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The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

Oral (similar to OECD 407), rat (m/f): NOAEL systemic = 103.6 mg/kg bw/day

Inhalation (9 days study), rat (m/ f): Vapor aerosol NOAEC systemic = 217.4 mg/m3 air, corresponding to 50.5 ppm

Dermal (9 days study, similar to OECD 410), rat (m/f): NOAEL systemic = 527 mg/kg bw/day

Dermal (9 days study, similar to OECD 410), rat (m/f): NOAEL local = 210.8 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1979-05-08 to 1979-06-05
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 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 03 Oct 2008
Deviations:
yes
Remarks:
no recovery group included, no detailed clinical observations, no FOB, clinical chemistry and haematology were limited to a few parameters, organ weight analysis and histopathology were limited to a few organs
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals, Zeist, The Netherlands
- Age at study initiation: Weanings were used
- Weight at study initiation: 75 ± 1.3 g (males) and 65.6 ± 1.2 g (females)
- Housing: 5/sex in suspended stainless steel cages fitted with wire mesh floors and fronts
- Diet: powdered stock diet, ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 0.4, 2 and 10%
- Amount of vehicle: 5 mL/kg bw
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily, 6 days/week
Dose / conc.:
0.02 other: mL/kg bw/day
Remarks:
corresponding to 20.72 mg/kg bw/day, dosel levels in mg/kg bw calculated with a density of 1036 mg/mL
Dose / conc.:
0.1 other: mL/kg bw/day
Remarks:
corresponding to 103.6 mg/kg bw/day, dosel levels in mg/kg bw calculated with a density of 1036 mg/mL
Dose / conc.:
0.5 other: mL/kg bw/day
Remarks:
corresponding to 518 mg/kg bw/day, dosel levels in mg/kg bw calculated with a density of 1036 mg/mL
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND FOOD EFFICIENCY: Yes
- Food consumption was measured weekly and the efficiency of food utilisation was calculated and expressed as grams weight gain per gram food consumed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples of blood were collected on study Day 22 by orbital puncture
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 5 rats per sex per group
- Parameters checked: haemoglobin concetration (Hb), packed cell volume (PCV), red blood cell count (RBC), white blood cell count (WBC), differential white blood cell count (eosinophils, basophils, neutrophils, lymphocytes and monocytes) and prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At autopsy on Day 28 blood samples were collected in heparinised tubes from the aorta of all rats.
- Anaesthetic used for blood collection: yes (ether)
- Animals fasted: Not specified
- How many animals: all rats
- Parameters checked: glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), alkaline phosphatase (ALP), total protein (TP), total bilirubin (BILIRU) and creatinine.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. On completion of 28 days of treatment, all rats were anaesthetized by ether, exsanguinated by cannulating the aorta and examined grossly for pathological changes.
- The following organ weights were determined for all animals: kidneys, liver, spleen, lungs, heart and testes.

HISTOPATHOLOGY: Yes. Samples of the organs mentioned above from the control and high dose group were fixed in 4% aqueous phosphate buffered formaldehyde solution. The samples were processed, embedded in Paraplast and processed for haematoxyline eosin staining. Additionally, the spleens of the rats from the mid and low dose groups were microscopically analysed.
Statistics:
Body weight and organ weight data were analysed statistically by the student's t-test. Haematological findings and blood biochemical values were evaluated by the Wilcoxon test.
Clinical signs:
no effects observed
Description (incidence and severity):
No substance related clinical signs were observed in any dose group.
Mortality:
no mortality observed
Description (incidence):
No deaths were observed in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
0.5 mL/kg bw/day corresponding to 518 mg/kg bw/day: Slightly, but significantly decreased body weight gain in the high dose group males (7.6%); and small, statistically not significant decrease in body weight gain in females (2.9%) at the end of the exposure period. Body weight gains in the high dose animals were also reduced compared to controls (9.7 and 6.1%), but not analysed statistically. The findings were consistent with reduced food efficiency in these animals and considered to be treatment-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food intake was comparable in all groups.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
0.5 mL/kg bw/day corresponding to 518 mg/kg bw/day: decreased in both sexes (statistically not significant, males: 0.34, contr. 0.37, females, 0.26, contr. 0.27), consistent with depressed body weight, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
0.5 mL/kg bw/day corresponding to 518 mg/kg bw/day: Reduced red blood cell count (statistically significant in females only) and reduced haemoglobin levels (statistically not significant) were observed in animals of both sexes (RBC count males: 5.7,contr. 6.1, females: 5.7, contr. 6.5; Hb: males: 8.4 mmol/l, contr. 8.9 mmol/l, females: 8.7 mmol/l, contr. 9.6 mmol/l). The findings were accompained by an increase in haematopoesis in the spleen and increased clinical chemistry parameters. The observations suggest that destruction of the red blood cells takes place and were considered to be treatment-related. Further, a statistically significant increase in white blood cell count was noted in males(16 x 10E9/L, contr. 11.8 x 10E9/L), the toxicological significance was unclear.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
0.1 mL/kg bw/day corresponding to 103.6 mg/kg bw/day: A slight increase (statistically not significant, 68.3 µmol/l compared to 63.3 µmol/l in control) was noted in creatinine levels of males. Without a clear dose-response and in the absence of findings in females the toxicological significance remained unclear.
0.5 mL/kg bw/day corresponding to 518 mg/kg bw/day: Total bilirubin levels were increased in animals of both sexes (males. 3.0 µmol/l, contr.1.9 µmol/l; females 2.8 µmol/l, contr. 2.0 µmol/l). The findings were consistent with haematological and histopathological findings in the spleen and considered to be treatment-related. In addition, a slight increase (statistically not significant, 69.1 µmol/l compared to 63.3 µmol/l in control)) was noted in creatinine levels of males. Without a clear dose-response and in the absence of findings in females the observation was considered not toxicologically relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
0.5 mL/kg bw/day corresponding to 518 mg/kg bw/day: The absolute weight of lungs was slightly decreased in females (statistically not significant, 0.824 g, contr. 0.904 g). Without any histopathological abnormalities the observation was considered to be incidental.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related changes were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
0.5 mL/kg bw/day corresponding to 518 mg/kg bw/day: Extramedullary haematopoesis and an increased amount of blood and brown pigment in the red pulp was found in the spleen of males and females. The observations were consistent with haematological and clinical chemistry findings and attributed to treatment.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
103.6 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
518 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
histopathology: non-neoplastic
Remarks on result:
other: dosel levels in mg/kg bw calculated with a density of 1036 mg/mL
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
518 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Based on the results of this study, the LOAEL for systemic toxicity was 0.5 mL/kg bw corresponding to 518 mg/kg bw/day in male and female rats. The NOAEL for systemic toxicity was 0.1 mL/kg bw/day, corresponding to 103.6 mg/kg bw/day in male and female rats.
Endpoint:
sub-chronic toxicity: oral
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
data reported as part of a review, no experimental details given
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
male
Route of administration:
oral: unspecified
Duration of treatment / exposure:
6 months
Dose / conc.:
4.4 mg/kg bw/day (actual dose received)
Dose / conc.:
44 mg/kg bw/day (actual dose received)
Dose / conc.:
88 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Post-exposure period: no data
Dose descriptor:
NOAEL
Effect level:
4.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effect observed
Critical effects observed:
not specified

Effects to redox processes and function of the liver, CNS-effects reported in study summary.

Conclusions:
The available information comprise only summary data but no experimental details. Based on the lack of data, it is not possible to derive a NOAEL for systemic toxicity.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
103.6 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Test material not fully characterised, particle size distribution (MMAD and GSD) not reported, no data of individual animals given, 13 days study with 9 days exposure instead of 28 day study with 5 days exposure/week
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Determination of systemic toxicity in rats upon repeated exposure via inhalation in a 13 day study.
- Short description of test conditions: Groups of 5 male and 5 female rats were exposed to the test item in whole body chambers at concentrations of 0.5, 5 and 50 ppm, corresponding to 0.00215, 0.0215 and 0.215 mg/L air or 2.15, 21.5 and 215 mg/m3 air. The animals were treated for 6 hours/day for 9 days (5 days in the first week and 4 days in the second week).
- Parameters analysed / observed: Physical observations, body weight, food consumption, haematology, clinical chemistry and urinalysis.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River laboratories (Kingston, New York, USA)
- Age at study initiation: 6 weeks
- Weight at study initiation: 177 g (males, range: 167 - 189 g) and 119 g (females, range 112 - 129 g)
- Fasting period before study: No
- Housing: Individually in stainless steel wire mesh cages
- Diet: Certified Rodent Diet No. 5002 (PMI Nutrition International, St. Louis, Missouri, USA), ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 34 - 75
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The air-alone controls were exposed in a 100 L chamber through which air was passed at 20 L/min. For the test item exposures, liquid test material was metered from a syringe pump on to the heated glass helix of a countercurrent volatization chamber. Air was fed through a flow meter into the volatization chamber countercurrent to the flow of the test item. The vapour-containing air was further diluted with additional air to achieve the desired concentration in the 200 L exposure chambers.

TEST ATMOSPHERE
Air was sampled 4 times each hour for measurement of the test item by means of an air pump drarwing chamber air into a 2-HMP coaed XAD-2 resin tube. The test item was extracted from the sampling tubes with carbon disulfide, and measured by a gas chromatography-flame ionization technique. Nominal concentrations were calculated based on a knowledge of the total amount of test item used and the air flow rates.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure concentrations were measured by gas chromatography (GC) four times per chamber per day. Mean chamber concentrations were 0.47 ± 0.05 ppm, 4.99 ± 1.1 ppm and 50.5 ± 5.5 ppm.
Duration of treatment / exposure:
6 h
Frequency of treatment:
6 hours/day for 9 days (5 days in first week, 4 days in second week)
Dose / conc.:
0.5 ppm (nominal)
Remarks:
corresponding to 2.15 mg/m3 air; corresponding to an analytical concentration of 0.47 ± 0.05 ppm or 2.0 mg/m3 air (dose levels in mg/m3 air were calculated as follows: [dose level in ppm x molecular weight] / 24.2)
Dose / conc.:
5 ppm (nominal)
Remarks:
corresponding to 21.5 mg/m3 air; corresponding to an analytical concentration of 4.99 ± 1.1 ppm or 21.5 mg/m3 air (dose levels in mg/m3 air were calculated as follows: [dose level in ppm x molecular weight] / 24.2)
Dose / conc.:
50 ppm (nominal)
Remarks:
corresponding to 215 mg/m3 air; corresponding to an analytical concentration of 50.5 ± 5.5 ppm or 217.4 mg/m3 air (dose levels in mg/m3 air were calculated as follows: [dose level in ppm x molecular weight] / 24.2)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE AND CLINICAL OBSERVATIONS: Yes
- Time schedule: during exposure and daily thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: 6 and 2 days before exposure and on study Days 7 and 11.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured for the week before exposures and for the 1st and 2nd exposure week.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected on the day following the final exposure from the retro-orbital sinus
- Anaesthetic used for blood collection: Yes (CO2/O2 anesthesia)
- Animals fasted: No
- How many animals: all animals
- Parameters examined: Hemoglobin (Hb), hematocrit (PCV), erythrocyte count, reticulocyte count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, total and differential leukocyte counts, prothrombin time and activated thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected on the day following the final exposure from the retro-orbital sinus
- Anaesthetic used for blood collection: Yes (CO2/O2 anesthesia)
- Animals fasted: No
- How many animals: all animals
- Parameters examined: glucose, total protein, albumin, globulin, urea nitrogen, creatinine, bilirubin (total, conjugated and unconjugated), Na+, K+, chloride, inorganic phosphorus, aspartate and alanine aminotransferases, alkaline phosphatase, lactate and sorbitol dehydrogenases, creatinine kinase and gamma-glutamyl transferase.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected over an 16 h interval following the final exposure
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters examined: appearance, specific gravity, osmolality, protein, glucose, ketones, pH, occult blood, bilirubin, urobilinogen, creatinine and N-acetyl-ß-D-glucosaminidase.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. After 9 exposures, all animals were sacrificed, selected organs were weighed and organ/body weight and organ/brain weight ratios calculated. The following organs were weighed: Adrenal glands, brain, kidneys, liver, lungs, ovaries and testes. Complete macroscopic postmortem examinations were conducted on all animals.

HISTOPATHOLOGY: Yes. Histopathological evaluation of selected tissues were conducted on all animals. Histology was conducted on the organs that were weighed and additionally on the heart, larynx, trachea, nasopharyngeal tissues, spleen and urinary bladder.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEC
Effect level:
50.5 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed
Remarks on result:
other: corresponding to 0.217 mg/L air or 217.4 mg/m3 air
Remarks:
(dose levels in mg/m3 air were calculated as follows: [dose level in ppm x molecular weight] / 24.2)
Key result
Critical effects observed:
no
Conclusions:
Based on the results of this study, the NOAEC for systemic toxicity was 50.5 ppm in male and female rats, corresponding to 217.4 mg/m3 air or 0.2174 mg/L air.
Endpoint:
short-term repeated dose toxicity: inhalation
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
mixed exposure with acrolein, test material not fully characterised, particle size distribution (MMAD and GSD) not reported, no data of individual animals given, 13 days study with 9 days exposure instead of 28 day study with 5days exposure/week, no data on food consumption, no urinalysis performed
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Determination of systemic toxicity in rats upon repeated exposure via inhalation in a 13 day study, followed by 28 days of recovery.
- Short description of test conditions: Groups of 20 Fisher 344 rats per sex were exposed to the test item in whole body chambers at concentrations of 25, 100 and 250 ppm, corresponding to 107.5, 430 and 1075 mg/m3 air. The animals were treated for 6 hours/day for 9 days (5 days in the first week and 4 days in the second week). Each 10 rats/sex were sacrificed the day following final exposure. The remaining animals were kept for a 28 days recovery period.
- Parameters analysed / observed: Physical observations, body weight, food consumption, haematology, clinical chemistry and urinalysis.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River laboratories (Raleigh, North Carolina, USA)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 121.7 - 137.4 g (males) and 103.0 - 115.0 g (females)
- Fasting period before study: No
- Housing: Individually in stainless steel wire mesh cages
- Diet: Certified Rodent Chow No. 5002 (Ralston, Purina, St. Louis, Missouri, USA), ad libitum
- Water: ad libitum
- Acclimation period: 12 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (F): 68 -76
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
All animals were exposed in stainless steel and glass chambers with an air flow rate of 325 L/min (15 air changes/h). Test item vapour was generated by metering liquid test item from a rotary pump into a stainless steel "J-tube" evaporator. Nitrogen was used to transfer the test item vapour into an air line entering the exposure chamber.

TEST ATMOSPHERE
Atmosphere samples for analysis were taken through a sampling tube in the center of each chamber. Test item vapour concentrations were measured once every 30 minutes from each chamber using a Foxburo-Miram 1A infrared gas analyser.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure concentrations were measured once every 30 minutes during exposure from each chamber by using gas chromatography (GC). Mean chamber concentrations were 23.6 ± 0.9 ppm, 96.8 ± 3.8 ppm and 246.2 ± 9.8 ppm.
Duration of treatment / exposure:
6 h
Frequency of treatment:
6 hours/day, 4 to 5 days/week for 2 weeks (total 9 exposures)
Dose / conc.:
25 ppm (nominal)
Remarks:
corresponding to 0.1075 mg/L air or 107.5 mg/m3 air; corresponding to an analytical concentration of 23.6 ± 0.9 ppm, 0.1016 mg/L air or 101.6 mg/m3 air (dose levels in mg/m3 air were calculated as follows: [dose level in ppm x molecular weight] / 24.2)
Dose / conc.:
100 ppm (nominal)
Remarks:
corresponding to 0.43 mg/L air or 430 mg/m3 air; corresponding to an analytical concentration of 96.8 ± 3.8 ppm, 0.417 mg/L air or 416.7 mg/m3 air (dose levels in mg/m3 air were calculated as follows: [dose level in ppm x molecular weight] / 24.2)
Dose / conc.:
250 ppm (nominal)
Remarks:
corresponding to 1.075 mg/L air or 1075 mg/m3 air; corresponding to an analytical concentration of 246.2 ± 9.8 ppm, 1.06 mg/L air or 1059.8 mg/m3 air (dose levels in mg/m3 air were calculated as follows: [dose level in ppm x molecular weight] / 24.2)
No. of animals per sex per dose:
Main group: 10
Recovery group: 10
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: 28 days
Observations and examinations performed and frequency:
CAGE SIDE AND CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: 5 days before exposure and on exposure days 1, 2, 5, 8 and 9. For recovery animals, body weight was further recorded on post exposure days 8, 15, 22 and 29.

FOOD CONSUMPTION AND COMPOUND INTAKE: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected on the day following the final exposure from the retro-orbital sinus into EDTA containing tubes.
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: No
- How many animals: all animals
- Parameters examined: Hemoglobin (Hb), hematocrit (PCV), erythrocyte count, reticulocyte count, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelet count, total and differential leukocyte counts, prothrombin time and activated thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected on the day following the final exposure from the retro-orbital sinus into dry serum separation tubes.
- Anaesthetic used for blood collection: Yes ((methoxyflurane)
- Animals fasted: No
- How many animals: all animals
- Parameters examined: glucose, total protein, albumin, globulin, urea nitrogen, creatinine, bilirubin (total, conjugated and unconjugated), Na+, K+, chloride, inorganic phosphorus, aspartate and alanine aminotransferases, alkaline phosphatase, creatinine kinase and lactate dehydrogenase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes. After 9 exposures, 10 rats per sex per group were necropsied on study Day 12, the day immediately following the last exposure. Another 10 rats per sex per group were designated as recovery animals and necropsied on study Day 40 in order to evaluate the progression or resolution of any tissue changes occurring for signs of moribundity or mortality. At each necropsy, the liver, lungs, spleen, brain, adrenals and kidneys from all rats and the testes from all males were weighed.

HISTOPATHOLOGY: Yes. Microscopic evaluation of tissues from all major organs systems was performed on the control and 250 ppm exposure groups and the respiratory tract on the 25 ppm and 100 ppm exposure groups at the interim necropsy. Microscopic evaluation of tissues from all recovery groups animals included the respiratory tract.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs of toxicity were observed in the 100 ppm and 250 ppm exposure groups, primarily during the exposure period. These signs included rales, alopecia, rough coat, and staining of the coat. The incidence of these signs progressively decreased during the recovery period. Additionally, accumulations of red material around the eye were observed immediately at the end of the exposure period (study day 11). These accumulations were absent on the following morning (study day 12). No eye changes were observed in the recovery animals.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant body weight gain reductions were observed in the 250 ppm rats during the exposure period. A large body weight loss was noted in males and females of the high dose group during study days 1-5. Body weights in the 250 ppm exposure groups quickly regained and by the end of the recovery period. The observations in the high dose group were attributed and considered to be toxicologically relevant. Reduced body weight gains (statistically not significant) were further observed in the 25 ppm and 100 ppm exposure groups, but as the reduction was minimally lower on some days only, the observation was considered not toxicologically relevant. At the end of the recovery period, body weights for all test item exposed groups of both sexes were similar to their respective controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematology results showed a small, not dose-related decrease in MCV for the 100 and 250 ppm groups of males, which was not present at the end of the recovery period. Lymphocyte changes were noted at the ends of the exposure and the recovery periods. Although there were increases of differential leukocyte counts in both males and females, this was only exposure concentration-related in females at the end of the recovery period. For absolute lymphocyte counts an exposure concentration relationship was seen in males and females, but not at the end of the recovery period. All alterations in haematology parameters were of minor degree and not clearly attributed to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the exposure period, decreased alkaline phosphatase, total protein, and globulin levels were observed in the 100 ppm male rats and 250 ppm exposure groups of both sexes. In the high exposure group male rats, elevated glucose and ALT levels were observed. In the female rats, reduced BUN values in all test item treated groups and reduced triglyceride and LDH levels were observed in the 250 ppm group. At the end of the recovery period, reduced BUN levels were observed in the 100 ppm and 250 ppm males and females. In the 250 ppm exposure groups, elevated alkaline phosphatase and reduced phosphorus and triglyceride levels were observed in the female rats. All test item treated males had elevated phosphorus levels. The observations were considered to be of minor degree and not clearly attributed to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute organ weight was reduced for liver and spleen in males and females of the high dose group at the end of the exposure period (please refer to Table 1 under "Any other information on results incl. tables"). The observation was in line with a generally reduced body weight at sacrifice and considered treatment-related. Increases in organ-to-body weight ratios (statistically not significant) were further noted in most of the other organs (brain, adrenals, kidneys and testes) in these exposure groups and considered to be the result of reductions in body weight and not the direct result of insult to these organs.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A number of nasal lesions in the anterior nasal passages were found in the respiratory tract of the animals of the 100 and 250 ppm groups (please refer to "Histopathological finings, non-neoplastic").
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment related histopathological lesions at the end of the exposure were confined to the respiratory tract. Nasal lesions were located in the anterior nasal passage and took the form mainly of squamous metaplasia but also minimal goblet cell hypertrophy. All animals of the 100 and 250 ppm dose group and 8/10 males and 3/10 females of the 25 ppm group were affected. At the end of the recovery period, there was partial to complete resolution of the anterior nasal squamous metaplasia, being present only in 1/10 males and 4/10 females of the high-concentration group and in 2/10 males in each of low and mid concentrations groups. Olfactory atrophy, with occasional small foci of necrosis, and associated with mild neutrophilic infiltration, was seen in all animals of the 250 ppm group, and 5 females of the 100 ppm group at the post-exposure sacrifice. These findings were still present in the high-concentration group at the recovery sacrifice. Mild to moderate squamous metaplasia of the laryngeal epithelium was seen in the 250 ppm males and females, but resolved at the end of the recovery period. Tracheal epithelial squamous metaplasia was seen at the end of the exposure period in 8/10 males and 3/10 females of the high-concentration group and minimal squamous metaplasia was present in the major bronchi in 8/10 males and in 3/10 females at the high concentration group. Histopathological evaluations of the recovery period indicated a partial to complete resolution of the lesions.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
25 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: corresponding to an analytical concentration of 23.6 ± 0.9 ppm, 0.1016 mg/L air or 101.6 mg/m3 air
Remarks:
(dose levels in mg/m3 air were calculated as follows: [dose level in ppm x molecular weight] / 24.2)
Key result
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: corresponding to an analytical concentration of 246.2 ± 9.8 ppm, 1.06 mg/L air or 1059.8 mg/m3 air
Remarks:
(dose levels in mg/m3 air were calculated as follows: [dose level in ppm x molecular weight] / 24.2)
Key result
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
other: corresponding to an analytical concentration of 96.8 ± 3.8 ppm, 0.417 mg/L air or 416.7 mg/m3 air
Remarks:
(dose levels in mg/m3 air were calculated as follows: [dose level in ppm x molecular weight] / 24.2)
Key result
Critical effects observed:
not specified

Table 1: Statistically significant findings in absolute organ weight changes

End of
exposure		 Tissue Sex Organ weight [g] in mean ± SD
0 ppm 23.6 ppm 96.8 ppm 246.2 ppm
Liver M 5.92 ± 0.09 5.82 ± 0.13 5.46 ± 0.16 5.12 ± 0.18**
F 4.06 ± 0.07 3.97 ± 0.05 4.02 ± 0.08 3.60 ± 0.08**
Spleen M 0.465 ± 0.004 0.422 ± 0.017 0.411 ± 0.09** 0.368 ± 0.009**
F 0.345 ± 0.004 0.341 ± 0.005 0.336 ± 0.002 0.312 ± 0.007**
Conclusions:
Evidence for local toxicity was observed at all exposure levels, which did not fully resolve within 28 days of recovery. Therefore a local LOAEC of 23.6 ± 10.9 ppm (analytical concentration) was determined, corresponding to 0.1016 mg/L air or 101.6 mg/m3 air.
Systemic toxicity, evident as reduced body weight gain, was observed for the high dose group only, therefore a LOAEC of 246.2 ± 9.8 ppm was derived, corresponding to 1.06 mg/L or 1059.8 mg/mg3 air. The NOAEC for systemic toxicity was 96.8 ± 3.8 ppm, corresponding to 0.417 mg/L air or 416.7 mg/m3 air.

Endpoint:
sub-chronic toxicity: inhalation
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
data reported as part of a review, no experimental details given
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
inhalation
Duration of treatment / exposure:
100 days
Dose / conc.:
0.006 mg/m³ air
Dose / conc.:
0.06 mg/m³ air
Dose / conc.:
0.6 mg/m³ air
No. of animals per sex per dose:
10
Control animals:
not specified
Dose descriptor:
NOAEC
Effect level:
0.006 mg/m³ air
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: no adverse effect observed
Critical effects observed:
not specified

Effects on redox processes and functions in the liver observed (reduced oxygen demand, reduced catalase activity), reduced blood levels of lactic and pyruvic acid observed, CNS effects noted. No further details were given in the study summary.

Conclusions:
The available information comprise only summary data but no experimental details. Based on the lack of data, it is not possible to derive a NOAEC for systemic toxicity.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
217.4 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate, reliable (Klimisch score 2) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The study was not performed according to modern standards. The dose levels were to high and caused severe local effects. Furthermore the skin was abraded in half of the animals. Purity not stated, too few animals, no statistical evaluation, no details on experimental procedure, type of coverage or frequency of observations, half of the animals were treated at abraded skin, body weight and food consumption were not recorded, few parameters in haematology, clinical chemistry and urinalysis investigated, time of death for individual animals unclear
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Repeated dose dermal toxicity
- Short description of test conditions: Male and female rabbits (6/sex/group) were dermally treated at 0.2, 0.4, 0.8 and 1.6 mL/kg bw/day for 3 consecutive weeks. The animals were exposed 5 days/week, 6 h exposure per day.
- Parameters analysed / observed: clinical signs, mortality, dermal reactions, gross pathology and histopathology.

Deficiencies when compared to OECD guideline 410:
The study was not performed according to modern standards. The dose levels were too high and caused severe local effects. Furthermore the skin was abraded in half of the animals. The analytical purity of the test material was not stated, too few animals, no statistical evaluation, no details on experimental procedure, type of coverage or frequency of observations, body weight and food consumption were not recorded, few parameters in haematology, clinical chemistry and urinalysis investigated and time of death for individual animals unclear.
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: Animals were treated in the dorsal region 24 h after preparation (shaving and scarification).
- Time intervals for shavings or clipplings: The animals were shaved and then closely shaved again. After 24 h, 12 superficial scarifications (3 cm length and spaced 2 cm apart) were made in half of the animals.

REMOVAL OF TEST SUBSTANCE
- Washing: The treated zones were washed with lukewarm water.
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied: 0.2, 0.4, 0.8 and 1.6 mL
Duration of treatment / exposure:
21 days
Frequency of treatment:
daily, 5 days/week
Dose / conc.:
0.2 other: mL/kg bw/day
Remarks:
corresponding to 207 mg/kg bw/day
Dose / conc.:
0.4 other: mL/kg bw/day
Remarks:
corresponding to 414 mg/kg bw/day
Dose / conc.:
0.8 other: mL/kg bw/day
Remarks:
corresponding to 829 mg/kg bw/day
Dose / conc.:
1.6 other: mL/kg bw/day
Remarks:
corresponding to 1658 mg/kg bw/day
No. of animals per sex per dose:
6
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Doses were selected based on the results of a preliminary 4-days range-finding study in which skin reactions were assessed for doses in the range of 1 and 2 mL/kg bw/day.
- No post-exposure recovery group included.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: No data

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before and at the end of treatment
- Parameters examined: leukocyte count
- How many animals: all surviving animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before and at the end of treatment
- Parameters examined: glucose, urea, bilirubin, alkaline phosphatase, serum glutamate pyruvic transferase, serum glutamate oxaloacetic transaminase
- How many animals: all surviving animals

URINALYSIS: Yes
- Time schedule for collection of urine: before and at the end of treatment
- Parameters examined: pH, protein, glucose, ketone bodies, occult blood
- How many animals: all surviving animals

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1.6 mL/kg bw/day: Paraplegia and loss of movement coordination were observed and considered as neurotoxic effects. The animals died within 48 h. The effects were considered adverse and related to treatment.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
A dose-related increase in the intensity of edema was observed. There was no perceptible difference between scarified and non-scarified animals. At 1.6 mL/kg bw/day 6/12 animals showed slight erythema. the observations were considered treatment-related and of toxicological relevance. Despite daily washings the cutaneous alteration induced a formation of a thick crust, cracked and dry, which persisted until study termination.
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality occured from the second day of dosing in all test item treated groups:
0.2 mL/kg bw/day: 2/12 animals died
0.4 mL/kg bw/day: 3/11 animals died
0.8 mL/kg bw/day: 10/12 animals died
1.6 mL/kg bw/day: 12/12 animals died
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight development was disturbed in the animals which died or had to be sacrificed.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematology of surviving animals (0.2 and 0.4 mL/kg bw/day) was not affected by treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was a slight, statistically not significant increase of uremia in the non-sacrificed treated animals. The effect was not considered treatment-related.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
1.6 mL/kg bw/day: Paraplegia and loss of movement coordination were observed and considered as neurotoxic effects. The animals died within 48 h. The effects were considered adverse and related to treatment.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The thickness of the skin (described as oedema) increased immediately after exposure in all animals; it was proportional to the applied dose. At high doses slight erythema was observed as well. Acanthosis, hyperkeratosis and dryness was observed up to the end of the treatment.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
LOAEL
Effect level:
ca. 0.2 other: mL/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
mortality
Remarks on result:
other: corresponding to 207 mg/kg bw/day
Critical effects observed:
not specified
Conclusions:
The test item repeatedly applied by cutaneous administration induced skin necrosis at all tested dose levels. At the highest dose (1.6 mL/kg bw/day) death occurred between study Day 2-4. Clinical signs at this dose level included paralysis and motion disorders. No clinical signs were seen at the lower dose levels. A death occurred also in the lowest dose group, a NOAEL could not be determined.
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted 12 May 1981
Deviations:
yes
Remarks:
limited study duration including 9 days treatment instead of 21/28 days, treatment 5 days/week instead of daily application, test material not fully characterised
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 165.3 - 195.5 g (males) and 182.5 - 216.0 g (females)
- Housing: individually in stainless steel wire mesh cages
- Diet: Certified Rodent Diet No. 5000 (PMI Nutrition International, St. Louis, Missouri), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 24 - 70

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: The doses were applied to the clipped dorsal trunk skin.
- Type of wrap if used: The treated areas were covered with a layer of 8-ply gauze and impervious plastic, which was secured with an adhesive elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: The test sites were wiped free of excess test material using gauze soaked in saline.
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount applied: 0.05, 0.2 and 0.5 mL/kg bw/day, corresponding to 52.7, 210.8 and 527 mg/kg bw/day
- Constant volume or concentration used: no

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 h
Frequency of treatment:
6 hours/day for 9 days (5 days in first week, 4 days in second week)
Dose / conc.:
0.05 other: mL/kg bw
Remarks:
corresponding to 52.7 mg/kg bw/day
Dose / conc.:
0.2 other: mL/kg bw
Remarks:
corresponding to 210.8 mg/kg bw/day
Dose / conc.:
0.5 other: mL/kg bw
Remarks:
corresponding to 527 mg/kg bw/day
No. of animals per sex per dose:
Main group: 10
Recovery group: 5 (control and high dose group only)
Control animals:
yes
Details on study design:
- Dose selection rationale: Dose levels were chosen based on a preliminary range-finding study, which was performed in 3 rats/sex with treatment for 5 consecutive days. 1/3 males and 1/3 females treated at 1.0 mL/kg bw/day died on study Day 4. There were no clinical signs of toxicity and no abnormalities in the Irwin Screen. Local skin irritation, seen at 0.5 and 1.0 mL/kg bw/day, was mainly mild erythema. Body weight losses at 0.5 mL/kg bw/day and 1.0 mL/ kg bw/day were significantly greater than for the control group. Except for the 1.0 mL/kg bw/day males, all groups gained weight by the fifth day of dosing. Based on these findings, it was considered that applied doses of 0.05, 0.2, and 0.5 mL/kg bw/day were appropriate for the full 9-day study.
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 28 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during the exposure period, once weekly during the recovery period
- Cage side observations included: local skin lesions and signs of toxic or pharmacological effects and mortality

DERMAL IRRITATION: Yes
- Time schedule for examinations: daily during the exposure period, once weekly during the recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: on study Days 1, 3, 5, 8 and 10 during the exposure period and on Day 15 and weekly thereafter during the recovery period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measured for a week prior to dosing, over Days 1-3, 3-5, 5-8 and 8-10 during the exposure period and for recovery animals over six daily intervals.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Prior to dosing, over Days 1-3, 3-5, 5-8 and 8-10 during the exposure period and for recovery animals over six daily intervals.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected before sacrifice from the retro-orbital venous plexus and used for chemical pathological measurements.
- Anaesthetic used for blood collection: no data
- Animals fasted: no data
- How many animals: all animals
- Parameters checked: hemoglobin concentration, hematocrit, erythrocyte count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet and reticulocyte counts, total and differential leukocyte counts, prothrombin time, and activated partial thromboplastin time.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected before sacrifice from the retro-orbital venous plexus and used for chemical pathological measurements.
- Anaesthetic used for blood collection: no data
- Animals fasted: no data
- How many animals: all animals
- Parameters checked: urea nitrogen, glucose, total protein, albumin, globulin, bilirubin, sodium, potassium, calcium, chloride, inorganic phosphorus, aspartate and alanine aminotransferases, alkaline phosphate, lactate and sorbitol dehydrogenases, creatine kinase, and y-glutamyl transferase.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected over a 2 h interval from trays under the animal’s cage, and over 16 h from animals in metabolism cages.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: Appearance, microscopy, pH, protein, ketones, bilirubin, occult blood, urobilinogen, glucose, creatinine, specific gravity, osmolality, and N-acetyl-ß-D-glucosaminidase (NAG) activity.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before dosing, after the fifth dosing and before necropsy. Recovery animals were also evaluated the day following the cessation of dosing and prior to sacrifice.
- Dose groups that were examined: all groups
- Type of test: Irwing Screen, functional observation battery
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Necroscopic examination was conducted on all animals sacrificed by CO2 inhalation at the ends of the dosing and recovery periods to detect any indications of gross pathological change. Complete macroscopic postmortem examinations were conducted on all animals.
- The following organs were removed for weighing: brain, adrenal glands, liver, kidneys, ovaries, and testes.

HISTOPATHOLOGY: Yes. Histopathological evaluation of selected tissues (brain, kidneys, nerves, skin, spinal cord and testes) were performed on controls and high dose animals. Tissues and organs were removed, fixed in 10% neutral-buffered formalin, and then processed for light microscopic examination.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Local signs of irritation were barely perceptible erythema in a few of the low-dose animals. Most mid- and high-dose animals showed very slight to slight erythema. By Day 18, all high-dose recovery animals were free of skin irritation.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains of the high-dose males and females were lower than for the controls over the treatment period. At the end of the dosing period (Day 12) high-dose males had mean body weights 7% lower than controls, and high-dose females 5% lower. Body weights for the mid- and low-dose groups were comparable to the controls. Gains during the recovery period were comparable for control and high-dose animals.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption per bw. in all treated groups of males was 10 to 15% (p<0.01) higher than in controls from Day 5 until Day 10 but not dose-related. The effect was considered to be non-adverse.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment related histopathologic findings in the skin of the high-dose animals consisted of an occasional trace to mild inflammatory cell infiltrates in the dermis. The effect was considered adverse.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mild to moderate hyperkeratosis and acanthosis were observed in all animals of all dose groups at the treated skin site and acute inflammatory cell infiltration was noted in high dose-groups animals only. The effects were considered treatment-related and toxicologically relevant.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
527 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed
Remarks on result:
other: corresponding to 0.5 mL/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
local
Effect level:
527 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
histopathology: non-neoplastic
Remarks on result:
other: corresponding to 0.5 mL/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
210.8 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed
Remarks on result:
other: corresponding to 0.2 mL/kg bw/day
Key result
Critical effects observed:
no
Conclusions:
Application of the test material to the skin of Sprague Dawley rats for a period of 9 days produced mild local skin reactions at 0.2 mL/kg bw/day (corresponding to 210.8 mg/kg bw/day) and mild to moderate local skin reactions at 0.5 mL/kg bw/day (corresponding to 527 mg/kg bw/day). The findings of the high dose group persisted until study Day 17. Based on these findings, a LOAEL for local toxicity of 527 mg/kg bw/day was derived and a NOAEL for local toxicity of 210.8 mg/kg bw/day. The NOAEL for systemic toxicity was 0.5 mL/kg bw/day (corresponding to 527 mg/kg bw/day).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
527 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate, reliable (Klimisch score 2) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose studies by oral, inhalative and dermal exposure are available.

 

Oral:

Oral repeated dose toxicity was investigated in a non-GLP study conducted similarly to OECD guideline 407 (79-0022-DKT). The test item was administered by oral gavage to groups of 10 rats/sex at dose levels of 0.02, 0.1 and 0.5 mL/kg bw/day, corresponding to 20.72, 103.6 and 518 mg/kg bw/day. The animals were treated for 28 days, 6 days/week. In addition, a group of 10 rats/sex received the vehicle (water) and served as control. No recovery group was included.

The animals were observed daily for mortality and clinical signs of toxicity; body weight and food consumption was recorded weekly. Blood samples for haematology and clinical chemistry were collected after 3 weeks of treatment and prior to sacrifice, respectively. At study termination, gross- and histopathological examination was performed on kidneys, livers, spleens, lungs, hearts and testes.

There was no mortality and no clinical sings of toxicity that were attributed to treatment. Body weight gain was slightly decreased in males (-7.6%) and females (-2.9%) of the high dose group (518 mg/kg bw/day). The findings were in line with reduced food efficiency in these animals and considered to be treatment-related.

Animals of the high dose group further showed a reduced red blood cell count and reduced haemoglobin levels, but the difference only reached statistical significance in females. In addition, white blood cell counts were increased in males of the high dose group. The findings were accompanied by an increase in haematopoesis in the spleen and increased clinical chemistry parameters and were considered to be treatment-related.

Changes in clinical chemistry parameters among animals of the high dose group included increased bilirubin levels in animals of both sexes (statistically significant) and a slight increase in creatinine levels of males (69.1 µmol/L compared to 63.3 µmol/L in control). The latter observation was also made for males of the low dose group (68.3 µmol/L compared to 63.3 µmol/L in control), but without a clear dose-response and in the absence of findings in females the toxicological significance remained unclear.

The organ weight analysis and the gross macroscopic examination showed no treatment-related findings. At the histopathological examination, extramedullary haematopoesis and an increased amount of blood and brown pigment in the red pulp was found in the spleen of males and females of the high dose group. The observations were consistent with haematological and clinical chemistry findings and attributed to treatment.

Based on the results of this study, the NOAEL for systemic toxicity was 0.1 mL/kg bw/day, corresponding to 103.6 mg/kg bw/day in male and female rats.

In addition, data from a review article on oral repeated dose toxicity are available (Dimitriew, 1981). Test item doses of 4.4, 44 and 88 mg/kg bw/day were orally applied to male rats for 6 months. The available information comprises only summary data but no experimental details and no detailed information on results, therefore the study was considered to provide supporting information. Based on the lack of data, no NOAEL was derived.

 

Inhalation route:

Two experimental studies on repeated dose toxicity via the inhalation route are available. Both studies investigate inhalation repeated dose toxicity over a treatment period of 9 days at different exposure levels, using test materials of different purity. The study with the higher test material purity, in which the concentration of the impurity acrolein were below the detection limit, was selected as key study (Ballantyne 2000 (1)).

The key study (non-guideline) was performed according to GLP but with a treatment-period far below the treatment duration recommended in current guidelines (Ballantyne 2000 (1)). Groups of 5 Sprague-Dawley rats per sex were exposed for 6 h/day for a total of 9 treatment days. The treatment was performed for 5 consecutive days, followed by 2 days rest and then an additional 4 days exposure. The test item was administered as a vapour in the breathing air of the animals and applied via whole body inhalation at dose levels of 0.5, 5 and 50 ppm, corresponding to analytical concentrations of 2.0, 21.5 and 217.4 mg/m3air. A similar constituted group of animals was exposed to the air control.

During exposure and daily thereafter, the animals were observed for mortality and clinical signs of toxicity. Individual body weights were recorded prior to exposure and on study Days 7 and 11 and food consumption was measured for the first and second exposure week. One day before sacrifice, blood was collected for haematology and clinical chemistry. In addition, urine was collected over a 16-h interval following the final exposure. At scheduled necropsy, all animals underwent gross pathological examination, organ weight analysis and histopathological examination.

There was no mortality and no clinical signs of toxicity were observed throughout the study period. In addition, no exposure-related effects were observed regarding haematology, clinical chemistry, urinalysis or macroscopic and microscopic examination. The NOAEC for systemic toxicity was 50.5 ppm, corresponding to 217.4 mg/m3air.

 

The second inhalation repeated dose toxicity study was performed in Fischer 344 rats under exposure conditions similar to those of the above-mentioned study (Ballantyne (1)) with test material of lower purity (Ballantyne 2000 (2)). Test item doses of 25, 100 and 250 ppm, corresponding to analytical concentrations of 101.6, 416.7 and 1059.8 mg/m3air were applied via whole body inhalation. Groups of 10 rats/sex/dose level were exposed for 6 h/day for a total of 9 treatment days. The treatment was performed for 5 consecutive days, followed by 2 days rest and then an additional 4 days exposure. Air-control animals and a group of 10 recovery animals/sex treated at the high dose level were included.

During exposure and daily thereafter, the animals were observed for mortality and clinical signs of toxicity. Individual body weights were recorded prior to exposure and on study Days 1, 2, 5, 8 and 9. For recovery animals, body weight was further recorded on post exposure days 8, 15, 22 and 29. Following the final exposure, blood was collected for the assessment of haematology and clinical chemistry parameters. After 9 exposures, 10 rats per sex per group were necropsied on study Day 12, the day immediately following the last exposure. Recovery animals were necropsied 28 days after the final exposure ended in order to evaluate the progression or reversal of any treatment-related changes.

Clinical signs of toxicity were observed in the 100 ppm and 250 ppm exposure groups, primarily during the exposure period. These signs included rales, alopecia, rough coat, and staining of the coat. The incidence of these signs progressively decreased during the recovery period. Additionally, accumulations of red material around the eye were observed immediately at the end of the exposure period (study Day 11). These accumulations were absent on the following morning (study Day 12). No eye changes were observed in the recovery animals.

Significant body weight gain reductions (> 10%) were observed in the 250 ppm rats during the exposure period. A large body weight loss was noted in males and females of the high dose group during study Days 1-5. Body weight in the 250 ppm exposure recovery group increased following the end of the exposure period and was similar to the body weight of the control group by the end of the recovery period. The observations in the high dose group were considered to be toxicologically relevant. Reduced body weight gains (statistically not significant) were further observed in the 25 ppm and 100 ppm exposure groups, but as the reduction was minimally lower on some days only, the observation was considered not toxicologically relevant. At the end of the recovery period, body weights for all test item exposed groups of both sexes were similar to their respective controls.

Hematology results showed a small, not dose-related decrease in MCV for the 100 and 250 ppm groups of males, which was not present at the end of the recovery period. Significantly increased levels of lymphocytes (absolute and differential) were noted at the end of the exposure and the recovery period. Although there were increases of differential leukocyte counts in both males and females, this was only exposure concentration-related in females at the end of the recovery period. For absolute lymphocyte counts, an exposure concentration relationship was seen in males and females, but not at the end of the recovery period. All alterations in haematology parameters were considered not to be  treatment-related.

At the end of the exposure period, significantly decreased alkaline phosphatase levels, total protein, and globulin levels were observed in the 100 ppm male rats and 250 ppm exposure groups of both sexes. In the high exposure group male rats, significantly elevated glucose and alanine aminotransferase levels were observed. In the female rats, significantly reduced blood urea nitrogen (BUN) values in all test item treated groups and reduced triglyceride and lactate dehydrogenase levels were observed in the 250 ppm group. At the end of the recovery period, significantly reduced BUN levels were observed in the 100 ppm and 250 ppm males and females. In the 250 ppm exposure groups, significantly elevated alkaline phosphatase and reduced phosphorus and triglyceride levels were observed in the female rats. All test item treated males had elevated phosphorus levels. The observations were not considered to be treatment-related, as they were reversible and/or the difference compared with the control was minimal.

Absolute organ weight was significantly reduced for liver and spleen in males and females of the high dose group at the end of the exposure period. The observation was in line with a generally reduced body weight at sacrifice and considered treatment-related. Increases in organ-to-body weight ratios (statistically not significant) were further noted in most of the other organs (brain, adrenals, kidneys and testes) in these exposure groups and considered to be the result of reductions in body weight and not the direct result of insult to these organs. At macroscopic and microscopic examination, a number of nasal lesions in the anterior nasal passages were found in the respiratory tract of the animals of the 100 and 250 ppm groups. Nasal lesions were located in the anterior nasal passage and took the form mainly of squamous metaplasia but also minimal goblet cell hypertrophy. All animals of the 100 and 250 ppm dose group and 8/10 males and 3/10 females of the 25 ppm group were affected. At the end of the recovery period, there was partial to complete resolution of the anterior nasal squamous metaplasia, being present only in 1/10 males and 4/10 females of the high-concentration group and in 2/10 males in each of low and mid concentrations groups. Olfactory atrophy, with occasional small foci of necrosis, and associated with mild neutrophilic infiltration, was seen in all animals of the 250 ppm group, and 5 females of the 100 ppm group at the post-exposure sacrifice. These findings were still present in the high-concentration group at the recovery sacrifice. Mild to moderate squamous metaplasia of the laryngeal epithelium was seen in the 250 ppm males and females, but resolved at the end of the recovery period. Tracheal epithelial squamous metaplasia was seen at the end of the exposure period in 8/10 males and 3/10 females of the high-concentration group and minimal squamous metaplasia was present in the major bronchi in 8/10 males and in 3/10 females at the high concentration group. Histopathological evaluations of the recovery period indicated a partial to complete resolution of the lesions.

In conclusion, effects indicating local toxicity was observed at all exposure levels, which did not fully resolve within 28 days of recovery. Therefore a local LOAEC of 25 ppm (nominal concentration) was determined, corresponding to 101.6 mg/m3 air. Systemic toxicity, evident as reduced body weight gain, was observed for the high dose group, therefore a LOAEC of 250 ppm was derived, corresponding to 1059.8 mg/mg3 air. The NOAEC for systemic toxicity was 100 ppm, corresponding to 416.7 mg/m3 air. The study was considered to provide supporting information because the test material contained the impurity acrolein, which was detected by analytical determination in the exposure chamber. Thus, it was not clear, whether the adverse effects observed were attributed to the test item or the irritating effects of the impurity.

 

In addition, data from a review article on inhalation repeated dose toxicity are available (Dimitriew, 1981). Groups of 10 rats/sex were exposed to test item doses of 0.006, 0.06 and 0.6 mg/m3air over a treatment period of 100 days. The available information comprises only summary data but no experimental details and no detailed information on results, therefore the study was considered to provide supporting information. Based on the lack of data, no NOAEC was derived.

 

Dermal route:

Two experimental studies on dermal repeated dose toxicity are available for the test substance and one less reliable study, which was not performed according to modern standards (73-0036-FKT).

A 9-day dermal repeated dose toxicity study was performed in accordance with GLP and using test material of known purity (Ballantyne 2000). In the 9 day dermal toxicity study, groups of 10 Sprague-Dawley rats per sex were exposed to the test item for 6 h/day for a total of 9 treatment days. The animals were exposed on 5 consecutive days, followed by 2 days rest and then 4 days exposure under occluded conditions and at dose levels of 0.05, 0.2 and 0.5 mL/kg bw/day, corresponding to 52.7, 210.8 and 527 mg/kg bw/day. A similar constituted group of animals remained untreated and served as control. For the control and high dose group, 5 rats/sex were additionally included as a recovery group. The animals were treated under the same conditions but observed for a 28-day recovery period following exposure.

The animals were inspected for mortality, clinical signs of toxicity and signs of dermal irritation, daily during the exposure period and once weekly during the recovery period. Body weights were recorded on study Days 1, 3, 5, 8 and 10 during exposure and on Day 15 and weekly thereafter during the recovery period. In addition, food and water consumption were monitored in 3-day intervals during exposure and in 6-day intervals during the recovery period. Prior to sacrifice, blood was collected for haematology and clinical chemistry and urine was sampled for 18 hours for urinalysis. In addition, a neurobehavioral test was performed prior to dosing, after the fifth dosing and before necropsy. Recovery animals were additionally evaluated the day following cessation of treatment.

All animals underwent complete macroscopic examination at scheduled necropsy and selected organs were weighed. Histopathological examination was performed in control and high dose group animals.

There were no signs of clinical toxicity or mortality in any animal that was considered to be related to treatment. Local signs of irritation at the test site were barely perceptible erythema in a few of the low-dose animals. Most mid- and high-dose animals showed very slight to slight erythema at the test site. By Day 18, all high-dose recovery animals were free of skin irritation.

Slight reductions in body weight gain were observed in males (- 7%) and females (- 5%) for the high dose group. Body weight gains during the recovery period were comparable for control and high-dose animals. The mean food consumption of males in all dose groups was 10 – 15% increased (statistically significant) from study Day 5 – 10 when compared to controls, but as a dose response relationship was lacking the effects were considered to be non-adverse.

There were no treatment-related effects on haematology or clinical chemistry and no adverse effects in neurobehavior. Treatment related histopathologic findings in the skin of the high-dose animals consisted of an occasional trace to mild inflammatory cell infiltrates in the dermis. In addition, mild to moderate hyperkeratosis and acanthosis were observed in all animals of all dose groups at the treated skin site and acute inflammatory cell infiltration was noted in high dose-groups animals only. The effects were considered treatment-related and toxicologically relevant.

Under the conditions of the study, the NOAEL for systemic toxicity was 0.5 mL/kg bw/day, corresponding to 527 mg/kg bw/day. The NOAEL for local toxicity was was 0.2 mL/kg bw/day, corresponding to 210.8 mg/kg bw/day.

 

In a supporting dermal repeated dose toxicity study (73-0036-FKT), 6 male and 6 female rabbits/group were dermally treated at 0.2, 0.4, 0.8 and 1.6 mL/kg bw/day, corresponding to 207, 414, 829 and 1658 mg/kg bw/day. The animals were exposed 5 days/week, 6 hours per day for 3 consecutive weeks. The dose levels were too high when compared to doses recommended in current OECD guidelines as they caused treatment-related mortality with deaths among all dose groups. At the highest dose (1.6 mL/kg bw/day) death occurred between study Day 2 - 4. Clinical signs at this dose level included paralysis and motion disorders. Animals of all dose levels showed skin necrosis at all tested dose levels. A NOAEL could not be derived.

Justification for classification or non-classification

The available information on oral repeated dose toxicity do therefore not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification. In addition, the available data on oral repeated dose toxicity do not meet the criteria for classification according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.

The available data on dermal and inhalation exposure did not reveal systemic treatment-related toxicity or toxicologically significant effects. Therefore, the available data on dermal and inhalation repeated dose toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 or according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations, and are therefore conclusive but not sufficient for classification.