Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
other: Compilation and interpretation of available data.
Adequacy of study:
key study
Study period:
2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Compilation and interpretation of available data.
Cross-referenceopen allclose all
Reason / purpose:
reference to other study
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
2009
Deviations:
yes
Remarks:
The temperature and humidity values deviated from the required range during the exposure and was in range 23.9-26.1ºC and 14.5-25.8%, respectively.
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Version / remarks:
2008
Deviations:
yes
Remarks:
The temperature and humidity values deviated from the required range during the exposure and was in range 23.9-26.1ºC and 14.5-25.8%, respectively.
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
1998
Deviations:
yes
Remarks:
The temperature and humidity values deviated from the required range during the exposure and was in range 23.9-26.1ºC and 14.5-25.8%, respectively.
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
CRL (WI) BR
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Rat, CRL (WI) BR of Wistar origin
Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during the test: Good conventional
Number of animals: 6 animals in the main study (3 male and 3 female)
Sex: Male and female. Female were nulliparous, non-pregnant
Age of animals: Young adult rats, 8-12 weeks old (at start of the study)
Planned body weight range at starting: The weight variation was exceed ± 20% of the mean weight for either sex
Acclimatization time: At least 5 days

Animal health: Only healthy animals were used. The breeder certified the healthy status.
Hygienic level during the test: Good conventional
Housing: Group caging (up to 3 animals, by sex, per cage)
Cage type: Polypropylene/polycarbonate (type III) with stainless steel mesh lids.
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: Above 10 air exchanges/hour by central air-condition system.
Housing/Enrichment: Rats are group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.

Feed: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
The food is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Water: Tap water from watering bottles ad libitum. The drinking water is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). The quality of the bedding material is guaranteed by the supplier. The bedding is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
3.65 µm
Geometric standard deviation (GSD):
2.84
Remark on MMAD/GSD:
During the technical phase of the study, it was intended to obtain particles of 1 to 4 µm MMAD.
Details on inhalation exposure:
Atmosphere generation: The test item was aerosolised using a Wright Dust Feeder II. (CH Technologies (USA), located at the top of the exposure chamber. Compressed air was supplied by means of an oil-free compressor and passed through a suitable filter system prior to introduction to the dust generator.

Animal Exposure Conditions
The animals were held in polycarbonate restraining tubes and exposed “nose-only” under dynamic air flow conditions, using an anodised aluminium Flow Past Exposure Chamber. (CR Equipment SA, Switzerland). Validation of the same type of system was published by J. Pauluhn. (1994).
The exposure unit is placed in closed hood in order to avoid cross-contamination and contamination of the laboratory environment.
The inhalation chamber used is modular and for this study was assembled from 4 identical vertical modules, with 8 animal ports in each module. The animals were distributed on one module (levels 1, counted from the top) of the exposure chamber.
Each module of the chamber consists of two concentric cylinders with inner diameter of 25 mm and 80 mm, respectively. The height of the modules is 110 mm. In this system the air carrying the test substance enters primary from above in the central cylinders and is distributed in each module through 8 radial tubes (diameter: 5 mm, length: 40 mm) to the animal ports, i.e. to the openings (diameter: 30 mm) on the wall of the outer cylinder where the animal restraining tubes can be attached. From the animal ports the air turns back into the outer cylinder towards the vacuum pump. In the particular set-up the radial tubes as well as the animal ports in the lowest module were closed by plastic stoppers. In such a way the air containing particles of the test item could flow out from the central plenum only through the 16 openings of the two upper levels. Here 6 animal ports were occupied by the animals, 1 by the Temperature-Humidity Sensor, 1 port was used for sampling on aerosol filters and the Cascade Impactor. All eight animal ports on the 2nd level (but not the radial tube) were closed by a plastic stopper.
The total volume of the exposure chamber was 2.24 L. The air flow rate of 19.3 L/min (1.2 L/min to each animal port) excluded re-breathing of the animals and ensured the continuous presence of fresh aerosol and of natural oxygen concentration in the breathing zones.
The air was supplied by an oil-free air compressor and was filtered in a two-stage filter set. The temperature of the air was regulated by a heat exchanger. The air was not humidified in order to avoid modification of particle size distribution.
The temperature and humidity values deviated from the required range during the exposure and was in range 23.9-26.1ºC and 14.5-25.8%, respectively. Reason for this change: Malfunction of the cooling system of compressor. Presumed effect on the study: This deviation is not considered to have affected the integrity and validity of the study.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
Gravimetrically.
Duration of exposure:
4 h
Remarks on duration:
The four-hour exposure period was not started until the theoretical chamber concentration equilibration, calculated according to Silver S. D. (1946), has been reached.
Concentrations:
Target concentration: 5 mg/L.
No. of animals per sex per dose:
3m + 3f.
Control animals:
no
Details on study design:
Particle Size Analysis:
Particle size analysis of generated atmospheres was performed using a 7-stage cascade impactor type 02-150 (IN-TOX Products, N.M., USA). During the exposure period samples were collected three times with one sample taken during the first hour of exposure and one during the last hour. The log-probit plots of the resulting data was used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and percentage < 4 μm (considered to be inhalable in the rat).
Chamber airflow rates, test atmosphere temperature and test atmosphere relative humidity were monitored continously.

Actual Test Atmosphere Concentrations
The actual concentration of generated atmosphere was measured at regular intervals during exposures by pulling a suitable, known volume of test atmosphere from the exposure chamber, through GF10 glass fibre filters (GE Healthcare Life Science, Whatman GmbH, Germany). Since it was proved that the concentration is stable within ±10% during the technical trials then at least 8 samples were taken during each exposure. Sampling was performed shortly after chamber equilibration and then uniformly distributed, at regular intervals. Samples were collected from a vacant animal exposure port (animals breathing zone). The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item.
The amount of material collected on glass fibre filters was determined gravimetrically.

Morbidity/Mortality were checked twice daily.
Clinical observations were performed after one, two and three hours exposure whilst the animals are still restrained. Following exposure clinical observations were performed at least twice on the day of exposure and then at least once daily for fourteen days.
Cage-side observations included changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Body weights were recorded on the day of exposure day 0 (prior to exposure) and days 1, 3, 7 and 14.
Necropsy/Pathology: A gross necropsy was performed on all animals euthanised by exsanguination following intra-peritoneal injection of pentobarbital solution at the end of the observation period.
A complete examination of the abdominal and thoracic cavities was made and special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity.
Histopathology: During necropsy no organs were found with macroscopic abnormalities therefore no organ has been preserved.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.24 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No animals died during the study.
Clinical signs:
other: Only dyspnoea (3/3♂, 3/3♀) and reddish staining around snout (3/3♂, 3/3♀) were recorded in the animals during and/or shortly after the exposure. Reddish staining around snout was also recorded on the day following exposure, however all animals recovered a
Body weight:
Normal bodyweight gain was noted in the exposed animals during two weeks observation period, with the exception of two female rats (2204, 2207) where slight bodyweight loss was observed occasionally during the first week of the observation period.
Gross pathology:
No test item-related macroscopic alterations were detected.
Other findings:
Test Atmosphere Concentration:
Target Concentration: 5 mg/L
Mean achieved Concentration: 5.24 mg/L
Standard Deviation: 0.12
Nominal Concentration: 12.44 mg/L

Particle Size Analysis:
Mean achieved concentration (mg/L): 5.24 mg/L
Mean Mass Median Aerodynamic Diameter (MMAD): 3.65 μm
Geometric Standard Deviation (GSD): 2.84
Inhalable Fraction (% < 4μm): 56.0 %

Test Chamber Environmental and Equilibration Data:
Mean Air Flow Application (Inner Plenum) (L/min): 30.2
Air Flow Out (Outer Cylinder) (L/min): 39.9
Temperature (°C): 25.1
Relative Humidity (%):16.2
Interpretation of results:
GHS criteria not met
Conclusions:
The acute inhalation LC50 of the test substance in Wistar rats is >5.24 mg/L.
Executive summary:

An acute inhalation toxicity study according to the OECD guideline 436 was performed. The only study group, consisting of 6 Wistar rats (3 males and 3 females), was exposed to the target concentration of 5 mg test substance/L as an aerosol. The animals were exposed for 4 hours using a nose-only exposure system, followed by a 14-day observation period. Aerosol concentration was measured gravimetrically. The particle size distribution of the test aerosol was determined regularly during the exposure period. Clinical observations and bodyweights were recorded throughout the study and at the end of the scheduled period the animals were euthanised and subjected to a gross examination post mortem.

 

Results:

Gravimetric results of the test atmosphere analysis: The mean achieved concentration in the study was 5.24 mg/L. The mass median aerodynamic diameter (MMAD) was 3.65 μm with a geometric standard deviation (GSD) 2.84.

Mortality: No animals died during the study.

Clinical observations: Only dyspnoea (3/3 m, 3/3 f) and fur stating by test item (3/3 m, 3/3 f) were recorded in the animals during and/or shortly after the exposure. All animals recovered and were symptom-free from Day 2 until the end of the observation period.

Dyspnoe and reddish staining around snout were reported in each of the last 8 reports of the same testing facility on acute inhalation toxicity, with partly quite different test items. It is therefore likely that these signs are not expressions of local or systemic toxicity of the test items but that they are signs of discomfort of the rats restricted in the inhalation tubes.

Bodyweight: Normal bodyweight gain was noted in the exposed animals during two weeks observation period, with the exception of two female rats (2204, 2207) where slight bodyweight loss was observed occasionally during the first week of the observation period.

Necropsy: No test item-related macroscopic alterations were detected.

The acute inhalation LC50 of the test substance in Wistar rats is >5.24 mg/L.

Reason / purpose:
reference to other study
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 19, 1994 to November 30, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study (OECD 401) with acceptable restrictions.
Qualifier:
according to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Animals:
Supplier: Forschungsinstitut für Versuchstierzucht, A-2325 Himberg
Age: approximately 8 weeks at time of administration
Environment:
Room temperature: average of 22 °C,
Humidity: average of 55 %,
Air exchange: 12 per hour,
Light artificial light from 6 a.m. to 6 p.m.,
Cages: Single caging in Makrolon cages type III,
Bedding material: Aspen wood chips, type "4 HV",
Feed: Altromin 1314 ff, gamma irradiated with 10 kGy 60Co, ad libitum,
Water: tap water, offered in Makrolon bottles with stainless steel canules, ad libitum,
Acclimatization: 6 days
Route of administration:
oral: gavage
Vehicle:
other: Carboxymethylcellulose, sodium salt
Details on oral exposure:
Peroral administration was performed within maximum 30 minutes after preparation of the test substance suspension once in the morning by stomach intubation using a metal gavage. The test substance suspension was stirred during the time of administration.
Doses:
2000 mg/kg body weight (20 ml/kg)
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
Observations in life: Behaviour, reactions and physical signs of the animals were observed 0-0.5, >0.5-1, >1-2, >2-4 and >4-6 hours after administration of the test substance (p.a.) and then at least once a day for a total of 2 weeks.

Body weight and body weight gain: Body weight was determined before administration, 7 days p.a. and 14 days p.a.
Body weight gain was calculated for each week of the study, i.e. 0-7 d p.a. and 7-14 d p.a.

Necropsy: All animals were sacrified by CO2 14 days p.a. and examined macroscopically in an attempt to detect possible residual lesions.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
All animals (5 males and 5 females) survived until the scheduled termination of 14 days p.a.
Clinical signs:
Males: 3/5 animals were normal during the whole obser vation period. In the affected males chromodacryorrhoea, an unspecific sign of general malaise, was noted on day of administration of the test substance for about 2 hours.
Females: All animals, i.e. 5 females, were normal during the whole observation period.
Body weight:
Males and females: At the scheduled examination terms, body weight and body weight gain were inconspicuous in all animals.
Gross pathology:
Males and females: All animals were normal at terminal necropsy.
Other findings:
Sex differences: Signs in life and post mortem findings revealed no differences between the sexes in the response to the test substance.
Interpretation of results:
GHS criteria not met
Conclusions:
The LD50 is above 2000 mg/kg bw.
Executive summary:

An acute oral toxicity study with rats was performed. The LD50 is above 2000 mg/kg bw.

Data source

Reference
Reference Type:
other: Compilation from available data.
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: Section R.7.12.2.1 of the 'Guidance on information requirements and chemical safety assessment'
Version / remarks:
2017
Deviations:
not specified
Principles of method if other than guideline:
Compilation and interpretation of available data.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Radiolabelling:
no

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
No relevant systemic toxic effects were noted in the acute oral and the acute inhalation toxicity studies.

Guidance for an oral absorption:
Based on the Section R.7.12.2.1 of the 'Guidance on information requirements and chemical safety assessment' some of the relevant physical-chemical properties for an oral absorption are:
- Ionic groups in the molecule are present and do not favour an absorption. The low pH of stomach may favour the absorption of the acidic molecule, but not of the contained amino-group.
- Molecular weights below 500 are favourable for absorption. 7-ACA has a molecular weight of 272.
- The water solubility of 7-ACA is low: 611 mg/L at 20 °C.
- The low n-octanol/water partition coefficient (log Pow <-3.4) for 7-ACA does not enable an extensive absorption by passive diffusion.
These data point to a low to moderate oral absorption - in agreement with no observed toxic effects in the available acute oral toxicity study.

Guidance for an inhalation absorption:
An acute inhalation toxicity study in rats with a MMAD of 3.65 µm did not show systemic toxic effects at the limit concentration of 5.24 mg/L, therefore no indication of an absorption was obtained. The relevant parameters for absorption at inhalation exposure are not very different to those for oral absorption, as described in the Section R.7.12.2.1 of the 'Guidance on information requirements and chemical safety assessment'.

Guidance for a dermal absorption:
For dermal absorption an even lesser absorption is predicted compared to the oral route, based on the following criteria:
- Molecular Weight: The range between 100 and 500 favours dermal uptake.
- n-octanol/water partition coefficient: For substances with log P values <0, poor lipophilicity will limit penetration into the stratum corneum and hence dermal absorption. Values <–1 suggest that a substance is not likely to be sufficiently lipophilic to cross the stratum corneum, therefore dermal absorption is likely to be low.
- Water Solubility: The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis
Details on distribution in tissues:
Distribution:
No indication from the available studies was obtained on the possible distribution of the substance in the body.

Bioaccumulation:
No relevant accumulation is expected, based on the low log Pow.
Details on excretion:
Excretion:
No relevant data are available, which provide evidence on the route of excretion.

Metabolite characterisation studies

Details on metabolites:
Metabolism:
No relevant differences occurred in the mutagenicity studies with and without the addition of a metabolising system. Therefore no indication of the importance of the metabolism of the test substance was obtained from these studies.

Applicant's summary and conclusion

Conclusions:
The toxicokinetic behaviour of the test item was estimated from available data.
No evidence of an oral absorption was obtained from the available acute toxicity tests. A low oral absorption is also predicted from the phys.-chem. properties of the substance.
No evidence of an inhalation absorption was obtained from the available acute toxicity test. The dermal absorption is predicted to be lower compared to the oral route.
No evidence of a distribution or of the way of excretion was obtained.
No evidence of metabolisation was obtained from the mutagenicity tests with and without metabolising systems.
No bioaccumulation is expected, based on the low log Pow.
Executive summary:

The toxicokinetic behaviour of the test item was estimated from available data.

No evidence of an oral absorption was obtained from the available acute toxicity tests. A low oral absorption is also predicted from the phys.-chem. properties of the substance.

No evidence of an inhalation absorption was obtained from the available acute toxicity test. The dermal absorption is predicted to be lower compared to the oral route.

No evidence of a distribution or of the way of excretion was obtained.

No evidence of metabolisation was obtained from the mutagenicity tests with and without metabolising systems.

No bioaccumulation is expected, based on the low log Pow.