Reaction mass of N,N-ethanediylbis- Hexadecanamide, N-[2-[(1-oxohexadecyl)amino]ethyl]-Octadecanamide, Hexadecanoic acid, 12-hydroxystearic acid, reaction products with ethylenediamine, N,N-ethanediylbis-Octadecanamide and Octadecanoic acid, 12-hydroxystearic acid, reaction products with ethylenediamine

Endpoint summary

Description of key information

oral (OECD 423): LD50 > 2000 mg/kg bw (limit test, rat)

inhalation (OECD 403): LC50 > 6.3 mg/L (limit test, rat; 4 h exposure; structural analogue)

dermal (OECD 402): LD50 > 2000 mg/kg bw (limit test, rabbit; structural analogue)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: HanRcc: WIST (SPF)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services CH-4414 Füllinsdorf
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12 weeks
- Fasting period before study: fasted for approximately 17 hours
- Housing: In groups of three in Makrolon type-4 cages with wire mesh tops and standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3433 rat/mouse mainte-nance diet, batch no. 001/06 (Provimi Kliba AG, CH-4303 Kaiseraugst/Switzerland) ad libitum.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf ad libitum
- Acclimation period: Under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 30.5. To: 22.06.2006

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

Number of animals per group: 3 females
Total number of animals: 6 females

Control animals:

yes

Details on study design:

DOSE FORMULATION:
The test item was pulverised into a fine powder at RCC Ltd Itingen with a centrifugal mill 1000 (retsch ZM 1000; Serie-Nr.: 30379024). The test item was mixed with liquid nitrogen approximately 10 minutes before milling and was added bit by bit to the centrifugal mill. Thereafter, the test item was dried during 12 hours in an exsiccator at room temperature. The dose formulations were made shortly before each dosing occasion using a magnetic stirrer and a spatula as homogenizers.
The test item reduced into a fine powder was weighed into a tared glass beaker on a suitable precision balance and the vehicle added (weight: volume). Homogeneity of the test item in the vehicle was maintained during administrantion using a magnetic stirrer.

TREATMENT:
The animals received a single dose of the test item by oral gavage administration at 2000 mg/kg body weight after being fasted for approximately 17 hours (access to water was permitted). Food was provided again approximately 3 hours after dosing. The application volume was 10 mL/kg body weight.
Rationale: Oral administration was considered to be an appropriate application method as it is a possible route of human exposure during manufacture, handling and use of the test item.

OBSERVATIONS:
- Mortability/Viability: Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15.
- Body weights: On test days 1 (prior to administration), 8 and 15.
- Clinical signs: Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1. Once daily during days 2-15. All abnormalities were recorded.

PATHOLOGY:
- Necropsy: All animals were killed at the end of the observation period by Carbon dioxide asphyxiation and discarded after macroscopic examinations were performed. No organs or tissues were retained.
- Statistical analysis: No statistical analysis was used.
- Data compilation: Body weights were on-line. Clinical signs were recorded on data sheets. Mortability/viability were compiled into the RCC Tox Computer System during recording and/or recorded on data sheets. Macroscopic findings were compiled into the RCC Tox Computer System during recording. The RCC Tox Computer System (RCC-Tox-Lims) had been validated with respect to data collection, storage and retrievability.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other:

Remarks:

after single oral administration to female rats, observed over a period of 14 days.

Mortality:

No deaths occurred during the study.

Clinical signs:

other: Slightly ruffled fur was noted in all animals of the first treated group from the 1-hour reading to test day 3. Hunched posture was also noted in the same animals from the 2- or 3-hour reading to the 5-hour reading nd slight sedation was also noted in two

Gross pathology:

No macroscopic findings were recorded at necropsy.

Interpretation of results:

other: not classified

Conclusions:

The median lethal dose of the test substance after single oral administration to female rats, observed over a period of 14 days is LD50 (female rat) > 2000 mg/kg body weight.

Executive summary:

Two groups, each of three female HanRcc:WIST (SPF) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight. The test item was diluted in vehicle (PEG 300) at a concentration of 0.2 g/mL and administered at a volume dosage of 10 mL/kg.

The animals were examined daily during the acclimatization period and mortality, viability and clinical signs were recorded. All animals were examined for clinical signs at approximately 30 minutes, 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test days 2 -15. Mortality/viability was recorded at approximately 30 minutes, 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2 -15. Body weights were recorded on day 1 (prior to administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

All animals survived until the end of the study period.

Slightly ruffled fur was noted in all animals of the first treated group from the 1 -hour reading to test day 3. Hunched posture was also noted in the same animals from the 2- or 3 -hour reading to the 5 -hour reading and slight sedation was also noted in two animals at the 3- and 5 -hour reading.

In the second treated group, slightly ruffled fur was noted in all animals from the 2- to the 5 -hour reading. Hunched posture was present in the same animals from the 1 -hour to the 3 -or 5 -hour reading. Slight sedation was noted in one animal at the 3 -hour reading and in the two remaining animals from the 2- to the 5 -hour reading.

The body weight of the animals was within the range commonly recorded for this strain and age. No macroscopic findings were recorded at necropsy.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

reliable without restriction

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

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 or test system 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

Key result

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.

Interpretation of results:

GHS criteria not met

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

6 300 mg/m³ air

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:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

yes

Remarks:

analytical purity of the test substance, 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 or test system 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

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

All animals survived until scheduled sacrifice.

Clinical signs:

other: No clinical signs of toxicity 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.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the result of the acute dermal toxicity study the LD50 is > 2000 mg/kg bw.

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

Thereis one limit dose study available for oral toxicity. Based on the results of this OECD 423 study the LD50 is at least greater than 2000 mg/kg bw.

Two studies are available for acute inhalation toxicity, which were performed with the structural analogue, Licowax C. 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) with the strucutral analogue, Licowax C. 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 classification or non-classification

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