - <= 861

Sex:

male/female

Dose descriptor:

LD50

Effect level:

775 mg/kg bw

Based on:

test mat.

95% CL:

>= 666 - <= 885

Mortality:

Animals died at dosages of 567 mg/kg and above. The deaths occurred on Days 2, 3 or 4.
One female destined to receive 450 mg/kg was found to be in an unsatisfactory condition before dosing on Day 1, and was humanely killed. No suitable replacement animal was available.

Clinical signs:

other: Ante mortem signs comprised lethary, unconsciousness, decreased motor activity, prone posture, ataxia, clonic convulsion, muscle tremor, bradypnoea, tachypnoea, hyperpnoea, hypopnoea, ungroomed appearance, pigmented orbital secretion and hunched posture.

Gross pathology:

Necropsy of the decedents revealed isolated incidences of altered stomach and jejunum contents, dark thymic lymph nodes and pale perineal staining. Necropsy of the surviving animals, on Day 15, revealed no significant macroscopic lesion.

Interpretation of results:

other: Classified according to EU criteria Category 4

Conclusions:

Under the conditions of the study, the combined acute oral LD50 for male and female CD rats was 775 mg/kg (95 % confidence limits of 666 – 885). The test material was considered acutely toxic via the oral route (Category 4).

Executive summary:

The acute oral toxicity of the test material was assessed according to OECD Test Guideline 401 and in compliance with GLP.

The study consisted of four groups of five male and five female CD rats. The animals were starved overnight prior to dosing. The test material was administered on Day 1 at dosages in the range 450 - 900 mg/kg, at a volume-dosage of 20 mL/kg in 0.5 % w/v methylcellulose in distilled water.

Mortality and signs of reaction to treatment were recorded during a subsequent 14-day observation period. Decedents and animals killed on Day 15 were subjected to necropsy.

The mortality distribution was as follows:

450 mg/kg group: 0/5 males and 0/4 females. One female was in unsatisfactory condition before dosing on Day 1; no suitable replacement was available so only four were dosed.

567 mg/kg group: 0/5 males and 1/5 females.

714 mg/kg group: 1/5 males and 0/5 females.

900 mg/kg group: 4/5 males and 5/5 females.

The deaths occurred on Days 2, 3 or 4.

Ante mortem signs comprised lethargy, unconsciousness, decreased motor activity, prone posture, ataxia, clonic convulsion, muscle tremor, breathing irregularities, ungroomed appearance, pigmented orbital secretion and hunched posture.

Signs of reaction to treatment in surviving animals were similar to those of the decedents with the exception of unconsciousness and clonic convulsion and with the addition of blanching and thin body conformation. The majority of animals were overtly normal on Day 6 and throughout the remainder of the observation period. Animals treated at 450 mg/kg were less severely affected than those treated at higher dosages.

The surviving animals generally achieved expected bodyweight gains.

Necropsy of the decedents revealed isolated incidences of altered stomach and jejunum contents, dark thymic lymph nodes and pale perineal staining. Necropsy of the surviving animals revealed no significant macroscopic lesion.

Under the conditions of this study the acute oral median lethal dosage (LD50) was:

Male: LD50 803 mg/kg, 95 % confidence limits of 710 – 896 (slope 83 degrees).

Female: LD50 756 mg/kg, 95 % confidence limits of 651 – 861 (slope 80 degrees).

Combined: LD50 775 mg/kg, 95 % confidence limits of 666 – 885 (slope 75 degrees).

Accordingly, under the conditions of the study, the test material was considered acutely toxic via the oral route (Category 4).

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 1994 to 29 April 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 81-1 (Acute Oral Toxicity)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 weeks old on arrival.
- Weight at study initiation: Selected animals were in a weight range from 306 to 365 g for the males and 185 to 226 g for the females on the day of treatment (i.e. within ± 20 % of the mean body weight).
- Fasting period before study: All animals were fasted overnight before test substance administration.
- Housing: Rats were housed individually in suspended stainless steel, wire mesh cages.
- Diet: Certified Rodent Pellet diet ad libitum.
- Water: Tap water from the municipal water supply, filtered and softened, ad libitum.
- Acclimation period: 13 days
- Method of randomisation in assigning animals to test and control groups: On the day before treatment, animals were assigned permanent identification numbers within groups using a randomisation procedure that ensures a similar body weight distribution among groups for each sex.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 15 %
- Air changes: 15 air changes per hour (average, not monitored).
- Photoperiod: 12-hour light, 12-hour dark cycles.

IN-LIFE DATES:
From: 16 March 1994
To: 20 April 1994

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % methylcellulose in distilled water

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

DOSAGE PREPARATION: The dosing formulation was prepared by suspending the test material in 0.5 % methylcellulose in distilled water to produce the required dosing concentration (w/w). The formulation was used as quickly as practicable after preparation.

Doses:

100, 180, 320 and 580 mg/kg

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were checked for clinical signs, moribundity and mortality approximately one hour after dosing and at least once more on Day 1. Thereafter, observed clinical signs were recorded at least once daily. The nature, onset, severity, reversibility and duration of clinical signs were recorded. Cages and cage-trays were inspected daily for evidence of ill-health, such as blood or loose faeces. In addition, animals were checked daily a second time for moribundity and mortality, except on weekends when they were checked once daily.
Each animal was weighed once during the acclimatisation period, on the day of test material administration then on Days 8 and 15.
- Necropsy of survivors performed: Yes. All animals were autopsied. At final sacrifice, surviving animals were anaesthetised by intraperitoneal injection of pentobarbital, then exsanguinated under deep anaesthesia before necropsy. Necropsy included macroscopic examination of abdominal and thoracic cavities, major organs and tissues. Significant macroscopic abnormalities were recorded.

Statistics:

LD50 values were calculated separately for each sex and for combined sexes from total mortality data using the method of Dragstedt-Lang.
For body weights and body weight gains, means and standard deviations were calculated.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

431 mg/kg bw

Based on:

test mat.

Mortality:

No deaths occurred at the 100, 180 and 320 mg/kg dose levels. All animals died at the 580 mg/kg dose level. All animals were found dead on Day 3 except one male which died on Day 8 and one female on Day 4.

Clinical signs:

other: There were no clinical signs in animals which received 100 mg/kg body weight. At 180 and 320 mg/kg, piloerection, reduced motor activity and hunched posture were observed on Days 1 and 2. From Day 3 all animals recovered and no clinical signs were observe

Gross pathology:

No treatment-related changes were observed at necropsy.

Mortality Levels

Dose Levels (mg/kg)	Number of Animals
	Males Dead / tested	Females Dead / tested	Combined Dead / tested
100	0 / 5	0 / 5	0 / 10
180	0 / 5	0 / 5	0 / 10
320	0 / 5	0 / 5	0 / 10
580	5 / 5	5 / 5	10 / 10

 

Interpretation of results:

other: Classified according to EU criteria Category 4

Conclusions:

Under the conditions of this study, the acute oral LD50 of the test material was determined to be 431 mg/kg bw in male and female Sprague-Dawley rats.

Executive summary:

The potential for the test material to cause acute toxicity via the oral route was investigated in a study conducted in accordance with the standardised guidelines OECD 401 and US EPA OPP 81-1 and in compliance with GLP.

The acute oral toxicity was assessed after a single oral administration of the test material at 100, 180, 320 and 580 mg/kg body weight in groups of five male and five female Sprague Dawley rats. All animals were observed daily tor fifteen days and their body weight measured weekly. At termination of the study period or when found dead, animals were subjected to a necropsy.

No animal died at 100, 180 and 320 mg/kg body weight. All animals died at 580 mg/kg. No clinical signs were noted at 100 mg/kg body weight. At 180 and 320 mg/kg body weight, piloerection, hunched posture and reduced motor activity were observed only during Days 1 and 2. At 580 mg/kg body weight, piloerection, dyspnoea, prostration, absence of traction and of grasping reflex, muscular atony, reduced motor activity and unconsciousness were the major clinical signs observed before the death of the animals. The body weight evolution of the animals which survived the study period was unaffected by treatment. No treatment-related changes were observed at necropsy.

Under the conditions of this study, the acute oral LD50 of the test material was determined to be 431 mg/kg bw in male and female Sprague-Dawley rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

431 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 January 1990 to 15 February 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Qualifier:

according to guideline

Guideline:

EPA OPP 81-3 (Acute inhalation toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

traditional method

Limit test:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The sub-sample of test material used during this study was transferred to an amber glass screw top container after milling.

Species:

rat

Strain:

other: CD strain (remote Sprague-Dawley origin)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: On arrival the rats were about 6 to 11 weeks old. At the time of dosing the animals were about seven to 12 weeks old.
- Weight at study initiation: On arrival the male rats were within the bodyweight range of 182 to 200 g and the females 191 to 212 g. At the time of dosing the male rats were within the bodyweight range 259 to 289 g and females from 243 to 254 g.
- Fasting period before study: Animals were not fed during the exposure procedure.
- Housing: The rats were housed five of one sex per cage. Each cage consisted of a high-density polypropylene body measuring 56 x 38 x 18 cm with a stainless-steel lid and floor. The cages were suspended above absorbent crepe paper which was changed daily.
- Diet: A commercially available complete pelleted rodent diet was fed ad libitum. This was an expanded diet which contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Drinking water was taken from the public supply, controlled by the East Anglian Water Company, Lowestoft, Suffolk, England and offered ad libitum to each cage in polyethylene bottles with sipper tubes.
- Acclimation period: 8 days
- Microbiological status when known: The animals had been bred under barriered conditions and travelled from the supplier to the animal-holding room in sealed boxes with filter protected air-vents.

ENVIRONMENTAL CONDITIONS
- Temperature: Target value 21° (18 to 25 °C)
- Humidity: Target value 55 % RH (40 to 70 % RH)
- Air changes: The room was kept at positive pressure with respect to the outside and had its own supply of filtered, fresh air which was passed to atmosphere and not re-circulated. Ventilation equipment was designed to provide approximately 15 air changes per hour.
- Photoperiod: Electric time switches regulated a lighting cycle of 12 hours of artificial light per day.

IN-LIFE DATES
From: 21 January 1990
To: 15 February 1990

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

snout only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

6.31 µm

Geometric standard deviation (GSD):

>= 2.39 - <= 10.23

Remark on MMAD/GSD:

The small percentage of inhalable particles was considered to be associated with the difficulties encountered during milling of the waxy, low melting point test material. The percentage of the atmosphere that was less than 4 μm EAD was 37 %.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure chamber consisted of 30 cm diameter aluminium alloy cylinder comprised of modules assembled to give a volume of ca. 60 litres. The cylinder incorporated three animal exposure sections each having 20 exposure ports (ADG Instruments Ltd, Codicote, Hitchin, Herts, England). The exposure chamber and generation apparatus were positioned in a large cabinet equipped with an extract fan exhausting to atmosphere through a collection filter.
- Exposure chamber volume: ca. 60 litres
- Method of holding animals in test chamber: Each rat was placed in an individual polycarbonate restraining tube so that only the snout protruded. The restraining tubes were attached to the chamber so that only the snout projected into the chamber.
- Source and rate of air (airflow): A dust feed mechanism (Wright, 1950, 1963) mounted into the top of the chamber, was used to produce the test atmosphere. The mechanism was designed to produce and maintain an atmosphere containing dust by suspending material scraped from the surface of a compressed powder in a stream of dry air. The concentration of dust suspended in the airstream could be varied by changing the gear ratio of the generator mechanism. Dry, oil free compressed air was passed through the mechanism to give a flow rate of 25 L/min.
- System of generating particulates/aerosols: It was not possible to generate atmospheres containing the test material as supplied. Mechanical milling by ultracentrifugal mill and by crossbeater mill resulted in the formation of a waxy solid on the milling screens. As a consequence of this characteristic of the test material, hand milling with a pestle and mortar was performed. Small quantities of the test material were ground gently with a pestle and mortar, the minimum effective pressure was applied to minimise the tendency of the test material to form a solid waxy film in the mortar. Waxy residues lining the pestle and mortar were scraped off and reground with a small quantity of fresh test material. The milled test material formed a solid cake during storage; therefore, the prepared material was brushed through a 150 μm test sieve before use to break up aggregates and ensure thorough mixing.
A Wright Dust Feed Mechanism powder canister was packed with previously milled test material to a pressure of 40 kg/cm^2 using a hydraulic press. Even density of packing was achieved by packing the canister in stages. The canister was weighed before use. A dust feed mechanism was positioned on top of the chamber and the gear ratio set to give the maximum practicable canister advance rate.
The powder canister of the Wright Dust Feed Mechanism was advanced until a trace of suspended dust could be seen in the chamber air. The generator gearing was then engaged and the generator motor switched on. After a 5.5 minute equilibration period, the theoretical time required for the concentration of aerosol to reach 90 % of the final value under the conditions of exposure employed (Silver and Arsenal, 1946), the exposure was timed for four hours. After four hours the generator was switched off and the rats were removed from the restraining tubes for examination.
- Method of particle size determination: Cascade Impactor
- Treatment of exhaust air: Exhaust air was drawn from the base of the chamber at a rate of 25 L/min and vented to atmosphere after first passing through an absolute filter.
- Temperature, humidity, pressure in air chamber: The chamber and generator air supplies were switched on and balanced to give a zero-pressure difference between the chamber and extract cabinet air. Wet and dry bulb thermometers located in the animal breathing zone were used to measure the temperature and humidity within the chamber. Readings were monitored frequently and recorded at 30-minute intervals throughout the exposure period. The mean chamber temperature and relative humidity during exposure were 22 °C and 53 % (RH), respectively.
- Respirable fraction determination: The fraction of the test atmosphere considered to be inhalable to the laboratory rat was calculated from the combined mass of test material collected on stages three to seven of Sierra Marple cascade impactor samples as a percentage of the total material collected by the device i.e. all particles with an Equivalent Aerodynamic Diameter of 6.0 μm or less.

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: The total aerosol concentration was determined on five occasions during the four-hour exposure. The atmosphere samples were withdrawn through a previously weighed Whatman GF/A glass fibre filter held in an open face filter holder. Samples were collected at an accurately calibrated flow rate of approximately four litres per minute. The filter and collected material were weighed to determine the atmosphere concentration of the test material.
- Time needed for equilibrium of exposure concentration before animal exposure: 5.5 minutes

VEHICLE
- Composition of vehicle: Dry, oil free compressed air.

TEST ATMOSPHERE
- Particle size distribution: The particle size distribution of the chamber atmosphere was measured once during each hour of the exposure period. The aerosol was characterised by drawing a continuous atmosphere sample at two litres per minute through a cascade impactor (Sierra Marple Model 296. Sierra Instruments Incorporated, Carmel Valley, California, U.S.A.) which was located in a spare animal exposure port.
At a sampling rate of two litres per minute the collection characteristics of the Sierra Marple Model 296 cascade impactor are:
Stage 1: Particles and droplets larger than 9.8 μm Equivalent Aerodynamic Diameter (EAD) and droplets.
Stage 2: Particles and droplets between 6.0 and 9.8 μm EAD
Stage 3: Particles and droplets between 3.5 and 6.0 μm EAD
Stage 4: Particles and droplets between 1.55 and 3.5 μm EAD
Stage 5: Particles and droplets between 0.93 and 1.55 μm EAD
Stage 6: Particles and droplets between 0.52 and 0.93 μm EAD
Stage 7: (Filter) Particles and droplets less than 0.52 μm EAD
The values of chamber concentration reported were determined by gravimetric analysis of five samples of chamber air taken during each exposure. Atmosphere concentrations are reported in terms of mg of test material per litre of chamber air.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 6.31 µm ± 3.92
The Mass Median Equivalent Aerodynamic Diameter (MMEAD) and geometric standard deviation of the test material present in the exposure atmosphere was calculated by linear regression of the mean cumulative percentage of the material collected on each Sierra Marple Cascade Impactor Stage (on a probability scale) against the logarithm of the manufacturers published stage cut points (μm) for the device.
The achieved chamber concentration was 0.87 mg/L (SD 0.07, 11.3 % generation efficiency). The relatively poor efficiency of atmosphere generation achieved during the exposure was considered to be associated with the waxy nature of the milled test material.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

One nominal concentration of 7.67 mg/L (actual 0.87 mg/L)

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed immediately before exposure, 15 and 30 minutes after the start of exposure and at 30 minute intervals for the remainder of the exposure period. Following return to their cages, the animals were observed at 30 minute intervals during the first three hours after exposure. Subsequently, they were examined twice daily until completion of 14 days of observation. The type, time of onset and duration of reactions to treatment were recorded. Each rat was weighed daily from the day of delivery until the end of the observation period.
- Necropsy of survivors performed: Yes. Rats that survived the observation period were placed under deep sodium pentobarbitone anaesthesia by intraperitoneal injection and killed by rapid exsanguination. All rats killed on completion of the observation period were subjected to necropsy with a minimum of delay.
- Macroscopic pathology: All animals were subjected to a detailed necropsy. The necropsy procedure included a review of the history of each animal and a detailed examination of the external features and orifices. The cranial cap was lifted and the brain dissected free of the meninges. The frontal and nasal elements were removed and any abnormality of the nasal passages, meninges, brain and pituitary recorded. The ventral abdominal skin was reflected to allow observation of the subcutaneous structures, in particular mammary glands and superficial lymph nodes. The abdominal viscera were examined in situ.
The entire gastro-intestinal tract was re-examined after removal. The stomach and caecum were opened along the greater curvature and rinsed in isotonic saline. After weighing the liver and kidneys were sectioned at intervals and the cut surfaces examined. The thorax was opened and the viscera examined before removal. The lungs were removed, dissected clear of surrounding tissue and weighed. Pleural surfaces were examined before inflation with 3 to 6 mL of buffered 4 % formaldehyde saline, injected slowly via the trachea. The trachea was then ligated.
- Organ weight: The organs specified were dissected free of adjacent fat and other contiguous tissue and the weights recorded: Lungs (with bronchi), liver and kidneys. Samples of the following tissues, together with macroscopic abnormalities, were preserved in buffered 4 % formaldehyde saline against possible future histological processing and microscopic examination: Larynx, lungs (with bronchi), liver and kidneys. Kidney weights wereseparately recorded for left and right sides. The recordings were sumed for presentation and before calculation of individual relative organ weights as a percentage of bodyweight.

Statistics:

Group mean values were calculated from individual values unless otherwise specified. Standard deviations were calculated where appropriate using the sample statistic.
Group mean and standard deviation are presented to the same level of accuracy as the individual values.
Standard deviations are not presented where the number of individual values is less than three.

Preliminary study:

An additional range-finding test was performed using a single group of two male and two female rats exposed to a nominal concentration of 2.85 mg/L. The results of this exposure, which are not reported in detail, indicated that the main study should be performed at the maximum practical chamber concentration.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 0.87 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

There were no deaths as a result of exposure.

Clinical signs:

salivation

Remarks:

Brown staining around the snout and wet fur were observed during the exposure period. These signs were considered consistent with excessive salivation.

Body weight:

Bodyweight was unaffected by treatment. The observed weight gain was in the expected range for rats of this age and strain.

Gross pathology:

There were no significant findings at necropsy. The lung, liver and kidney weights for all animals were within the expected ranges for rats of this age and strain.

Other findings:

Test material around the snout was recorded for most animals at the end of the exposure period. A reduction in respiratory rate was seen in some animals. The signs observed were considered to be consistent with exposure to a slightly irritant particulate atmosphere.
During the three hours immediately following the exposure, test material around the snout, brown staining around the snout and wet fur were recorded for most animals. Brown staining around the snout persisted after the exposure for up to three days in male rats and up to four days in females. Subsequently, all rats were normal in behaviour and appearance.

Particle Size Distribution

The mean proportion by weight of particles of inhalable size (< 6 μm Equivalent Aerodynamic Diameter) derived from the cascade impactor samples was:

Percent < 6 μm E.A.D. ± SD: 42.6 ± 9.2

Percent < 4 μm E.A.D.: 36.9

Mass Median E.A.D. ± geometric SD: 6.31 ± 3.92 μm

The small percentage of inhalable particles was considered to be associated with the difficulties encountered during milling of the waxy, low melting point, test material.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the study, there were no deaths following the exposure for four hours of five male and five female rats to the maximum chamber concentration that could be generated using the methods described in this report.
The median lethal chamber concentration for four hours' exposure (LC50 4-hour) is therefore greater than 0.87 mg per litre of air.

Executive summary:

The acute inhalation toxicity of the test material was assessed according to EPA APP 81-3 and equivalent to OECD Test Guideline 403 and in compliance with GLP by exposing a single group of five male and five female rats, for a continuous period of four hours, to an atmosphere containing the maximum practicable concentration of the test material.

The acute toxicity was investigated by exposing a single group of five male and five female albino rats to an atmosphere containing the maximum practicable concentration of test material. The test group was subjected to a single four-hour, continuous snout-only exposure. Signs of reaction to treatment were recorded during a subsequent 14-day observation period. The animals were sacrificed at the end of the observation period and were subjected to a detailed necropsy.

The nominal concentration of the test material was 7.67 mg/L, with the actual concentration being 0.87 mg/L. The relatively poor efficiency of atmosphere generation and the small percentage of inhalable particles (< 6.0 μm EAD) achieved during the exposure were considered to be associated with the difficulties encountered during milling of the waxy, low melting point test material. The Mass Median EAD of the atmosphere generated was 6.31 μm with a geometric standard deviation of 3.92 μm. The percentage of the atmosphere that was less than 4 μm EAD was 37 %.

There were no deaths during the study. Reduced respiratory rate, test material around the snout, brown staining around the snout and wet fur were recorded during the exposure period. Signs evident immediately following the exposure were test material around the snout, brown staining around the snout and wet fur; brown staining around the snout persisted for up to four days in some rats. Subsequently, all animals were normal in appearance and behaviour. Bodyweight gain was unaffected by treatment and there were no macroscopic findings that were attributed to treatment. Examination of the absolute and bodyweight-relative organ weights did not reveal any changes that were attributed to treatment.

Under the conditions of this study, there were no deaths following the exposure for four hours of five male and five female rats to the maximum chamber concentration that could be generated using the methods described in this report.

The median lethal chamber concentration for four hours' exposure (LC50 4-hour) is therefore greater than 0.87 mg per litre of air.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 September 1977 to 22 September 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 7 male and 7 female rats were exposed to the test material for 4 hours (whole body exposure) at 2.21 g/m^3 air dust concentration. A control group was exposed to the same conditions without the addition of the test material. Animals were observed for 14 days following test material exposure.

GLP compliance:

no

Remarks:

This study pre-dates the inception of GLP

Test type:

fixed concentration procedure

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD (a caesarian derived strain of Sprague-Dawley origin)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: animals were housed in polypropylene wire bottom cages, suspended on a mobile rack. Each cage held 7 rats of like sex.
- Diet: ad libitum
- Water: ad libitum tap water
- Acclimation period: a minimum of 3 days prior to exposure

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55-70 %

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

EQUIPMENT
- The dust of the powder was produced using a Wright Dust generator.
- In this electronically operated device, a scraper blade removes the powder from a pre-packed canister. The powder is removed at a steady rate which is determined by selection of different gear ratios on the generator. Dry compressed air passed into the canister along a groove cut in the scraper blade, blows out the loose powder as it is scraped off, after passing through a jet and impinging on a baffle plate (to break up any aggregates), the air containing the powder finely-dispersed, passes via an inlet column (3 cm in diameter; 25 cm in length) into the exposure chamber.
- The exposure chamber is constructed from 0.63 cm thick Perspex sheet. The internal volume of the chamber is approximately 100 litres. Galvanised metal grilles divided each chamber into 8 separate compartments, into which the animals were placed immediately prior to exposure. Each chamber is of a square section and fitted with a pyramidal top.

METHOD
- A portion of the test material was packed into a canister of a Wright Dust Generator with the aid of a hydraulic bench press. The pressure employed in packing the powder was 0.4 ton. Packing was accomplished in stages to ensure an even density within the canisters. Each canister was placed in position on a Wright dust generator mounted on a stand below the inlet column, connecting with a central hole at the base of the exposure chamber. The exit nozzle of the dust generator passed through a rubber diaphragm covering the end of the column nearest the dust generator. The diaphragm formed an airtight seal around the exit nozzle of the dust generator and contained the dust within the column until it passed into the exposure chamber. As the dust passed up this column the velocity of the air flow was reduced so that the larger particles deposited out within the column and only the smaller particles passed into the exposure chamber.
- The animals were then placed within the compartments of the exposure chambers and the position of each individual animal was noted. The flow of compressed air through each generator was regulated at 20 L/minute prior to packing of the canister.
Each exposure was started by disengaging the gears on the Wright dust generator and manually advancing the loaded canister up onto the scraper blade until a trace of dust was seen to emerge into the chamber. The gears were then re-engaged and the mechanism switched on. The gear ratio used to generate the dust was 1:6 (increased speed).
- The dust dispersed evenly throughout each chamber, and exited through a series of small holes drilled below animal level around the base of each chamber. The chambers were positioned inside larger glass walled extraction chambers, approximately 1 m^3 in volume. Each extraction chamber was equipped with an externally sited extract fan venting to the atmosphere through a collecting filter. The dust emerging from the holes around the base of the chamber was drawn off as it emerged. The dust of both samples was generated continuously throughout the 4 hour exposure period.
- The conditions under which exposure of the control group took place was identical to those used for the test group with the exception that an empty canister was placed on the generator.

ESTIMATION OF THE DUST CONCENTRATION WITHIN THE CHAMBER
-Samples were taken during exposure in order to estimate the total atmosphere concentration of each of the test substances. A total of 7 samples were withdrawn from the atmosphere generated of the dust of the test material during the 4 hour exposure period.
-A gravimetric method was used employing an open face filter collection system. For each sample a pressed glass fibre filter (Whatman GF/A-3.7 cm diameter) was placed in a filter holder. The total area of exposed filter available for collection was 7 cm^2. The holder together with the filter was placed over a 5 cm^2 round hole in the front face of the chamber wall, secured using an airtight seal. Air was drawn through the filter at a rate of approximately 1.76 L/minute using a vacuum pump (Edwards RB 5). The volume of each sample taken from the atmosphere of the test material was a standard 5 litres.
Each filter was weighed before and after sampling in order to calculate by difference the weight of dust collected. Knowing the weight of dust collected during each sample time, and the total volume of chamber air drawn through the filter, the chamber concentration of each substance was calculated. The results of each individual sample taken during each of the exposures were averaged to give the mean concentration of the test material within each chamber.
The amount of test material present on each filter was estimated using an analytical method supplied by the test material supplier.

ANALYTICAL METHOD
Methylation
-Test material dissolved in ethyl ether, acetone or dichloromethane, is methylated using diazomethane and chromatographed as its methyl ester.

Gaseous phase chromatography
-Inject 2 µL of the solution obtained above under the following conditions:
Apparatus: Pye Unicam GCV
Column: Glass, 2 metre x 5 mm i.d.
Phase: NPGS (neopentylglycolsuccinate) 5 % on EMBACEL 60/100 mesh
Temperature: Injector 180 °C, Detector 230 °C, Column 180 °C
Carrier gas: Nitrogen (flow rate 60 cm^3/minute)
Detector: Flame ionization (hydrogen flow rate 66 cm^3/minute, air at 500 cm^3/minute)
Recorder: 1 mV full range

Calculations
The calculation is done by measuring the surface area by one or other of the following methods:
-by comparison with a standard solution of methylated 2-(4-chloro 2-methyl phenoxy) propionic acid prepared and injected under the same conditions as the test preparation (external standard);
-by the internal standard method with 2-(2,4-dichlorophenoxy) propionic acid added to the test solution before methylation.

The method has a limit of sensitivity of about 30 µg/mL, ie. 60 ng per injection of 2 µL
The test material (isomer D) and L of 2-(4-chloro 2-methylphenoxy) propionic acid are not separated by this method.

ESTIMATION OF THE PARTICLE SIZE DISTRIBUTION
-The particle size distribution of each dispersed sample was estimated during the 4 hour exposure period using an Anderson Mini-Sampler (2000 Inc. Salt Lake City, Utah, USA).
-This device consists of 4 metal impaction stages and a back-up glass fibre filter (Whatman GF/C-2.5 cm diameter) housed within an aluminium can 2.8 cm in diameter, 3.1 cm in length. Each impaction plate is 2.5 cm in diameter. The sampling rate is a standard 1.4 L/minute and the collection characteristics at this flow rate are listed below:
Plate 1: particles greater than 5.5 µm diameter
Plate 2: particles between 3.5 and 5.5 µm diameter
Plate 3: particles between 2.0 and 3.5 µm diameter
Plate 4: particles between 0.3 and 2.0 µm diameter
Filter: particles below 0.3 µm diameter
-A single sample was taken during each exposure through a 5.0 cm^2 round hole in the exposure chamber wall. An airtight seal was made around the chamber wall and chamber air was drawn through the mini-sampler by a Casella general purpose sampler, Mark II, model C. Each stage of the sampling device was cleaned and weighed immediately prior to use, and after sampling reweighed. The weight of dust collected on each stage was calculated by difference.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Mean concentration of airborne dust within the exposure chamber was 2.21 ± 0.14 g/m^3.
Estimated nominal concentration was 6.48 g/m^3

No. of animals per sex per dose:

7 animals per sex per dose group

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All rats were observed at frequent intervals throughout the 4 hour exposure periods. Frequent checks on appearance and behaviour were made subsequently during the 14 day post exposure period. All animals were observed at least twice daily during this time.
- Necropsy of survivors performed: yes. Immediately following exposure, 2 males and 2 females from the control group and group 2 (mecoprop) were anaesthatised by intraperitoneal injection of pentobarbitone sodium. The subclavian blood vessels were exposed, cut and the animals allowed to bleed out. The respiratory tract of these animals was subjected to detailed macroscopic examination. The lungs were dissected out and weighed in order to calculate the lung to body weight ratio of each animal.
- Clinical signs including body weight: All rats were weighed immediately prior to exposure (day 0), the day following exposure (day 1) and subsequently on days, 3, 7, 10 and 14 post exposure. From the individual bodyweights the group mean bodyweight at each of these stages during the study was calculated. The amount of food consumed by each cage of rats was measured daily, and the average amount consumed by a single rat in each cage was calculated.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.21 other: g/m3

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Control group: no deaths
Test material treated group: 1 dead

Clinical signs:

irregular respiration

Body weight:

Slightly lower bodyweights were recorded on day 1 for male rats exposed to the test material. On day 3 lower bodyweights were recorded for female rats exposed to the test material.
The bodyweight gain of all animals from day 3 onwards of the observation period was similar and considered normal.
One male lost 34 g over the final four days of the post-exposure period. The appearance of this animal at the end of the observation period was emaciated but no macroscopic change was seen to account for this condition.

Gross pathology:

Lung to bodyweight ratios
-The lung to bodyweight ratio of the male rat that died during the study was considered abnormally high, 1.68. This was considered a result of the presence within the lungs of large amounts of oedematous fluid noted post mortem.
-The lung to bodyweight ratios of all other animals killed either immediately following exposure or at the end of the 14 day post exposure observation period were considered normal.

Macroscopic pathology
- The lungs of the male rat that died during the study were severely haemorrhaged and the presence of massive amounts of oedematous fluid was noted. The stomach was filled with gas, presumably ingested as a result of gasping.
- Other incidental findings not regarded as significant, included haemorrhage of the thymus in one male, the emaciated condition of one rat which lost a large amount of weight, small red spots not exceeding 1 mm in diameter in the lungs of rats in all groups and the appearance in the lungs of some animals of purplish areas considered due to inadequate bleeding out prior to post mortem examination.

Other findings:

Clinical signs:
- Non-specific reactions, typified by licking of the inside of the mouth and blinking of the eyes were seen during the first 5-10 minutes of exposure.
- Rats showed severe reactions to the presence of dust, typified by modified respiration (panting) that developed into gasping as the exposure progressed. By the end of the 4 hour exposure period one male rat had died and all other rats were either gasping or had moderate difficulty in breathing. All rats were cold to the touch when removed from the chamber at the end of the exposure period. All surviving animals had recovered overnight following exposure and were normal during the remainder of the 14 day observation period.

Food Consumption:
- Less food was consumed overnight following exposure by both male and female animals. Thereafter food consumption was considered normal over the remainder of the 14 day observation period.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this study, the acute inhalation LC50 was > 2.21 g/m^3.

Executive summary:

The acute inhalation toxicity of the test material was investigated in CD Sprague Dawley rats.

Groups of 7 male and 7 female rats were exposed to the test material for 4 hours (whole body exposure) at 2.21 g/m^3 air dust concentration. A control group was exposed to the same conditions without the addition of the test material. Animals were observed for 14 days following test material exposure.

The amount of test material in the chamber was estimated using gas chromatography analysis. The mean concentration of airborne dust within the exposure chamber was 2.21 ± 0.14 g/m^3. The estimated nominal concentration was 6.48 g/m^3. The mean measured airborne concentration was 34 % of the nominal concentration, this discrepancy can be accounted for by the fact that a significant proportion of the dispersed dust was deposited around the end of the connecting column, this proportion of the generated dust consists of particles too large to remain airborne.

One animal died during the exposure and all other animals treated with the test material showed severe reactions to the presence of dust, typified by modified respiration (panting) that developed into gasping as the exposure progressed. All surviving animals had recovered overnight following exposure and were normal during the remainder of the 14 day observation period.

Under the conditions of this study, the acute inhalation LC50 was > 2.21 g/m^3.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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:

24 January 1990 to 25 March 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 81-2 (Acute Dermal Toxicity)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: CD strain (remote Sprague-Dawley origin)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Animals were approximately eight weeks old on arrival. On the day prior to dosing the animals were approximately ten weeks old.
- Weight at study initiation: Bodyweight was recorded on the day prior to dosing and was in the range 246 - 264 g for males and 216 - 237 g for females.
- Housing: The animals were housed in stainless steel grid cages measuring 54 x 33 x 20 cm. The grid floors ensured rapid removal of waste material to undertrays which were cleaned as necessary. Five animals of the same sex were housed in each cage. The cages were suspended in mobile stainless steel racks. On completion of dosing, the animals were accommodated individually in cages measuring 45 x 28 x 20 cm. The dressings were removed 24 hours after administration. The animals were then returned to their original cages.
- Diet: A commercially-available complete pelleted rodent diet was fed without restriction. The diet contained no added antibiotic or other chemotherapeutic or prophylactic treatment.
- Water: Animals had free access to tap water supplied in a single bottle per cage and re-filled as required. The water was supplied by the East Anglian Water Company.
- Acclimation period: At least 6 days.
- Microbiological status when known: The animals had been bred under barriered conditions and travelled from the supplier to the animal-holding room in sealed boxes with filter protected air-vents

ENVIRONMENTAL CONDITIONS
- Temperature: Target value 21 °C (18 to 25 °C)
- Humidity: Target value 55 % relative humidity (40 to 70 %)
- Air changes: All rooms were kept at slight positive pressure relative to the outside and each had its own filtered air supply giving approximately 15 complete air changes per hour without re-circulation.
- Photoperiod: Electric time-switches regulated a lighting cycle of 12 hours of artificial light per day.

IN-LIFE DATES
From: 1 March 1990
To: 15 March 1990

Type of coverage:

occlusive

Vehicle:

water

Details on dermal exposure:

TEST SITE
- Area of exposure: The dorsum between the limb-girdles was clipped free of hair as close to the skin as possible using Oster small animal clippers. The exposed application site of each rat was examined before the animal was allocated to study.
- Type of wrap if used: The gauze patch was placed on the closely-clipped dorsum and was occluded with aluminium foil. The foil was kept in place and protected by a bandage of waterproof plaster wrapped twice around the trunk of the body with sufficient tension to ensure the dose remained securely in place.

REMOVAL OF TEST SUBSTANCE
- Washing: The dermal site was gently wiped with wet disposable towels to remove residual test material.
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied: The dose was determined for each animal according to its bodyweight on the morning of dosing. The dosage was calculated and expressed gravimetrically in terms of the material as received.
- For solids, paste formed: No. The test material was placed on a gauze patch and moistened by application of 0.2 mL distilled water immediately before dosing. This was intended to ensure good contact between the test material and the skin surface and to simulate the maximum adverse conditions of the product in use.

VEHICLE
- Amount(s) applied: 0.2 mL

Duration of exposure:

24 hours

Doses:

2 000 mg/kg

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

not required

Details on study design:

- Duration of observation period following administration: The test was terminated on Day 15
- Frequency of observations and weighing: Three separate inspections were made during the first hour after administration and two further inspections during the remainder of Day 1. From Day 2 onwards, the animals were inspected twice daily (morning and afternoon). Each dermal application site was examined at the morning observation. The type, time of onset and duration of reactions to treatment were recorded. The bodyweight of each animal was recorded on the day before dosing and on Days 1, 8 and 15.
- Necropsy of survivors performed: Yes. Animals were killed at termination of the study by carbon dioxide inhalation. All rats were weighed and thoroughly examined at necropsy for abnormality of tissues or organs. All body cavities were opened, larger organs were sectioned and the gastro-intestinal tract was opened at intervals for examination of the mucosal surfaces. Any abnormalities were described or the normal appearance of major organs was confirmed
- Other examinations performed: Each dermal application site was examined in situ, and in macroscopic section to obtain more information on dermal effects of treatment.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There was no death.

Clinical signs:

other: There was no systemic or local sign of reaction to treatment.

Gross pathology:

Necropsy, on Day 15, revealed no significant macroscopic lesion.

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of this study, the acute percutaneous median lethal dosage (LD50) of the test material was greater than 2 000 mg/kg.

Executive summary:

The acute percutaneous toxicity of the test material was investigated in a group of five male and five female CD rats in accordance with the standardised guideline OECD 402 and in compliance with GLP.

The test material was applied to the closely-clipped dorsum of each animal on Day 1 at a limit dosage of 2 000 mg/kg, and was covered by an occlusive dressing for 24 hours. Systemic or local signs of reaction to treatment were recorded during a subsequent 14-day period of observation. The animals were killed on Day 15 and subjected to necropsy.

There was no death. There was no systemic or local sign of reaction to treatment. The animals achieved expected bodyweight gains. Necropsy, on Day 15, revealed no significant macroscopic lesion.

Under the conditions of this study, the acute percutaneous median lethal dosage (LD50) of the test material was greater than 2 000 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Acute Toxicity, Oral Route: Dange (1994)

The potential for the test material to cause acute toxicity via the oral route was investigated in a study conducted in accordance with the standardised guidelines OECD 401 and US EPA OPP 81-1 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The acute oral toxicity was assessed after a single oral administration of the test material at 100, 180, 320 and 580 mg/kg body weight in groups of five male and five female Sprague Dawley rats. All animals were observed daily tor fifteen days and their body weight measured weekly. At termination of the study period or when found dead, animals were subjected to a necropsy.

No animal died at 100, 180 and 320 mg/kg body weight. All animals died at 580 mg/kg. No clinical signs were noted at 100 mg/kg body weight. At 180 and 320 mg/kg body weight, piloerection, hunched posture and reduced motor activity were observed only during Days 1 and 2. At 580 mg/kg body weight, piloerection, dyspnoea, prostration, absence of traction and of grasping reflex, muscular atony, reduced motor activity and unconsciousness were the major clinical signs observed before the death of the animals. The body weight evolution of the animals which survived the study period was unaffected by treatment. No treatment-related changes were observed at necropsy.

Under the conditions of this study, the acute oral LD50 of the test material was determined to be 431 mg/kg bw in male and female Sprague-Dawley rats.

Acute Toxicity, Oral Route Supporting Study: Cummins (1990)

The acute oral toxicity of the test material was assessed according to OECD Test Guideline 401 and in compliance with GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The study consisted of four groups of five male and five female CD rats. The animals were starved overnight prior to dosing. The test material was administered on Day 1 at dosages in the range 450 - 900 mg/kg, at a volume-dosage of 20 mL/kg in 0.5 % w/v methylcellulose in distilled water.

Mortality and signs of reaction to treatment were recorded during a subsequent 14-day observation period. Decedents and animals killed on Day 15 were subjected to necropsy.

The mortality distribution was as follows:

450 mg/kg group: 0/5 males and 0/4 females. One female was in unsatisfactory condition before dosing on Day 1; no suitable replacement was available so only four were dosed.

567 mg/kg group: 0/5 males and 1/5 females.

714 mg/kg group: 1/5 males and 0/5 females.

900 mg/kg group: 4/5 males and 5/5 females.

The deaths occurred on Days 2, 3 or 4.

Ante mortem signs comprised lethargy, unconsciousness, decreased motor activity, prone posture, ataxia, clonic convulsion, muscle tremor, breathing irregularities, ungroomed appearance, pigmented orbital secretion and hunched posture.

Signs of reaction to treatment in surviving animals were similar to those of the decedents with the exception of unconsciousness and clonic convulsion and with the addition of blanching and thin body conformation. The majority of animals were overtly normal on Day 6 and throughout the remainder of the observation period. Animals treated at 450 mg/kg were less severely affected than those treated at higher dosages.

The surviving animals generally achieved expected bodyweight gains.

Necropsy of the decedents revealed isolated incidences of altered stomach and jejunum contents, dark thymic lymph nodes and pale perineal staining. Necropsy of the surviving animals revealed no significant macroscopic lesion.

Under the conditions of this study the acute oral median lethal dosage (LD50) was:

Male: LD50 803 mg/kg, 95 % confidence limits of 710 – 896 (slope 83 degrees).

Female: LD50 756 mg/kg, 95 % confidence limits of 651 – 861 (slope 80 degrees).

Combined: LD50 775 mg/kg, 95 % confidence limits of 666 – 885 (slope 75 degrees).

Under the conditions of the study, the combined acute oral LD50 for male and female CD rats was 775 mg/kg (95 % confidence limits of 666 – 885).  The test material was considered acutely toxic via the oral route (Category 4).

Acute Toxicity, Inhalation Route: Cracknell (1990)

The acute inhalation toxicity of the test material was assessed according to EPA APP 81-3 and equivalent to OECD Test Guideline 403 and in compliance with GLP by exposing a single group of five male and five female rats, for a continuous period of four hours, to an atmosphere containing the maximum practicable concentration of the test material. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The acute toxicity was investigated by exposing a single group of five male and five female albino rats to an atmosphere containing the maximum practicable concentration of test material. The test group was subjected to a single four-hour, continuous snout-only exposure. Signs of reaction to treatment were recorded during a subsequent 14-day observation period. The animals were sacrificed at the end of the observation period and were subjected to a detailed necropsy.

The nominal concentration of the test material was 7.67 mg/L, with the actual concentration being 0.87 mg/L. The relatively poor efficiency of atmosphere generation and the small percentage of inhalable particles (< 6.0 μm EAD) achieved during the exposure were considered to be associated with the difficulties encountered during milling of the waxy, low melting point test material. The Mass Median EAD of the atmosphere generated was 6.31 μm with a geometric standard deviation of 3.92 μm. The percentage of the atmosphere that was less than 4 μm EAD was 37 %.

There were no deaths during the study. Reduced respiratory rate, test material around the snout, brown staining around the snout and wet fur were recorded during the exposure period. Signs evident immediately following the exposure were test material around the snout, brown staining around the snout and wet fur; brown staining around the snout persisted for up to four days in some rats. Subsequently, all animals were normal in appearance and behaviour. Bodyweight gain was unaffected by treatment and there were no macroscopic findings that were attributed to treatment. Examination of the absolute and bodyweight-relative organ weights did not reveal any changes that were attributed to treatment.

Under the conditions of this study, there were no deaths following the exposure for four hours of five male and five female rats to the maximum chamber concentration that could be generated using the methods described in this report.

The median lethal chamber concentration for four hours' exposure (LC50 4-hour) is therefore greater than 0.87 mg per litre of air.

Acute Toxicity: Inhalation Route: Coombs (1977)

The acute inhalation toxicity of the test material was investigated in CD Sprague Dawley rats. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Groups of 7 male and 7 female rats were exposed to the test material for 4 hours (whole body exposure) at 2.21 g/m^3 air dust concentration. A control group was exposed to the same conditions without the addition of the test material. Animals were observed for 14 days following test material exposure.

The amount of test material in the chamber was estimated using gas chromatography analysis. The mean concentration of airborne dust within the exposure chamber was 2.21 ± 0.14 g/m^3. The estimated nominal concentration was 6.48 g/m^3. The mean measured airborne concentration was 34 % of the nominal concentration, this discrepancy can be accounted for by the fact that a significant proportion of the dispersed dust was deposited around the end of the connecting column, this proportion of the generated dust consists of particles too large to remain airborne.

One animal died during the exposure and all other animals treated with the test material showed severe reactions to the presence of dust, typified by modified respiration (panting) that developed into gasping as the exposure progressed. All surviving animals had recovered overnight following exposure and were normal during the remainder of the 14 day observation period.

Under the conditions of this study, the acute inhalation LC50 was > 2.21 g/m^3.

Acute Toxicity, Dermal Route: Cummins (1990)

The acute percutaneous toxicity of the test material was investigated in a group of five male and five female CD rats in accordance with the standardised guideline OECD 402 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was applied to the closely-clipped dorsum of each animal on Day 1 at a limit dosage of 2 000 mg/kg, and was covered by an occlusive dressing for 24 hours. Systemic or local signs of reaction to treatment were recorded during a subsequent 14-day period of observation. The animals were killed on Day 15 and subjected to necropsy.

There was no death. There was no systemic or local sign of reaction to treatment. The animals achieved expected bodyweight gains. Necropsy, on Day 15, revealed no significant macroscopic lesion.

Under the conditions of this study, the acute percutaneous median lethal dosage (LD50) of the test material was greater than 2 000 mg/kg.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does require classification with respect to acute toxicity via the oral route (Category 4).

The substance does require classification with respect to acute toxicity via the dermal route.