Registration Dossier

Administrative data

Description of key information

Oral (2-year feeding study): NOAEL = 1000 mg/kg bw/day (2-year feeding study)

Inhalation (28-day, OECD 412): NOAEC = 60 mg/m3 air

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:
supporting study
Study period:
22 Dec 2009 - 29 Mar 2010
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study, but with significant deviations. Since this study was a range-finder for a reproductive toxicity study, only 3 animals per sex and dose were used in the test and major endpoints for the evaluation of systemic toxicity such as haematology, histopathology and clinical biochemistry were not addressed. Thus, this study is insufficient for the evaluation of systemic toxicity after repeated exposure.
Qualifier:
according to
Guideline:
other: OECD 421
Deviations:
yes
Remarks:
only 3 animals per sex per dose level, no haematology, no clinical biochemistry, no histopathology
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 200-260 g (males) and 140-200 g (females)
- Housing: animals were housed in groups of up to three in open macrolon cages type 2000P (TechniPlast).
- Diet: maintenance diet for rats and mice, No. 1324 TPF, ad libitum.
- Water: autoclaved community tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The suspension of the test item in corn oil was prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): As the test item’s solubility in water is poor, corn oil was used as an organic solvent. Pre tests showed that the solubility of the test item in corn oil was sufficient. Corn oil is well investigated as vehicle for toxicity studies, and it is explicitly recommended in the OECD 421.
- Amount of vehicle: 4 mL
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly and once before beginning of application

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly and once before beginning of application. Females during pregnancy: Day 0, 7, 14, 20, within 24 h post parturition and Day 4 post partum.

FOOD CONSUMPTION: Yes
- Time schedule: once weekly

WATER CONSUMPTION: Yes
- Time schedule: twice weekly
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: laboured breathing, increased salivation and bleeding at the mucous membranes
Mortality:
mortality observed, treatment-related
Description (incidence):
1000 mg/kg bw/day: laboured breathing, increased salivation and bleeding at the mucous membranes
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):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
At Day 1, all animals treated with the high dose of the test item wiped their mouth in the cage bedding material immediately after application for approximatel 30 seconds, indicating discomfort. From Day 1 to approximately Day 6, all animals from the high dose group showed laboured breathing, increased salivatio and in two instances bleedings at the mucous membranes of mouth and nose. No bleedings or laboured breathing were observed after week one of application. Other than these, no clinical / behavioural signs, stereotypies, or behavioural reactions were observed.

BODY WEIGHT AND WEIGHT GAIN
No effects on body weight gain were observed in animals of the low- and high-dose group. The mean body mass and the mean relative body mass gain of male rats treated with the high dose were slightly reduced in week one when compared to the vehicle control group, but a net body mass loss did not occur. As a tendency, that effect was present but reduced during week two of the pre-mating phase. However, since this tendency diminished towards the end of the in-life phase, a clear indication for toxicity of the test item at the high dose could not be substantiated.

FOOD CONSUMPTION
No changes in food consumption of the animals were observed.

WATER CONSUMPTION AND COMPOUND INTAKE
No changes in daily mean relative water consumption of the animals were apparent.

NECROPSY FINDINGS
No irregular findings indicating toxicological effects of the test item were detected at necropsy (no further information).
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Critical effects observed:
not specified
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Principles of method if other than guideline:
2-year chronic oral toxicity study in 200 rats.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Albino Sherman Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 100-150 g
- Housing: animals were housed together (no further information)
- Diet: animal colony diet supplemented twice weekly with fresh meat, ad libitum
- Water: ad libitum
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
The substance powder was mixed intimately with the diet in the desired concentrations.

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
24 months
Frequency of treatment:
7 days/week
Dose / conc.:
0.05 other: % nominal in the diet
Remarks:
The concentration of test material in the low dose group was increased to 2% after 6 months
Dose / conc.:
0.2 other: % nominal in the diet
Dose / conc.:
1 other: % nominal in the diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: dosage levels, to be fed for two years, were premised on a maximum possible exposure in humans.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes (no further information)

BODY WEIGHT: Yes, at end of Week 1 and 2

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 30, Day 90, 6 months and 24 months
- Parameters: red and white blood cell count, haemoglobin content and differential count

OTHER
FERTILITY ASSESSMENT: Yes (no further information)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Liver, spleen, heart, lungs, stomach, large intestine, small intestine, adrenal glands, gonads, pancreas and brain.
HISTOPATHOLOGY: Yes. Liver, spleen, heart, lungs, stomach, large intestine, small intestine, adrenal glands, gonads, pancreas and brain.
Rats were killed at 1, 3, 6 and 24 months for necropsy and tissues collected for microscopic examinations (no further information)
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):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
2% and 1 % in the diet: at 24 months minor hyperplasia of the stratified squamous epithelium with excess keratin formation of the cardiac mucosa of the stomach
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: At 1, 3 and 6 months, no significant differences were observed in mortality.

BODY WEIGHT AND WEIGHT GAIN: At 1, 3 and 6 months, no significant differences were observed in body weight gain.

HAEMATOLOGY: At 1, 3 and 6 months, no significant differences were observed in haematology.

GROSS PATHOLOGY: At 24 months, the only consistent difference that could be attributed to the test article was minor hyperplasia of the stratified squamous epithelium with excess keratin formation of the cardiac mucosa of the stomach in rats receiving the highest exposure to the test article (group 1: 2% in the diet after 6 months, group 3: 1% in the diet).

OTHER FINDINGS: No significant differences were observed in fertility in the 2 year study.
Dose descriptor:
NOAEL
Effect level:
>= 2 other: % in the diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14 Feb 1995 - 17 May 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(adopted 12 May 1981)
Deviations:
no
Qualifier:
according to
Guideline:
other: FDA (United States Food and Drug Administration): Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food, 1986, Appendix II page 19 "Subchronic Oral Toxicity Studies."
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Albino rats (Outbred) VAF/Plus CD (Sprague-Dawley derived) (Crl:CD BR)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. New York, USA
- Age at study initiation: 43 days
- Weight at study initiation: 197 g (175-219, males), 156 g (140-178, females)
- Housing: in group of two per cage per sex in elevated stainless steel wire mesh cages (during acclimatisation period, individually thereafter)
- Diet: Certified Rodent Diet, No. 5002 (Neal) (PMI Feeds, Inc., St. Louis, NO, USA), ad libitum
- Water: via automated watering systems, ad libitum
- Acclimation period: at leasrt 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25 (desired), 20-24 (actual); monitored and recorded twice daily
- Humidity (%): 40-70 (desired), 22-64 (actual); monitored and recorded once daily
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(sterile, distilled)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Appropriate amounts of test substance were mixed in the vehicle weekly to yield dosing solutions of 3, 10, and 25 mg/mL. Individual doses were adjusted by most recent weekly body weight. After mixing, dosing solutions werre filter sterilised (0.2 µm filter) and distributed into sterile vials for individual use. Vials were stored under refrigeration. Fresh dosing solutions were prepared onnce weekly.

VEHICLE
- Amount of vehicle (if gavage): constantly 10 mL/kg bw/dose
- Lot/batch no. (if required): 50988
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine stability, homogeneity, and concentration of the test substance with carriers under conditions of this study were performed and the results were presented in the study period.
Prior to initiation of the study batches of low- and high-concentration dose solutions were prepared. Three samples each from the top, middle and bottom portion of each mixture were taken for analysis.
Stability of the test material in the dose solution was determined for at least 2 weeks. Samples were stored at room temperature and for the first 4 days and then refrigerated for the remaining stability test intervals. Duplicate samples of the low- and high-concentration dose solutions were assayed 4, 7, and 14 days after preparation.
All 3 dose levels were assayed weekly for the first 4 weeks (one sample per concentration and 2 samples were analysed). Subsequent assays were performed at monthly intervals for the remainder of the study.
Duration of treatment / exposure:
91 or 92 days (depending on the day of sacrifice)
Frequency of treatment:
once daily (7 days/week)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon
- Cage side observations included: mortality and gross signs of toxicologic or pharmacologic effects

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pretest and weekly thereafter
- Detailed clinical observations included: detailed physical examinations for signs of local or systemic toxicity, pharmacologic effects and palpation for tissue masses (behaviour: aggressiveness, increased or decreased activity; respiration: nasal discharge and rales; ocular: chromodacryorrhea, excessive lacrimation and percent specify; appearence: alopecia, ano-genital staining and general condition; gastrointestinal: abdominal shape and faecal consistency, palpation for tissue masses)

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest, weekly during treatment and a fasted body weight just prior to necropsy

FOOD CONSUMPTION:
- Time schedule for examinations: weekly, beginning one week prior to treatment
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretest (08 Feb 1995) and at termination (15 May 1995)
- Ophthalmoscopic observations included: lids, lacrimal apparatus and conjunctiva examined grossly; cornea, anterior chamber, lens, vitreous humor, retina and optic disc examined by indirect ophthalmoscopy (Opticyl 1% was used to induce mydriasis)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 1 (20 Feb 1995) and at termination (15/16 May 1995)
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: HGB (haemoglobin), HCT (haematocrit), RBC (erythrocyte count), PLT (platelet count), MCV (mean corpuscular volume), MCH (mean corpuscular haemoglobin), MCHC (mean corpuscular haemoglobin concentration), PT (prothrombin time), APTT (activated partitial thromboplastin time), WBC (total leucocyte count), differential lecucyte and erythrocyte morphology, absolute leucocytes and segmented neutrophils, absolute lymphocytes, absolute segmented neutrophils

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Week 1 (20 Feb 1995) and at termination (15/16 May 1995)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: AST (aspartate amino transferase (SGOT)), ALT (alanine aminotransferase (SGPT)), ALKP (alkaline phosphatase), BUN (blood urea nitrogen), CREAT (creatinine), GLU (glucose), CHOL (cholesterol (enzymatic)), TRIG (triglycerides), T PROT (total protein), ALB (albumin), GLOB (globulin), A/G (albumin/globulin ratio), T BILI (total bilirubin), Na+ (sodium), K+ (potassium), Cl- (chloride), Ca2+ (calcium), PHOS (inorganic phosphorus), GGT (gamma-glutamyl transferase)

URINALYSIS: Yes
- Time schedule for collection of urine: urine was collected over a 16 h interval
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No (neither were the animals water deprived)
- Parameters examined: volume, specific gravity

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Complete macroscopic examinations were performed on all animals including animals which died prior to study termination. The macroscopic postmortem examination included examination of the external surfaces and all orifices; the external surfaces of the brain and spinal cord; the organs and tissues of the cranial, thoracic, abdominal and pelvic cavities and neck; and the remainder of the carcass. Animals were fasted prior to scheduled sacrifice.

HISTOPATHOLOGY: Yes
- All tissues listed in the following were examined for all animals in the control- and high-dose groups. In addition, stomachs were examined for all animals in low- and mid-dose groups. Organs were preserved in 10% netral buffered formalin (eyes were placed in glutaraldehyde/paraformaldehyde initially and then retained in formalin). Lungs and urinary bladder were infused with formalin to ensure fixation. Tissues were stained with Haematoxilin and Eosin for analyses. The organs examined were: adrenal glands (2), aorta (thoracic), bone (sternum/femur with articular surface (2)), brain (medulla/pons. cerebrum and cerebellum), epididymides (2), oesophagus, eyes with optic nerve (2), heart, intestine (caecum, colon, duodenum, ileum, jejunum, rectum), kidneys, lacrimal gland (extraorbital (2)), liver (2 sections), lungs (with with mainsteam bronchi (2)), lymph nodes (mesenteric and submandibular), mammary gland, nerve (sciatic), ovaries (2), pancreas, pituitary gland, prostate gland, salivary glands (submandibular (2)), seminal vesicle (2), skeletal muscle (biceps femoris), spinal cord (cervical, thoracic, lumbar), spleen, stomach (glandular and non-glandular (2)), testes with epididymides (2), thymic region, thyroid, parathyroid glands (2), trachea, urinary bladder, uterus (body/horns with cervix (2), vagina, gross lesions, tissue masses. (The numbers in parentheses indicate the number of organs/sections examined.)

ORGAN WEIGHTS: Yes
- Organs were weighed and organ/body weight and organ/brain weight ratios were calculated for the following: adrenal glands, brain, heart, kidneys, liver, ovaries, pituitary gland, spleen, stomach (rinsed with saline), testes with epididymides, thyroid/parathyroid glands (weighed post fixation). Organs were weighed for all surviving animals at the scheduled sacrifice interval. Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other contiguous tissues in uniform manner. Organs were weighed as soon as possible after dissection in order to avoid drying. Paired organs were weighed together.
Statistics:
Body weight, body weight change from Week 0, food consumption, haematology and clinical chemistry parameters, terminal organand body weights and organ/body weight and organ/brain weight rations were analysed. Mean values of all dose groups were compared to control at each time interval
Statistical evaluation was not performed when the standard deviation for the control group or more than one group was zero, due to lack of variance. If a standard deviation for one treated group was zero, the variancies of the remaining groups were tested using parametric and non-parametric procedures.
Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure, if needed. First, Bartlett's test was performed to determine if groups had equal variance. If the variances were equal, parametric procedures were used, if not non-parametric procedures were used. The parametric procedures were the standard one-way ANOVA using the F distribution to assess significance. If significant differences among the means were identified, Dunnett's test was used to determine which means were significantly different from the control. If a non-parametric procedure for testing equality of means was needed, the Kruskal-Hallis test was used, and if differences were indicated a summed rank test (Dunn) was used to determine which treatments differed from control.
A statistical test for trend in the dose levels was also performed. In the parametric case (i.e. equal variances) standard regression techniques with a test for trend and lack of fit were used. In the non-parametric case Jonckheare's test for monotonic trend was used.
The test for equal variances (Bartlett's) was conducted at the 1% two-sided risk level. All other statistical tests were conducted at the 5% and 1% two sided risk-level.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
100, 250 mg/kg bw/day: statistically significant decreased body weight gain in males, 7% and 9% decreased body weights at termination; females lower body weight gain (not statistically significant), 4% body weights at termination for both dose-groups
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
100, 250 mg/kg bw/day: increased absolute stomach weight, and stomach/body and stomach/brain weight ratios in both males and females; all values statistically significant (except absolute stomach weight in mid-dose (100 mg/kg bw/day females)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
100, 250 mg/kg bw/day: increased stomach wall thickness and yellow discolouration of non-glandular gastric mucosa in both males and females
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
100, 250 mg/kg bw/day: squamous cell hyperplasia, hyperkeratosis/parakeratosis, inflammation and oedema of the non-glandular gastric mucosa, increased incidence and severity in both male and females
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- No apparent test material-related mortality occurred. 1/15 high-dose males (250 mg/kg bw/day) was found dead on Day 66. The cause of death determined from microscopic examination of tissues was congestion, haemorrhage and oedema of the lungs, suggesting a possible misdosing of the animal. One mid-dose animal (100 mg/kg bw/day) was killed via gavage accident on Day 4 and replaced by another animal. All other animal survived throughout the study.
- There were no test material-related abnormalities observed during the study. Abnormal observations were the type commonly found in laboratory animals or occurred sporadically and were not dose-related.

BODY WEIGHT AND WEIGHT GAIN
- Test material-related effects on body weights were found in the mid-and high-dose males (100 and 250 mg/kg bw/day). In these two groups, body weight gain from Week 0 was decreased throughout the study period; these decreases were statistically significant from control control values at 5 of 13 intervals (Weeks 2, 3, 4, 9 and 11) in the mid-dose males and at 8 of 13 intervals (Weeks 1, 2, 3, 9, 11, 12 and 13) in the high-dose males. Mean body weights were also lower than control values, but these changes were statistically significant on only a few occasions. At Week 13, mean body weight was decreased 7% and 9% in the mid- and high-dose males, respectively.
- In the mid- and high-dose females (100 and 250 mg/kg bw/day), mean body weight gain from Week 0 and mean body weight were only slightly lower than control values and none of these decreases were statisticaly significant. At week 13, mean body weight was decreased 4% in both the mid (100 mg/kg bw/day)- and high-dose females (250 mg/kg bw/day).
- Mean body weights and body weight gain in the low-dose males and females (30 mg/kg bw/day) were considered comparable to values of the control group.

FOOD CONSUMPTION
- There were slight, but statistically significant, decreases in food consumption values of the high-dose males (250 mg/kg bw/day) in Week 1 and in the mid- and high-dose females (100 and 250 mg/kg bw/day) in Week 2. Subsequent food consumption values of all groups were considered comparable to the control values or displayed normal variability.

OPHTHALMOSCOPIC EXAMINATION
- There were no ocular abnormalities that could be attributed to administration of the test material.

HAEMATOLOGY
- Values for haematology parameters for males and females in all dose groups at Week 1 were considered comparable to control values.
- At termination, there were slight, but statistically significant increases in mean values for heamoglobin and haematrocrit in the high-dose males (250 mg/kg bw/day) compared to control values. In addition, there were statistically significant increases in white blood cell count in the high-dose males and in neutrophil count in the mid-dose (100 mg/kg bw/day) and high-dose males, which may be reflective of the gastric inflammation seen microscopically. These increases in haematology parameters appeared to be dose-related, but values were within the normal range and differences were not considered toxicologically relevant.
- for haematology parameters in the low-dose males (30 mg/kg bw/day) and females of all dose-groups at study termination were considered comparable to control values.

CLINICAL CHEMISTRY
- Values for clinical chemistry parameters in all dose groups at Week 1 were considered comparable to control values.
- At termination, there were slight decreases in values for glucose and triglycerides in the high-dose (250 mg/kg bw/day) females. Although these decreases were statistically significant compared to control values, values for treated animals were within the normal range and the decreases were decreases were not considered toxicologically significant.
- Values for clinical chemistry parameters in the high-dose males (250 mg/kg bw/day) and mid- and low dose males and females (100 and 30 mg/kg bw/day)at termination were considered comparable to the control values.

URINALYSIS
- There was no evidence of of toxicity of the test material based on urinalysis data.

ORGAN WEIGHTS
- There were increases in the absolute weight of the stomach and stomach/body weight and stomach/brain weight ratios in the mid- and high-dose groups (100 and 250 mg/kg bw/day). All values, with the exception of the absolute weight of the stomach in the mid-dose females, were statistically significant from control animals. The increases correlated with postmortem macroscopic findings of stomach thickening in most animals of these dose-groups.
- There were statistically significant increases in the absolute and relative weights of the adrenals in the high dose males. Absolute and relative adrenal weights were alos slightly increased in the mid-dose males and mid- and high-dose females, but only the only the adrenal/body weight ratios were statistically significant increased relative to control values. The adrenals were macroscopically and microscopically unremarkable and the iincreases in adrenal weights were not considered toxicologically relevant.
- The liver/body weight ratio of the high-dose females was increased significantly compared to the control value. This finding was not considered toxicologically relevant in the absence of microscopic evidence of hepatic lesions.
- Other statitically significant changes in organ weight values were not considered test material related since they were consisitent with low body weight or were not dose-related.

GROSS PATHOLOGY
- There was an apparent treatment-related increase in stomach wall thickness and yellow discolouration of non-glandular gastric mucosa in the mid- and high-dose groups (100 and 250 mg/kg bw/day) which was consistent with increased stomach weight and microscopic evidence of test-aterial related effects in these groups. A moderate increase in stomach wall thickness was also observed in 2/15 low-dose males, however, there was no correlative increase in stomach weights in these animals. Furthermore, microscopic findings (inflammation, hyperplasia and hyperkeratosis) were seen in only 1 of these 2 males and was not considered a test material-related effetc. Tab. 2 presents the incidence of the above changes.
- A small number of other macroscopic findings were noted in various organs and tissues examined, bearing no obvious relationship to exposure of the animals to the test item.

HISTOPATHOLOGY: NON-NEOPLASTIC
- The only treatment-related microscopic changes were present in the fore-stomachs of the treated animals. These changes were characterised by squamous cell hyperplasia, hyperkeratosis/parakeratosis, inflammation and oedema of the non-glandular gastric mucosa. The incidence and severity of these changes were increased in both male and female animals of the mid- and high-dose groups (100 and 250 mg/kg bw/day) compared to the findings of the control group. The microscopic findings in the stomachs of animals in the low-dose groups (30 mg/kg bw/day) were comparable to the findings of the control group. Tab. 3 presents the incidence and severity of the morphologic findings in the stomach.
- Microscopic examinations of other selected organs and tissues revealed no evidence of test material associated toxicity.
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects on the forestomach (increased incidence and severity of squamous cell hyperplasia, hyperkeratosis/parakeratosis, inflammation and oedema of the non-glandular gastric mucosa in both males and females)
Dose descriptor:
LOAEL
Remarks:
local effects
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects on the forestomach (increased incidence and severity of squamous cell hyperplasia, hyperkeratosis/parakeratosis, inflammation and oedema of the non-glandular gastric mucosa in both males and females)
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Decreased body weight gain in 100 and 250 mg/kg bw/day dose groups was <10% and therefore considered to be not adverse.
Critical effects observed:
not specified

Tab. 1: Body weights: % differences from mean control body weight

 

Males

Females

Group

I

II

III

IV

I

II

III

IV

Dose (mg/kg bw/day)

control

30

100

250

control

30

100

250

Total number of Rats in group

15

15

15

15

15

15

15

15

Week

-1

0.0

1.7

1.1

2.5

0.0

0.3

-0.7

-0.7

0

0.0

0.4

-0.2

0.1

0.0

-0.3

-0.5

-0.6

1

0.0

0.4

-1.9

5.5

0.0

-1.0

-1.0

-0.4

2

0.0

-0.5

-6.0

-6.6

0.0

-1.0

-3.4

-1.4

3

0.0

-0.5

-6.0

-5.6

0.0

-1.1

-2.5

-1.7

4

0.0

-0.2

-7.9

-5.0

0.0

-1.4

-2.5

-2.8

5

0.0

0.3

-6.3

-4.9

0.0

-1.1

-1.3

-1.5

6

0.0

0.2

-6.6

-5.5

0.0

-1.2

-4.0

-3.1

7

0.0

-1.0

-6.7

-6.5

0.0

-0.4

-3.2

-2.5

8

0.0

-0.2

-6.0

-7.1

0.0

-0.1

-4.4

-4.2

9

0.0

1.5

-7.9

-8.6

0.0

0.7

-2.9

-4.3

10

0.0

0.7

-7.0

-7.9

0.0

-0.4

-4.0

-5.3

11

0.0

-0.3

-9.7

-8.2

0.0

0.7

-4.6

-5.1

12

0.0

-0.1

-7.7

-8.7

0.0

-1.1

-4.8

-4.8

13

0.0

0.5

-7.2

-8.6

0.0

-0.4

-4.2

-4.2

                                                             

Tab. 2: Selected macroscopic Stomach Findings

 

Males

Females

Group

I

II

III

IV

I

II

III

IV

Dose (mg/kg bw/day)

control

30

100

250

control

30

100

250

Total number of Rats in group

15

15

15

15

15

15

15

15

Number of rats with thick stomach walls

0

2

4

6

0

0

3

7

Number of rats with yellow discolouration in stomach

0

0

7

10

1

1

7

11

Tab. 3: Incidence and severity of the macroscopic findings in the stomach

 

Males

Females

Group

I

II

III

IV

I

II

III

IV

Dose (mg/kg bw/day)

control

30

100

250

control

30

100

250

Total number of Rats in group

15

15

15

15

15

15

15

15

Number of Stomachs examined

15

15

15

15

15

15

15

15

Forestomach:

Acute/subacute inflammation

Slight

0

1

7

6

0

0

3

6

Moderate

2

0

1

2

1

0

2

2

Total

2

1

8

8

1

0

5

8

Forestomach: submucosa oedema

Slight

0

0

3

1

0

0

0

2

Moderate

1

0

3

1

5

0

3

3

Moderately severe

0

0

1

0

0

0

0

0

Total

1

0

7

2

5

0

3

5

Forestomach:

Squamous cell hyperplasia

Slight

1

1

4

1

1

0

9

0

Moderate

1

0

6

6

0

0

2

3

Moderately severe

0

0

0

8

0

0

2

12

Total

2

1

10

15

1

0

13

15

Forestomach:

Hyperkeratosis/

parakeratosis

Slight

2

1

4

1

1

0

9

0

Moderate

0

0

6

7

0

0

2

6

Moderately severe

0

0

0

7

0

0

2

9

Total

2

1

10

15

1

0

13

15

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable (Klimisch score 1 and 2) studies from a source substance with similar structures and intrinsic properties. Read-across is justified based on common precursors and metabolic degradation products and consistent trends in environmental fate, ecotoxicological and toxicological profile. The selected studies are thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Mar - 9 Jul 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bundesamt für Sicherheit im Gesundheitswesen, Wien, Austria
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation:
- Fasting period before study: animals were fasted overnight prior to administration.
- Housing: animals were housed in groups of 3 per sex in the dose range finding study. In the main study, animals were housed in groups of 2-3 per cage per sex in Makrolon cages Type IV with wire mesh lids.
- Diet: Ssniff R/M-H maintenance diet for rats and mice (V1534-300, Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, acidified with HCL to pH ≥ 3, ad libitum
- Acclimation period: about 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 (average dose range finding study), 19.8 (average main study)
- Humidity (%): 59.1 (average dose range finding study), 56.6 (average main study)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: ethanol
Remarks on MMAD:
MMAD / GSD: MMAD: 1.11, 1.15 and 1.22 µm (low-, mid- and high-dose)
GSD: 1.68-2.57
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: head nose only exposure unit (TSE-Systems GmbH, Bad Homburg, Germany)
- Method of holding animals in test chamber: animal exposure cage
- Source and rate of air: compressed air supply or compressed air generator
- Method of particle size determination: the size distribution of the aerosol particles was analysed with a cascade impactor (Berner-Impaktor, Type LPI40,06/2, Hauke KG, Gmunden, Austria)

TEST ATMOSPHERE
- Brief description of analytical method used: the aerosol concentration in the breathing zone was determined gravimetically.
- Samples taken from breathing zone: yes

VEHICLE
- Justification for use and choice of vehicle: the original high viscous liquid could not be pressed through the aerosol nozzle. Therefore the test substance was mixed with ethanol to decrease the viscosity. In the high-dose group the concentration was 10%. It was the target to have the same concentration of ethanol in all dose groups and in the control group. Therefore different concentrations of ethanol in the test substance were used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The aerosol concentration in the breathing zone was determined gravimetrically. An accurately measured volume of air from the inhalation devices was sucked through a pre-weighed filter with cotton wool. The filters were dried before and after the sampling by pressing dry air through them. From the weight difference and the volume the actual aerosol concentration was calculated.
Duration of treatment / exposure:
4 weeks (main study)
4 weeks and 2 weeks post-exposure observation period (satellite control and high-dose test group)
Frequency of treatment:
6 h/day, 5 days/week
Dose / conc.:
1.2 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)
Dose / conc.:
4 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)
Dose / conc.:
11.6 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)
Dose / conc.:
1.3 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)
Dose / conc.:
4.3 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)
Dose / conc.:
12.9 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)
Dose / conc.:
0.006 mg/L air (nominal)
Dose / conc.:
0.02 mg/L air (nominal)
Dose / conc.:
0.06 mg/L air (nominal)
Dose / conc.:
6 mg/m³ air (nominal)
Dose / conc.:
20 mg/m³ air (nominal)
Dose / conc.:
60 mg/m³ air (nominal)
No. of animals per sex per dose:
3 (preliminary study)
5 (main study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose ranges are based on the results of the dose range finding study. In the high-dose group (0.6 mg/L) and mid-dose group (0.19 mg/L) all animals died on the day of administration. Therefore, the dose of 0.06 mg/L, which was the low-dose in the dose range finding study, was chosen as high-dose for the main study. The low dose was set at 10% of the high dose; the mid-dose was interpolated geometrically.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were carefully observed for general signs and the health status before, during and after the exposure; on days without exposure once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice per week

FOOD CONSUMPTION:
- Food consumption per cage in weekly intervals in all animals. Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 (main study groups) and Day 43 (satellite groups)
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: Yes
- How many animals: All animals of main study and satellite groups.
- Parameters checked: red blood cell count (RBC), haemoglobin concentration (Hb), haematrocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean cell volume (MCV), reticulocyte count (% of erythrocytes), white blood cell count (WBC), platelet count, differential white blood cell count (% of the different cell species), prothombin time (Quick) as indicator of blood clotting capacity

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (main study groups) and Day 43 (satellite groups)
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: Yes
- How many animals: All animals of main study and satellite groups.
- Parameters checked: aspartate aminotransferase, alanine aminotrasferase, albumin, alkaline phosphatase, bilirubin, calcium, chloride, cholesterol, creatinine, gamma glytamyl transferase, globulin, glucose, phosphorus, potassium (K+), sodium (NA+), total protein, triglycerides, urea
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Animals were killed and subjected to gross pathological examination immediately after death.
HISTOPATHOLOGY: Yes. Gross lesions, tissue masses or tumours, adrenal glands, brain (including cerebrum, cerebellum and pons), eyes, heart, kidneys, larynx (3 levels, including the base of the epiglottis), liver, lungs (all lobes at one level, including main bronchi), lymph nodes (hilar region of the lungs), nasopharyngeal tissues (4 levels), oesophagus, ovaries, seminal vesicles, spinal cord (cervical, thoracal, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid glands, trachea (longitudinal section through the carna and 1 transverse section), uterus
Statistics:
Analysis of variance followed by the Scheffe-test was used for all data with means and standard deviations with comparison of more than two groups. The t-test was used for all data with means and standard deviations for comparison of two groups only. H-test of Kruskal and Wallis followed by the test of Nemenyi counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled. Chi2-test was used for counted events. Fisher´s exact test was used for counted events, if the Chi2-test was not applicable.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
mid- and high dose males and females and female control group: chromodakryorrhoea; high dose satellite group males and low- and high-dose group females: reduced well-being
Mortality:
mortality observed, treatment-related
Description (incidence):
mid- and high dose males and females and female control group: chromodakryorrhoea; high dose satellite group males and low- and high-dose group females: reduced well-being
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
high-dose groups: generally lower body weight gain
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:
effects observed, treatment-related
Description (incidence and severity):
high-dose males: increased MCV, high-dose males after recovery: reduced WBC, lymphocytes; mid-dose females: decreased reticulocyte count, non adverse
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
low-dose females: decreased chloride, increased phosphor; high-dose females increased AP and ALT; low-dose females: increased ALT, all females: decreased CHOL during dosing, increased at the end of the recovery period
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
significant changes in organ weights are summarized in Table 3 under "Any other information on results incl. tables".
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
adaptive responses to the deposits of the test substance and the resulting irritation (squamous metaplasia and epithelium proliferation and submucous acute inflammation at the basis of the epiglottis).
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived until the end of the study period. Chromodakryorrhoe occurred in male animals of the mid and high dose groups. This finding was also observed in females of the mid- and high-dose and control group. The findings may be caused by the stress during the administration procedure.
Signs of reduced well-being were noted in male animals of the high dose satellite group and in females of the low and high dose group. Stress can also be the cause for these findings. The effects were seen during the administration period and were not observed during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
Body weight and body weight gain were lower in the high dose groups. In the mid dose group an effect was observed only in male animals and only at the end of the administration period (see Table 1 under “Any other information on results incl. tables”).

FOOD CONSUMPTION
No significant effects on food consumption were noted during the study period.

HAEMATOLOGY
Changes in the MCV, WBC and lymphocytes in male animals of the high dose groups were observed. In female animals of the mid-dose group reticulocyte counts were significantly reduced. The effects may be random events, because no other parameters are impaired (see Table 2 under “Any other information on results incl. tables”).

CLINICAL CHEMISTRY
No significant effects on clinical chemistry parameters were observed in male animals.
In females of the low-dose group increased phosphor, decreased cholesterol and increased AST was apparent. In the mid-dose group the cholesterol level was decreased as well. In high-dose females chloride level was decreased and normal at the end of the recovery period. Cholesterol was decreased during the dosing period and increased at the end of the recovery period in high-dose females. AP and ALT were statistically significant increased in the high dose group and normal after the recovery period. The changes of cholesterol in all females dosing groups may be caused by the test substance. However, corresponding damages of the liver were not found in the animals at necropsy, histopathology and in organ weights.
All other parameters and statistically calculated significances do not show a dose response relationship and/or are very small changes and are therefore assumed to be random events without toxicological importance (see Table 2 under “Any other information on results incl. tables”).

ORGAN WEIGHTS
Different significant changes in organs weights in the mid- and high-dose male animals were observed (see Table 3 under “Any other information on results incl. tables”). The changes in testes were also seen in the high-dose satellite group and can therefore be considered as not reversible. Changes in spleen were noted in the high-dose satellite group only. The author assumed that such an effect may be test substance related, and occur only after the end of the administration period.
In female animals relevant changes were noted in the high-dose group only. The changed parameters and statistically calculated significances in female animals do not show a dose-relationship. Therefore, the changes are considered to be random without toxicological importance.
In general the increase in relative organ weights may be associated with the reduced body weight and body weight gain.

GROSS PATHOLOGY
No test substance related changes were observed at the gross examination.

HISTOPATHOLOGY: NON-NEOPLASTIC
The test substance aerosol caused several effects in the respiratory system, in lungs and bronchi. Most effects were adaptive responses to the deposits of the test substance and the resulting irritation (squamous metaplasia and epithelium proliferation and submucous acute inflammation at the basis of the epiglottis). In addition focal early stage of fibrosis was noted at the alveoli walls mainly in the groups treated with the test substance. Due to the high amount of the test substance deposits in the lungs especially in the mid and high dose groups these changes may be explained by overloading the tissue and not necesarrily imply an intrinsic toxicity of the test material. The author considered an intrinsic toxicity as unlikely as the test material is insoluble and the shape of the particles is not fibrous.
Some other findings were considered as unspecific and of no toxicological relevance. However, most findings were observed in the high-dose groups.

OTHER FINDINGS
RESULTS OF THE DOSE RANGE FINDING STUDY
All animals of the mid and high dose group died on Day 1 acutely still during the exposition and/or directly thereafter (aerosol concentration 0.19 mg/L and 0.6 mg/L, respectively). No indication for a test substance related alteration in animals of the low dose group was noted (aerosol concentration 0.06 mg/L)). Body weight was lower in the high dose groups. In the mid dose group an effect was observed only in male animals and only at the end of the administration period. There were no statistically significant changes in the low dose groups in body weight, feed consumption and at necropsy.

AEROSOL CONCENTRATION
The mean actual aerosol concentrations were 0.006, 0.02 and 0.059 mg aerosol per liter air for the low, mid- and high-dose groups. The values were converted to the inhaled amount (dose) using the equation: minute_volume (L/min) = body weight (kg)^0.75 x 0.373 (Guyton´s equation) for the breathing volume of the animals.
The calculation gave doses of 11.6/12.9, 4.0/4.3 and 1.2/1.3 mg/kg/day for the high-, mid- and low-dose groups (males/females).
The inhaled amount of ethanol in every group is expected to be about 1/10 of the test substance amount (1 mg/kg/day in the high-dose group).


Dose descriptor:
NOEC
Effect level:
< 0.006 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects on the respiratory system with indications for a local irritation
Dose descriptor:
NOAEC
Effect level:
> 0.06 mg/L air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: effects on organ weight without corresponding effects in histopathological examination and without dose-relationship
Critical effects observed:
not specified

Table 1. Body weight (g) presented as mean ± SD (males and females).

Dose Level

Body weight (g) on Days / Males

 

1

12

15

19

22

26

28

33

36

40

42

Control

205 ± 8.73

221 ± 13.3

218 ± 14.1

232 ± 14.0

229 ± 13.2

240 ± 13.0

237 ± 14.6

-

-

-

-

Control satellite

203 ± 3.21

245 ± 2.45

215 ± 2.92

227 ± 2.88

225± 1.82

236 ± 1.95

232 ± 0.84

246 ± 3.11

254 ± 5.39

265 ± 5.89

265 ± 6.06

Low-dose

200 ± 9.18

212 ± 9.42

210 ± 8.5

221 ±10.4

216 ± 7.29

226 ± 7.76

221 ± 8.79

-

-

-

-

Mid-dose

200 ± 6.46

210 ± 5.07

204 ± 4.76

219 ± 4.83

212 ± 4.47

225 ± 4.97

216 ± 5.63*

-

-

-

-

High-dose

200 ± 9.5

206 ± 8.7

200 ± 9.07

209 ± 9.18*

200 ± 8.41*

211 ± 8.94*

202 ± 7.23*

-

-

-

-

High-dose satellite

202 ± 7.96

207 ± 8.83

199 ± 9.36±

212 ± 9.64*

202 ± 8.58*

213 ± 10.8*

203 ± 8.29*

223 ± 10.4*

234 ± 12.1*

247 ± 13.1*

247 ± 16.4

Dose Level

Body weight (g) on Days / Females

 

1

22

26

28

33

36

40

42

Control

140 ± 3.13

149 ± 4.49

153 ± 4.51

152 ± 5.41

-

-

-

-

Control satellite

137 ± 3.16

146 ± 4.1

152 ± 5.59

150 ± 4.87

158 ± 5.5

162 ± 6.38

168 ± 7.8

166 ± 9.13

Low-dose

138 ± 4.27

142 ± 9.15

146 ± 9.2

143 ± 9.02

-

-

-

-

Mid-dose

141 ± 4.82

145± 2.95

151 ± 2.7

147 ± 4.04

-

-

-

-

High-dose

138 ± 4.87

136 ± 5.1*

142 ± 4.3*

136 ± 4.97*

-

-

-

-

High-dose satellite

140 ± 6.19

138 ± 5.85*

141 ± 8.17*

138 ± 5.97*

148 ± 6.91*

153 ± 6.8

164 ± 7.3

162 ± 6.73

*: significant different from control group p < 0.05

Table 2. Mean haematological data and clinical chemistry (only significant changes are given).

Parameter

 

Results of statistical calculation

Males

 

Females

Control

Control satellite

High dose

High dose satellite

Control

Control satellite

Low dose

Mid dose

High dose

High dose satellite

MCV (mean corpuscular volume fL)

47.3 ± 0.95

46.9 ± 0.17

49.3 ± 1.33*

47.1 ± 0.83

-

-

-

-

-

-

WBC (white blood cell count, 1/nL)

7.82 ± 1.73

6.12 ± 1.3

5.66 ± 0.49

4.89 ± 0.49*

-

-

-

-

-

-

Lymphocytes (% of leukocytes or n 1/nL)

5.37 ± 1.28

4.42 ± 1.32

3.56 ± 0.47

3.21 ± 0.24*

-

-

-

-

-

-

Reticulocyte count (%), males

-

-

-

-

3.69 ± 0.36

-

-

2.58 ± 0.33*

-

-

Chlorid (mmol/L)

-

-

-

-

99.9 ± 0.72

99.7 ± 1.19

-

-

98.2 ± 0.77*

100 ± 1.07

Phosphor (mmol/L)

-

--

-

-

2.53 ± 0.23

-

3.09 ± 0.43*

-

-

-

Cholesterol (mmol/L)

-

-

-

-

2.37 ± 0.15

2.34 ± 0.06

1.90 ± 0.26*

1.87 ± 0.27*

1.83 ± 0.14*

2.6 ± 0.2*

AP (alkaline phosphatase, U/L)

-

-

-

-

118 ± 5.34

100 ± 7.6

-

-

138 ± 7.97*

103 ± 4.04

AST (aspartate aminotransferase, U/L)

-

-

-

-

92.2 ± 2.68

91.4 ± 7.47

105 ± 6.88*

-

-

-

ALT (alanin aminotransferase, U/L)

-

-

-

-

61.4 ± 6.43

64.4 ± 2.88

-

-

75.4 ± 7.99*

62.2 ± 1.79

*: significant different from control group p < 0.05

Table 3. Mean organ weights. Only significant changes are given.

Parameter

Results of statistical calculation

Males

Females

Control / Control satellite

Mid dose

High dose

High dose satellite

Control / Control satellite

High dose

High dose satellite

Absolute organ weights (mg)

Testes

2283 ±185

1966 ± 162

2716 ± 123*

2771 ± 42.4 *

2651 ± 204*

-

-

-

Spleen

512 ± 66.7

645 ± 48.4

-

-

531 ± 59.2*

-

-

-

Lung

-

-

-

-

1173 ± 118

926 ± 76.9

-

1104 ±154*

Thymus

291 ± 60.7

313 ± 51.5

186 ± 17.9*

188 ± 17.9*

261 ± 51.9

-

-

-

Organ weight/body weight ratios (mg/g)

Brain

7.86± 0.55

7.42 ± 0.19

5.66 ± 0.37*

9.0 ± 0.23*

7.61 ± 0.44

11.2 ± 0.74

10.4 ± 0.31

12.9 ± 0.46*

10.6 ± 0.46

Lung

-

-

-

-

7.69 ± 0.68

5.57 ± 0.47

-

6.79 ± 0.86*

Heart

3.42 ± 0.43

3.51 ± 0.22

-

3.97 ± 0.23*

3.68 ± 0.35

3.88 ± 0.11

3.38 ± 0.26

4.27 ± 0.18*

3.81 ± 0.25

Testes

9.64 ± 0.43

7.41 ± 0.19

12.6 ± 0.76*

13.7 ± 0.3*

10.8 ± 1.13*

-

-

-

Spleen

2.17 ± 0.29

2.43 ± 0.16

-

-

2.14 ± 0.18*

-

-

-

Kidneys

6.86 ± 0.65

6.65 ± 0.62

-

8.01 ± 0.53*

6.6 ± 0.52

8.08 ± 0.41

6.43 ± 0.35

-

7.28 ± 0.66*

Thymus

1.22 ± 0.18

0.86 ± 0.06*

-

-

-

-

-

Organ weight/brain weight ratios (mg/g)

Heart

-

-

-

-

-

-

-

Spleen

277 ± 41.1

325 ± 23.5

-

-

282 ± 24.5*

-

-

-

Thymus

157 ± 32.7

159 ± 24.9

99.4 ± 8.15*

103 ± 23.9*

139 ± 29.0

-

-

-

Kidneys

-

 

-

-

719 ± 31.3

618 ± 20.1

-

685 ± 53.9*

Testes

1231 ± 92.5

1000 ± 93.8

1456 ± 75.4*

1522 ± 41.8*

1415 ± 47*

-

-

-

*: significant different from control group p < 0.05

Conclusions:
The used concentration range of the study was chosen due to a range finding test. The aerosol of the test substance caused effects at several sites of the respiratory system at concentrations of 0.006 to 0.060 mg/L with indications for a local irritation. The alterations may be explained by the amounts of inert material deposited within the respiratory system. In the lungs and bronchi the most prominent finding was a focal early stage of fibrosis. Due to the high amount of test substance deposits in the lungs especially in the mid and high dosed groups these changes may be explained by simply overloading the tissue and do not necessarily imply an intrinsic toxicity of the test material. An intrinsic toxicity is unlikely as the test material is insoluble and the shape of the particles is not fibrous.
Alterations in clinical biochemistry were only seen in female animals. Beside this and the increased absolute and relative organ weights of testes in male animals there was no other substantially sex difference in the response to the test substance. Furthermore, the differences did not show a dose relationship and no correlating effects on the respective organ were seen in the histopathological examinations.
The observed effects were at most mild in severity, never became life threatening, but persisted partly until the end of the recovery period. Furthermore, due to the self-warning characteristics (immediate irritant reactions to the test substance if inhaled), high substance concentrations of inhalation may be avoided.
The No-observed-effect-concentration (NOEC) for local irritation of the test material via inhalation was smaller than 0.006 mg/L. There was however no life threatening systemic effect present up to the highest concentration tested. Therefore, the systemic NOAEC for the test material will be > 0.06 mg/L.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
60 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
The available study is reliable without restrictions (Klimisch score 1), adequate and was performed with the registered substance. It is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Mar - 9 Jul 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bundesamt für Sicherheit im Gesundheitswesen, Wien, Austria
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation:
- Fasting period before study: animals were fasted overnight prior to administration.
- Housing: animals were housed in groups of 3 per sex in the dose range finding study. In the main study, animals were housed in groups of 2-3 per cage per sex in Makrolon cages Type IV with wire mesh lids.
- Diet: Ssniff R/M-H maintenance diet for rats and mice (V1534-300, Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, acidified with HCL to pH ≥ 3, ad libitum
- Acclimation period: about 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 (average dose range finding study), 19.8 (average main study)
- Humidity (%): 59.1 (average dose range finding study), 56.6 (average main study)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: ethanol
Remarks on MMAD:
MMAD / GSD: MMAD: 1.11, 1.15 and 1.22 µm (low-, mid- and high-dose)
GSD: 1.68-2.57
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: head nose only exposure unit (TSE-Systems GmbH, Bad Homburg, Germany)
- Method of holding animals in test chamber: animal exposure cage
- Source and rate of air: compressed air supply or compressed air generator
- Method of particle size determination: the size distribution of the aerosol particles was analysed with a cascade impactor (Berner-Impaktor, Type LPI40,06/2, Hauke KG, Gmunden, Austria)

TEST ATMOSPHERE
- Brief description of analytical method used: the aerosol concentration in the breathing zone was determined gravimetically.
- Samples taken from breathing zone: yes

VEHICLE
- Justification for use and choice of vehicle: the original high viscous liquid could not be pressed through the aerosol nozzle. Therefore the test substance was mixed with ethanol to decrease the viscosity. In the high-dose group the concentration was 10%. It was the target to have the same concentration of ethanol in all dose groups and in the control group. Therefore different concentrations of ethanol in the test substance were used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The aerosol concentration in the breathing zone was determined gravimetrically. An accurately measured volume of air from the inhalation devices was sucked through a pre-weighed filter with cotton wool. The filters were dried before and after the sampling by pressing dry air through them. From the weight difference and the volume the actual aerosol concentration was calculated.
Duration of treatment / exposure:
4 weeks (main study)
4 weeks and 2 weeks post-exposure observation period (satellite control and high-dose test group)
Frequency of treatment:
6 h/day, 5 days/week
Dose / conc.:
1.2 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)
Dose / conc.:
4 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)
Dose / conc.:
11.6 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)
Dose / conc.:
1.3 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)
Dose / conc.:
4.3 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)
Dose / conc.:
12.9 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)
Dose / conc.:
0.006 mg/L air (nominal)
Dose / conc.:
0.02 mg/L air (nominal)
Dose / conc.:
0.06 mg/L air (nominal)
Dose / conc.:
6 mg/m³ air (nominal)
Dose / conc.:
20 mg/m³ air (nominal)
Dose / conc.:
60 mg/m³ air (nominal)
No. of animals per sex per dose:
3 (preliminary study)
5 (main study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose ranges are based on the results of the dose range finding study. In the high-dose group (0.6 mg/L) and mid-dose group (0.19 mg/L) all animals died on the day of administration. Therefore, the dose of 0.06 mg/L, which was the low-dose in the dose range finding study, was chosen as high-dose for the main study. The low dose was set at 10% of the high dose; the mid-dose was interpolated geometrically.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were carefully observed for general signs and the health status before, during and after the exposure; on days without exposure once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice per week

FOOD CONSUMPTION:
- Food consumption per cage in weekly intervals in all animals. Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 (main study groups) and Day 43 (satellite groups)
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: Yes
- How many animals: All animals of main study and satellite groups.
- Parameters checked: red blood cell count (RBC), haemoglobin concentration (Hb), haematrocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean cell volume (MCV), reticulocyte count (% of erythrocytes), white blood cell count (WBC), platelet count, differential white blood cell count (% of the different cell species), prothombin time (Quick) as indicator of blood clotting capacity

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (main study groups) and Day 43 (satellite groups)
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: Yes
- How many animals: All animals of main study and satellite groups.
- Parameters checked: aspartate aminotransferase, alanine aminotrasferase, albumin, alkaline phosphatase, bilirubin, calcium, chloride, cholesterol, creatinine, gamma glytamyl transferase, globulin, glucose, phosphorus, potassium (K+), sodium (NA+), total protein, triglycerides, urea
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Animals were killed and subjected to gross pathological examination immediately after death.
HISTOPATHOLOGY: Yes. Gross lesions, tissue masses or tumours, adrenal glands, brain (including cerebrum, cerebellum and pons), eyes, heart, kidneys, larynx (3 levels, including the base of the epiglottis), liver, lungs (all lobes at one level, including main bronchi), lymph nodes (hilar region of the lungs), nasopharyngeal tissues (4 levels), oesophagus, ovaries, seminal vesicles, spinal cord (cervical, thoracal, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid glands, trachea (longitudinal section through the carna and 1 transverse section), uterus
Statistics:
Analysis of variance followed by the Scheffe-test was used for all data with means and standard deviations with comparison of more than two groups. The t-test was used for all data with means and standard deviations for comparison of two groups only. H-test of Kruskal and Wallis followed by the test of Nemenyi counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled. Chi2-test was used for counted events. Fisher´s exact test was used for counted events, if the Chi2-test was not applicable.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
mid- and high dose males and females and female control group: chromodakryorrhoea; high dose satellite group males and low- and high-dose group females: reduced well-being
Mortality:
mortality observed, treatment-related
Description (incidence):
mid- and high dose males and females and female control group: chromodakryorrhoea; high dose satellite group males and low- and high-dose group females: reduced well-being
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
high-dose groups: generally lower body weight gain
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:
effects observed, treatment-related
Description (incidence and severity):
high-dose males: increased MCV, high-dose males after recovery: reduced WBC, lymphocytes; mid-dose females: decreased reticulocyte count, non adverse
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
low-dose females: decreased chloride, increased phosphor; high-dose females increased AP and ALT; low-dose females: increased ALT, all females: decreased CHOL during dosing, increased at the end of the recovery period
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
significant changes in organ weights are summarized in Table 3 under "Any other information on results incl. tables".
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
adaptive responses to the deposits of the test substance and the resulting irritation (squamous metaplasia and epithelium proliferation and submucous acute inflammation at the basis of the epiglottis).
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived until the end of the study period. Chromodakryorrhoe occurred in male animals of the mid and high dose groups. This finding was also observed in females of the mid- and high-dose and control group. The findings may be caused by the stress during the administration procedure.
Signs of reduced well-being were noted in male animals of the high dose satellite group and in females of the low and high dose group. Stress can also be the cause for these findings. The effects were seen during the administration period and were not observed during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
Body weight and body weight gain were lower in the high dose groups. In the mid dose group an effect was observed only in male animals and only at the end of the administration period (see Table 1 under “Any other information on results incl. tables”).

FOOD CONSUMPTION
No significant effects on food consumption were noted during the study period.

HAEMATOLOGY
Changes in the MCV, WBC and lymphocytes in male animals of the high dose groups were observed. In female animals of the mid-dose group reticulocyte counts were significantly reduced. The effects may be random events, because no other parameters are impaired (see Table 2 under “Any other information on results incl. tables”).

CLINICAL CHEMISTRY
No significant effects on clinical chemistry parameters were observed in male animals.
In females of the low-dose group increased phosphor, decreased cholesterol and increased AST was apparent. In the mid-dose group the cholesterol level was decreased as well. In high-dose females chloride level was decreased and normal at the end of the recovery period. Cholesterol was decreased during the dosing period and increased at the end of the recovery period in high-dose females. AP and ALT were statistically significant increased in the high dose group and normal after the recovery period. The changes of cholesterol in all females dosing groups may be caused by the test substance. However, corresponding damages of the liver were not found in the animals at necropsy, histopathology and in organ weights.
All other parameters and statistically calculated significances do not show a dose response relationship and/or are very small changes and are therefore assumed to be random events without toxicological importance (see Table 2 under “Any other information on results incl. tables”).

ORGAN WEIGHTS
Different significant changes in organs weights in the mid- and high-dose male animals were observed (see Table 3 under “Any other information on results incl. tables”). The changes in testes were also seen in the high-dose satellite group and can therefore be considered as not reversible. Changes in spleen were noted in the high-dose satellite group only. The author assumed that such an effect may be test substance related, and occur only after the end of the administration period.
In female animals relevant changes were noted in the high-dose group only. The changed parameters and statistically calculated significances in female animals do not show a dose-relationship. Therefore, the changes are considered to be random without toxicological importance.
In general the increase in relative organ weights may be associated with the reduced body weight and body weight gain.

GROSS PATHOLOGY
No test substance related changes were observed at the gross examination.

HISTOPATHOLOGY: NON-NEOPLASTIC
The test substance aerosol caused several effects in the respiratory system, in lungs and bronchi. Most effects were adaptive responses to the deposits of the test substance and the resulting irritation (squamous metaplasia and epithelium proliferation and submucous acute inflammation at the basis of the epiglottis). In addition focal early stage of fibrosis was noted at the alveoli walls mainly in the groups treated with the test substance. Due to the high amount of the test substance deposits in the lungs especially in the mid and high dose groups these changes may be explained by overloading the tissue and not necesarrily imply an intrinsic toxicity of the test material. The author considered an intrinsic toxicity as unlikely as the test material is insoluble and the shape of the particles is not fibrous.
Some other findings were considered as unspecific and of no toxicological relevance. However, most findings were observed in the high-dose groups.

OTHER FINDINGS
RESULTS OF THE DOSE RANGE FINDING STUDY
All animals of the mid and high dose group died on Day 1 acutely still during the exposition and/or directly thereafter (aerosol concentration 0.19 mg/L and 0.6 mg/L, respectively). No indication for a test substance related alteration in animals of the low dose group was noted (aerosol concentration 0.06 mg/L)). Body weight was lower in the high dose groups. In the mid dose group an effect was observed only in male animals and only at the end of the administration period. There were no statistically significant changes in the low dose groups in body weight, feed consumption and at necropsy.

AEROSOL CONCENTRATION
The mean actual aerosol concentrations were 0.006, 0.02 and 0.059 mg aerosol per liter air for the low, mid- and high-dose groups. The values were converted to the inhaled amount (dose) using the equation: minute_volume (L/min) = body weight (kg)^0.75 x 0.373 (Guyton´s equation) for the breathing volume of the animals.
The calculation gave doses of 11.6/12.9, 4.0/4.3 and 1.2/1.3 mg/kg/day for the high-, mid- and low-dose groups (males/females).
The inhaled amount of ethanol in every group is expected to be about 1/10 of the test substance amount (1 mg/kg/day in the high-dose group).


Dose descriptor:
NOEC
Effect level:
< 0.006 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects on the respiratory system with indications for a local irritation
Dose descriptor:
NOAEC
Effect level:
> 0.06 mg/L air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: effects on organ weight without corresponding effects in histopathological examination and without dose-relationship
Critical effects observed:
not specified

Table 1. Body weight (g) presented as mean ± SD (males and females).

Dose Level

Body weight (g) on Days / Males

 

1

12

15

19

22

26

28

33

36

40

42

Control

205 ± 8.73

221 ± 13.3

218 ± 14.1

232 ± 14.0

229 ± 13.2

240 ± 13.0

237 ± 14.6

-

-

-

-

Control satellite

203 ± 3.21

245 ± 2.45

215 ± 2.92

227 ± 2.88

225± 1.82

236 ± 1.95

232 ± 0.84

246 ± 3.11

254 ± 5.39

265 ± 5.89

265 ± 6.06

Low-dose

200 ± 9.18

212 ± 9.42

210 ± 8.5

221 ±10.4

216 ± 7.29

226 ± 7.76

221 ± 8.79

-

-

-

-

Mid-dose

200 ± 6.46

210 ± 5.07

204 ± 4.76

219 ± 4.83

212 ± 4.47

225 ± 4.97

216 ± 5.63*

-

-

-

-

High-dose

200 ± 9.5

206 ± 8.7

200 ± 9.07

209 ± 9.18*

200 ± 8.41*

211 ± 8.94*

202 ± 7.23*

-

-

-

-

High-dose satellite

202 ± 7.96

207 ± 8.83

199 ± 9.36±

212 ± 9.64*

202 ± 8.58*

213 ± 10.8*

203 ± 8.29*

223 ± 10.4*

234 ± 12.1*

247 ± 13.1*

247 ± 16.4

Dose Level

Body weight (g) on Days / Females

 

1

22

26

28

33

36

40

42

Control

140 ± 3.13

149 ± 4.49

153 ± 4.51

152 ± 5.41

-

-

-

-

Control satellite

137 ± 3.16

146 ± 4.1

152 ± 5.59

150 ± 4.87

158 ± 5.5

162 ± 6.38

168 ± 7.8

166 ± 9.13

Low-dose

138 ± 4.27

142 ± 9.15

146 ± 9.2

143 ± 9.02

-

-

-

-

Mid-dose

141 ± 4.82

145± 2.95

151 ± 2.7

147 ± 4.04

-

-

-

-

High-dose

138 ± 4.87

136 ± 5.1*

142 ± 4.3*

136 ± 4.97*

-

-

-

-

High-dose satellite

140 ± 6.19

138 ± 5.85*

141 ± 8.17*

138 ± 5.97*

148 ± 6.91*

153 ± 6.8

164 ± 7.3

162 ± 6.73

*: significant different from control group p < 0.05

Table 2. Mean haematological data and clinical chemistry (only significant changes are given).

Parameter

 

Results of statistical calculation

Males

 

Females

Control

Control satellite

High dose

High dose satellite

Control

Control satellite

Low dose

Mid dose

High dose

High dose satellite

MCV (mean corpuscular volume fL)

47.3 ± 0.95

46.9 ± 0.17

49.3 ± 1.33*

47.1 ± 0.83

-

-

-

-

-

-

WBC (white blood cell count, 1/nL)

7.82 ± 1.73

6.12 ± 1.3

5.66 ± 0.49

4.89 ± 0.49*

-

-

-

-

-

-

Lymphocytes (% of leukocytes or n 1/nL)

5.37 ± 1.28

4.42 ± 1.32

3.56 ± 0.47

3.21 ± 0.24*

-

-

-

-

-

-

Reticulocyte count (%), males

-

-

-

-

3.69 ± 0.36

-

-

2.58 ± 0.33*

-

-

Chlorid (mmol/L)

-

-

-

-

99.9 ± 0.72

99.7 ± 1.19

-

-

98.2 ± 0.77*

100 ± 1.07

Phosphor (mmol/L)

-

--

-

-

2.53 ± 0.23

-

3.09 ± 0.43*

-

-

-

Cholesterol (mmol/L)

-

-

-

-

2.37 ± 0.15

2.34 ± 0.06

1.90 ± 0.26*

1.87 ± 0.27*

1.83 ± 0.14*

2.6 ± 0.2*

AP (alkaline phosphatase, U/L)

-

-

-

-

118 ± 5.34

100 ± 7.6

-

-

138 ± 7.97*

103 ± 4.04

AST (aspartate aminotransferase, U/L)

-

-

-

-

92.2 ± 2.68

91.4 ± 7.47

105 ± 6.88*

-

-

-

ALT (alanin aminotransferase, U/L)

-

-

-

-

61.4 ± 6.43

64.4 ± 2.88

-

-

75.4 ± 7.99*

62.2 ± 1.79

*: significant different from control group p < 0.05

Table 3. Mean organ weights. Only significant changes are given.

Parameter

Results of statistical calculation

Males

Females

Control / Control satellite

Mid dose

High dose

High dose satellite

Control / Control satellite

High dose

High dose satellite

Absolute organ weights (mg)

Testes

2283 ±185

1966 ± 162

2716 ± 123*

2771 ± 42.4 *

2651 ± 204*

-

-

-

Spleen

512 ± 66.7

645 ± 48.4

-

-

531 ± 59.2*

-

-

-

Lung

-

-

-

-

1173 ± 118

926 ± 76.9

-

1104 ±154*

Thymus

291 ± 60.7

313 ± 51.5

186 ± 17.9*

188 ± 17.9*

261 ± 51.9

-

-

-

Organ weight/body weight ratios (mg/g)

Brain

7.86± 0.55

7.42 ± 0.19

5.66 ± 0.37*

9.0 ± 0.23*

7.61 ± 0.44

11.2 ± 0.74

10.4 ± 0.31

12.9 ± 0.46*

10.6 ± 0.46

Lung

-

-

-

-

7.69 ± 0.68

5.57 ± 0.47

-

6.79 ± 0.86*

Heart

3.42 ± 0.43

3.51 ± 0.22

-

3.97 ± 0.23*

3.68 ± 0.35

3.88 ± 0.11

3.38 ± 0.26

4.27 ± 0.18*

3.81 ± 0.25

Testes

9.64 ± 0.43

7.41 ± 0.19

12.6 ± 0.76*

13.7 ± 0.3*

10.8 ± 1.13*

-

-

-

Spleen

2.17 ± 0.29

2.43 ± 0.16

-

-

2.14 ± 0.18*

-

-

-

Kidneys

6.86 ± 0.65

6.65 ± 0.62

-

8.01 ± 0.53*

6.6 ± 0.52

8.08 ± 0.41

6.43 ± 0.35

-

7.28 ± 0.66*

Thymus

1.22 ± 0.18

0.86 ± 0.06*

-

-

-

-

-

Organ weight/brain weight ratios (mg/g)

Heart

-

-

-

-

-

-

-

Spleen

277 ± 41.1

325 ± 23.5

-

-

282 ± 24.5*

-

-

-

Thymus

157 ± 32.7

159 ± 24.9

99.4 ± 8.15*

103 ± 23.9*

139 ± 29.0

-

-

-

Kidneys

-

 

-

-

719 ± 31.3

618 ± 20.1

-

685 ± 53.9*

Testes

1231 ± 92.5

1000 ± 93.8

1456 ± 75.4*

1522 ± 41.8*

1415 ± 47*

-

-

-

*: significant different from control group p < 0.05

Conclusions:
The used concentration range of the study was chosen due to a range finding test. The aerosol of the test substance caused effects at several sites of the respiratory system at concentrations of 0.006 to 0.060 mg/L with indications for a local irritation. The alterations may be explained by the amounts of inert material deposited within the respiratory system. In the lungs and bronchi the most prominent finding was a focal early stage of fibrosis. Due to the high amount of test substance deposits in the lungs especially in the mid and high dosed groups these changes may be explained by simply overloading the tissue and do not necessarily imply an intrinsic toxicity of the test material. An intrinsic toxicity is unlikely as the test material is insoluble and the shape of the particles is not fibrous.
Alterations in clinical biochemistry were only seen in female animals. Beside this and the increased absolute and relative organ weights of testes in male animals there was no other substantially sex difference in the response to the test substance. Furthermore, the differences did not show a dose relationship and no correlating effects on the respective organ were seen in the histopathological examinations.
The observed effects were at most mild in severity, never became life threatening, but persisted partly until the end of the recovery period. Furthermore, due to the self-warning characteristics (immediate irritant reactions to the test substance if inhaled), high substance concentrations of inhalation may be avoided.
The No-observed-effect-concentration (NOEC) for local irritation of the test material via inhalation was smaller than 0.006 mg/L. There was however no life threatening systemic effect present up to the highest concentration tested. Therefore, the systemic NOAEC for the test material will be > 0.06 mg/L.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
6 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
The available study is reliable without restrictions (Klimisch score 1), adequate and was performed with the registered substance. It is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity oral

Sodium N-lauroylsarcosinate (CAS 137-16-6) was tested for chronic oral toxicity in a 2-year study in 200 rats (CIR, 2001). Groups of 25 Wistar rats per sex and dose were given 0.05, 0.2 and 1% of the test material in the diet, 7 days/week for 24 months. The concentration of the test material in the low dose group was increased to 2% (equiv. to 1000 mg/kg bw/day) after 6 months. A concurrent negative control group receiving the plain diet was included. At one, three and six months no significant differences were observed in mortality, body weight gain and haematology. At the end of the study period, the only consistent difference observed in the gross pathology was minor hyperplasia of the stratified squamous epithelium with excess keratin formation of the cardiac mucosa of the stomach in rats receiving the highest exposure to the test article (group 1: 2%; group 3: 1% in the diet). Furthermore, fertility assessment did not show any significant differences (no further information). Based on the lack of adverse effects, a NOAEL of 1000 mg/kg bw/day (m, f) was identified in this study.

Sodium N-lauroylsarcosinate (CAS 137-16-6) was tested for subchronic repeated dose oral toxicity in a 90-day study, conducted according to OECD test guideline 408, and in compliance with GLP (Huntington, 1997). Groups of 15 Sprague-Dawley-derived outbred albino rats per sex and dose received the test item at doses of 30, 100 and 250 mg/kg bw/day on 7 days per week for 91 or 92 days, respectively (depending on scheduled sacrifice). Concurrent negative control animals received the vehicle only (sterile, distilled water). No treatment-related mortality or clinical signs of toxicity were observed throughout the study period. Statistically significant decreased body weight gain with 7 and 9% decreased body weights at study termination compared to controls was observed for the mid- and high-dose males (100 and 250 mg/kg bw/day, respectively). In contrast, females of the same dose groups showed recognisable, but statistically not significant lower body weight gain with 4% decreased body weights at necropsy compared to controls. Since animals still gained weight, and the decrease of body weight in comparison to the controls was <10%, this effect is considered to be not adverse. At necropsy, increased absolute stomach weights, stomach/body and stomach/brain weight ratios were noted in both males and females of the 100 and 250 mg/kg bw/day dose groups. All differences were statistically significant, except for absolute stomach weight in mid-dose females. The effect was associated with increased stomach wall thickness and yellow discolouration of non-glandular gastric mucosa. Histopathological examination revealed increased incidence and severity of squamous cell hyperplasia, hyperkeratosis/parakeratosis, inflammation and oedema of the non-glandular gastric mucosa of both males and females in these dose groups. No effects were observed in low-dose animals (30 mg/kg bw/day). Since this effect was noted in a dose-related manner, it is considered to be treatment-related. However, the effects reported were localised to the stomach only, reflecting the irritant characteristics of the test substance, and no further signs of systemic toxicity were observed in any of the animals in any dose-group throughout the study period. Thus, the NOEL of this study is 30 mg/kg bw/day, the LOAEL for local effects in the stomach is 100 mg/kg bw/day, and the systemic NOAEL was set to ≥ 250 mg/kg bw/day.

In summary, both available oral repeated dose toxicity studies revealed local effects to the stomach only. Since the chronic study is the one with the longest study duration, and considering the lack of any systemic effects in both chronic and subchronic studies, the overall NOAEL for systemic toxicity for Sodium N-lauroylsarcosinate was set to 1000 mg/kg bw/day.

In addition, a repeated dose toxicity study via the oral route of N-methyl-N-(C18-(unsaturated)alkanoyl)glycine was carried out equivalent to OECD guideline 407 (vivo Science, 2010). Groups of 3 Wistar rats were given 50, 250 and 1000 mg/kg bw/day of the test material in corn oil by gavage. A negative group receiving the vehicle corn oil was included in the study. Since all animals receiving the high dose of the test item showed signs of toxicity during the first two days of application, application of the high dose was ceased after Day 2 and animals were sacrificed on Day 10 of application. In comparison to the vehicle group, the animals of the high dose group showed mild discomfort at the beginning of administration including increased salivation, laboured breathing and occasional bleeding of mucous membranes from nose and mouth. The observed effects waned after the first week of application. Within the low and mid dose groups, no mortality was observed within the study period. No abnormal clinical signs or altered behaviour were observed in the remaining groups. No changes in body weight, food and water consumption were apparent in the low and mid dose groups. During the necropsy, no changes were observed that were definitively treatment-related (no further information). Due to the possibly locally irritating effects of the test material in the respiratory tract in high-dose animals, the treatment with 1000 mg/kg bw/ day was ceased on Day 2 of application. Thus, based on the lack of systemic toxicity in the mid dose group a NOAEL of 250 mg/kg bw/day (m, f) was identified in this study.

Repeated dose toxicity inhalation

A repeated dose toxicity study via the inhalation route of N-methyl-N-(C18-(unsaturated)alkanoyl)glycine was carried out according to OECD guideline 412 under GLP conditions (Seibersdorf, 2010).

Groups of 5 Fischer rats per sex and dose were exposed to 0.006, 0.02 and 0.06 mg/L of the test material for 6 h/day nose only, 5 days/week for 4 weeks. A concurrent negative control group receiving the vehicle ethanol only was included. Furthermore, additional satellite control and high-dose groups with 5 animals per sex were included in the study for investigating the reversibility of possible effects after a 14-day post-exposure recovery period. The used concentration range of the study was chosen based on the results of a previous range finding test.

All animals survived until the end of the study period. Body weight and body weight gain were lower in the high dose groups. In the mid dose male group an effect was observed at the end of the administration period. No significant effects on food consumption were noted during the study period. Alterations in clinical biochemistry were only seen in female animals; the cholesterol value was decreased during the dosing period and increased at the end of the recovery period in high-dose females. The changes of cholesterol in all females dosing groups may be caused by the test substance. However, corresponding damages of the liver were not found in the animals at necropsy, histopathology and in organ weights. Increased absolute and relative organ weights of testes in male animals were observed. However, the differences did not show a dose relationship and no correlating effects on the respective organ were seen in the histopathological examinations. In addition, no test substance related changes were observed at the gross examination. The aerosol of the test substance caused effects at several sites of the respiratory system at concentrations of 0.006 to 0.060 mg/L with indications for a local irritation. The alterations may be explained by the amounts of inert material deposited within the respiratory system. In the lungs and bronchi the most prominent finding was a focal early stage of fibrosis. Due to the high amount of test substance deposits in the lungs especially in the mid and high dosed groups these changes may be explained by overloading the tissue and do not necessarily imply an intrinsic toxicity of the test material. An intrinsic toxicity is unlikely as the test material is insoluble and the shape of the particles is not fibrous.

In summary, the observed effects were at mostly mild in severity, never became life threatening, but persisted partly until the end of the recovery period. Furthermore, due to the self-warning characteristics (immediate irritant reactions to the test substance if inhaled), high substance concentrations of inhalation may be avoided.

The No-observed-effect-concentration (NOEC) for local irritation of the test material via inhalation was smaller than 0.006 mg/L. There was however no life threatening systemic effect present up to the highest concentration tested. Therefore, the systemic NOAEC for the test material will be > 0.06 mg/L.

Justification for classification or non-classification

Based on read-across from the structurally related substance Sodium N-lauroylsarcosinate and substance specific data, the available data on repeated dose toxicity via the oral and inhalation route do not meet the criteria for classification according to Regulation (EC) No 1272/2008 and are therefore conclusive but not sufficient for classification.