Registration Dossier

Administrative data

Description of key information

oral (OECD 401): LD50 > 5000 mg/kg bw (limit test, rat)
inhalation (OECD 403): LC50 > 6.3 mg/L (limit test, rat; 4 h exposure)
dermal (OECD 402): LD50 > 2000 mg/kg bw (limit test, rabbit)

Key value for chemical safety assessment

Acute toxicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
5 000 mg/kg bw
Quality of whole database:
The available information comprises adequate and reliable (Klimisch score 2) studies. The information from these independent sources is consistent and provides sufficient weight of evidence for hazard assessment leading to an endpoint conclusion in accordance with Annex XI, 1.2, of Regulation (EC) No 1907/2006. Therefore, the available information as a whole is sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (analytical purity of test substance not specified, only 4 days acclimatisation time, particle size of 5.2 µm MMAD in accordance to former guideline)
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
analytical purity of test substance not specified, only 4 days acclimatisation time, particle size of 5.2 µm MMAD in accordance to former guideline
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc.
- Age at study initiation: young adult
- Weight at study initiation: 180-300 g
- Housing: All animal housing and care conformed to AAALAC standards and to those published in the "Guide for the CareI and Use of Laboratory Animals," NIH Publication N0. 85-23. The animals were individually housed in suspended stainless steel wire mesh bottom cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 4 days


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Rochester type inhalation chamber
- Exposure chamber volume: 270 L
- Method of holding animals in test chamber: The animals were individually housed during the exposure in wire mesh cages without access to food or water.
- Source and rate of air: High pressure external air source, 75 L / min
- System of generating particulates/aerosols: Particle generator (Model FD-100. Unifab Corporation, Kalamazoo. Michigan)
- Method of particle size determination: Particle size analysis was performed once per hour during the exposure using an Anderson 2000 impactor (Model 20-800). Stages one to eight of the impactor, and the final filter stage were fitted with pre-weighed glass fiber filters. A known volume of chamber air (30 L) was drawn through the impactor and the change in weight of each filter was then determined and recorded.
- Treatment of exhaust air: Air treatment system which consisted of a HEPA filter, a charcoal filter and a water scrubber
- Temperature, humidity, pressure in air chamber: The test atmosphere temperature, relative humidity and percent oxygen content, and the air flow rate to the chamber were recorded at approximately 30 minute intervals during the exposure. Average temperature, relative humidity, and oxygen content of the test atmosphere were 21°C, 56.8% and 21.0%, respectively.

TEST ATMOSPHERE
The inhalation chamber was maintained at a slightly negative pressure at all times during operation. Air flow rate to the chamber was monitored continuously during the exposure using calibrated Dwyer air flow meters (Dwyer Instruments, Inc.).
- Brief description of analytical method used: The average actual concentration of the test atmosphere was determined by gravimetric sampling. At the time of theoretical chamber equilibration a test atmosphere sample was drawn from the breathing zone of the chamber (5 L) through a pre·weighed glass fiber filter. The change in weight of the filter (mg) was determined and this value was divided by the volume of test atmosphere sampled (5 L) to yield the actual test atmosphere concentration. Additional gravimetric samples were obtained at approximately 30 minute intervals during the exposure. The average actual concentration of the test atmosphere was calculated for the exposure based on the initial and subsequent concentration analyses.
- Samples taken from breathing zone: yes
- MMAD: The mass median aerodynamic diameter of the generated particles was 5.2 µm and the standard geometric deviation was 1.7. Approximately 87% of the particles were less than 10 µm in size.



Analytical verification of test atmosphere concentrations:
yes
Remarks:
by gravimetric sampling
Duration of exposure:
240 min
Concentrations:
The average actual test atmosphere concentration was determined to be 6.3 mg/L. The calculated nominal concentration was 27 mg/L. Thus, the average actual concentration was approximately 23.3% of the calculated nominal test atmosphere concentration.
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Mortality checks were performed twice daily, a minimum of 5 hours apart. The animals were observed for outward signs of toxicity 3 times on study day 1 (post exposure) and once daily thereafter for the duration of the study (day 15). Due to the density of the test atmosphere, an accurate observation of the study animals could not be performed during the actual exposure. Individual body weights were determined and recorded on study days 1, 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, histopathology
Statistics:
not determined
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 6.3 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
240 min
Mortality:
1 male, necropsy findings suggest that death was caused by asphyxiation
Clinical signs:
other: Labored breathing and/or rales, dark material around nose or mouth, decreased activity, urine stain, trashing (in cage), for details see table 1 in remarks
Body weight:
Decreased body weight gain and/or weight loss were observed in both the male and female animals. No net change in mean body weight was observed in the male rats between days 1 and 15. In the female animals, mean body weight was decreased approximately 5% during the period of the study.
Gross pathology:
Necropsy examinations of the surviving animals revealed yellow material in the stomach (4 females), pale lungs (one male) and
multifocal dark red foci on the lungs (one male). The significance of the above findings, if any was not determined in this study.
Other findings:
- Histopathology: No microscopic lesions were observed in the kidneys or liver of any animal.

Table 1: Clinical signs during the 4 day observation period after exposure; because of the death of one male, 4 males and 5 females (9 animals) were examined

 

Incidence of animals exhibiting finding / number of total animals on day

Finding

1

2

3

4

Labored breathing and/or rales

7 / 9

7 / 9

3 / 9

2 / 9

Dark material around nose or mouth

3 / 9

4 / 9

1 / 9

0 / 9

Decreased activity

0 / 9

3 / 9

0 / 9

0 / 9

Urine stain

0 / 9

1 / 9

0 / 9

0 / 9

Trashing (in cage)

1 / 9

0 / 9

0 / 9

0 / 9

Based on the results of this study, the acute inhalation LC50 of ACRAWAX C in rats is estimated to be greater than 6.3 mg/L.

According to the criteria laid down in Regulation (EC) 1272/2008 and Directive 67/548/EEC ACRAWAX C does not have to be classified for acute toxicity via the inhalation route.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
Value:
6 300 mg/m³
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (analytical purity of the test substamce, humidity and temperature are not specified in the study report)
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
yes
Remarks:
analytical purity of the test substamce, humidity and temperature are not specified in the study report
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Gota-Frisco Farms
- Age at study initiation: young adult, not further specified
- Weight at study initiation: 2.0 - 3.0 kg, weight variation did not exceed ± 20 % of the group mean of each sex
- Housing: Individually housing in suspended stainless steel wire mesh bottom cages, conform to AAALAC standards and those published in the "Guide for the care and use of laboratory animals" NIH publication No. 85-23
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Minimum of 5 days


ENVIRONMENTAL CONDITIONS

- Photoperiod (hrs dark / hrs light): 12/12


Type of coverage:
semiocclusive
Vehicle:
water
Details on dermal exposure:
TEST SITE
- Area of exposure: 12 x 20 cm
- % coverage: 10 %
- Type of wrap if used: A tubular stockinette sleeve and an adjustable harness were placed around the animal’s trunk. Nonirritating tape was used to secure both the gauze dressing and stockinette sleeve.

REMOVAL OF TEST SUBSTANCE
- Washing: yes, with distilled water
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- For solids, paste formed: yes, the dressing was moistened with distilled water at a rate of 1 mL/g of test article to ensure good contact of the test article with the skin


Duration of exposure:
24 h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for pharmacotoxic signs three times on the day of dosing and once daily thereafter for the duration of the study (15 days). Mortality checks were performed twice daily, a minimum of 5 hours apart. Individual body weights were determined and recorded on Days 1, 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: Histopathology: At study termination (day 15), the heart, kidneys, liver, lungs and stomach were excised from each animal and preserved. After complete fixation, the liver and kidneys from each rabbit were embedded in paraffin, sectioned at 3-5 mm in thickness, stained with hematoxylin and eosin, and then examined microscopically by an SIB Pathologist.
Statistics:
no data
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Mortality:
All animals survived until scheduled sacrifice.
Clinical signs:
No clinical signs of toxicity were noted during the study.
Body weight:
No treatment related body-weight changes were noted during the study.
Gross pathology:
Necropsy examinations revealed the kidneys of two males to have a pitted capsular surface. No other gross abnormalities were noted.
Other findings:
- Histopathology: Microscopic examination of the liver revealed minimal chronic multifocal inflammation in three animals (one male and two females) and minimal acute exudative multifocal inflammation in one animal (male). In three of the ten test animals, chronic multifocal inflammation of the liver and kidneys occurred together.

- Other observations: The occurrence of the above pathological findings cannot be definitely attributed to treatment with the test article as similar lesions are commonly seen at this incidence level in the liver and kidneys of untreated stock laboratory rabbits.

Acrawax C is judged to be nonlethal and nontoxic when administered by the dermal route at 2000 mg/kg body weight to New Zealand albino rabbits.

Acrawax C does not have to be classified for acute toxicity via the dermal route according to the criteria laid down in Regulation (EC) 1272/2008 and Directive 67/548/EEC.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, of Regulation (EC) No 1907/2006.

Additional information

There are three studies available for oral toxicity, two limit tests and a study with three doses ranging from 1350 to 15380 mg/kg bw. Although all of these study reports are acceptable for assessment, each of them has some minor flaws. The study by Lilja, 1981, was a limit test conducted with 10 animals at a dose of 5000 mg/kg bw, but administered in a high dose volume of 15 mL/kg bw of a non-aqueous vehicle, the study by Til and Spanjers, 1983, was conducted with 20 animals receiving a dose of 5000 mg/kg bw, but with an even higher dose volume of 20 mL/kg bw of a non-aqueous vehicle. In the study by Nham and Harrison, 1976, three dose levels of 1350, 4556 and 15380 mg/kg bw were administered, but only 4 animals (2 of each sex) were included in the respective groups.

However, there was no mortality observed in any of these studies even up to the highest administered dose of 15380 mg/kg bw, no clinical signs were observed except a slight hypoactivity within 5 minutes after application described in the study by Nham and Harrison, body weights were not affected, and no apparent gross tissue or organ abnormalities were detected by pathology in any of the animals necropsied 14 days after treatment. Therefore, it is reasonable to conclude that the LD50 is at least greater than 5000 mg/kg bw.

Two studies are available for acute inhalation toxicity. In the key study (Siglin, 1987) 10 animals (5 per sex) were exposed whole body to a fixed gravimetrically determined concentration of 6.3 mg/L for 4 hours. The mass median aerodynamic diameter of the generated particles was determined to be 5.2 ± 1.7 µm, which is acceptable considering the technical standards and the requirements of the guideline at the time of the study. No mortality occured during exposure or the 14-day observation period, therefore the LC50 was determined to be greater than 6.3 mg/L. In the supporting study (Rusch, 1979) 10 animals were exposed to a nominal exposure concentration of 58.2 mg/L for 1 hour only, which was not analytically confirmed. No mortality occurred under the conditions of this study, either. During exposure, no treatment-related abnormalities were observed, while clear signs of a reversible irritant effect were reported in the post-treatment observation period. According to Haber's Law one could calculate a nominal LC50 > 14.55 mg/L for a 4-hour exposure, but as analytical confirmation of the actual concentration is missing, the lack of mortality in this study can only support the view of an LC50 > 6.3 mg/L.

There are data for acute dermal toxicity available from a limit test conducted with rabbits (Siglin, 1986). In this study 10 animals (5 per sex) were exposed to a single dose of 2000 mg/kg bw under semiocclusive conditions for 24 hours. No mortality occurred, and no clinical signs were observed in the 14-day observation period. Pathological and histopathological findings in the liver and the kidneys were observed in some animals at necropsy that were assumed to be not treatment-related and to be common for untreated stock laboratory rabbits, as well. Therefore, the acute dermal LD50 was assumed to be greater than 2000 mg/kg bw.


Justification for selection of acute toxicity – oral endpoint
Hazard assessment is based on the weight of evidence from all available studies.

Justification for selection of acute toxicity – inhalation endpoint
The selected study is the most adequate and reliable study with the lowest dose descriptor.

Justification for selection of acute toxicity – dermal endpoint
There is only one study available.

Justification for classification or non-classification

The available data on the acute toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.