Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Endpoint summary

Description of key information

The acute oral and inhalation toxicity studies indicate that the substance is of low  toxicity if swallowed (rat LD0 > or = 2000 mg/kg bw) or inhaled (rat LC0 > or = 5050 mg/m3).
No information regarding the acute dermal toxicity of the test substance is available. 

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:

From 25 July 2012 to 21 September 2012

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)

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Approximately 8 weeks old on the day of treatment
- Mean body weight at study initiation: 207 g (range: 191 g to 225 g)
- Fasting period before study: Yes, fasted overnight before treatment. Food was given approximately 4 h after administration of the test substance.
- Housing: The animals were housed by three from the same group in polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: Tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: At least 5 or 8 d before the treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 07 August 2012 to 05 September 2012.

DEVIATION: The temperature recorded in the animal room was sometimes outside of the target ranges specified in the study plan. However, this deviation was considered not to have compromised the validity or integrity of the study.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose aqueous

Details on oral exposure:

VEHICLE
The solubility assay first started at the concentration of 200 mg/mL, and the first choice vehicle was drinking water treated by reverse osmosis.
Since a heterogeneous suspension at 200 mg/mL was obtained with drinking water treated by reverse osmosis, the test substance was tested with a 0.5% methylcellulose aqueous solution.
A homogenous suspension was obtained at the concentration of 200 mg/mL in a 0.5% methylcellulose aqueous solution.

DOSAGE PREPARATION (if unusual): The test substance was administered as a homogeneous suspension in the vehicle. The test substance was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
Dose formulations preparations were prepared extemporaneously on the day of each administration.
The dose formulations were stored at room temperature and delivered to the study room in brown flasks.

CLASS METHOD (if applicable):
- Rationale for the selection of the starting dose:
Since no relevant toxicity data were available for the estimation of a lethal dose-level and any existing data were taken into account by the Sponsor, the starting dose level was 300 mg/kg for ethical reasons.
After treatment at the starting dose-level, the next higher dose-level of 2,000 mg/kg was tested.

ADMINISTRATION:
The dose formulations were administered once by gavage, using a plastic syringe fitted with a plastic gavage tube. The quantity of dose formulation administered to each animal was adjusted according to the bodyweight recorded on the day of the dose administration.
A constant dosage-volume of 10 mL/kg was used. The dose formulations were stirred continuously throughout the dosing procedure.

Doses:

300 and 2000 mg/kg bw.

No. of animals per sex per dose:

3 females per treatment step.

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Clinical observations: At least once during the first 30 minutes, periodically during the first 4 hours, then once a day, at approximately the same time.
- Morbidity and mortality: Frequently during the hours following administration, then at least once a day until the end of the observation period, including weekends and public holidays.
- Bodyweight: Just before treatment on Day 1; then on Days 8 and 15.
- Necropsy of survivors performed: Yes (macroscopic). The main organs included the digestive tract, heart, kidneys, liver, lungs, pancreas, spleen and any other organs with obvious abnormalities. All gross observations were recorded individually for each animal.

Statistics:

none

Key result

Sex:

female

Dose descriptor:

LD0

Effect level:

>= 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No unscheduled deaths occurred during the study.

Clinical signs:

other: No clinical signs were observed in any animals.

Gross pathology:

There were no macroscopic post-mortem findings.

Table 7.2.1/1: Mean body weight changes (g) in treated animals during the observation period compared to laboratory historical control data

Sex	Female
Group	Laboratory Historical control data	1	2	3
Dose-level (mg/kg bw)	0	300	2000	2000
Body weight
Day 1	219	196	206	219
Day 8	264	241	246	263
Day 15	284	256	261	274
Body weight change
Day 1	+44	+45	+40	+44
Day 8	+20	+15	+15	+11
Day 15	+64	+60	+55	+55

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the oral LD0 of the test substance was equal or higher than 2,000 mg/kg bw in rats.

Executive summary:

The substance was tested for acute oral toxicity according to the OECD 423 guideline and in compliance with Good Laboratory Practice.

The test item was administered once by gavage to 3 groups of 3 fasted female rats under a dosage-volume of 10 mL/kg bw. The test item was prepared in a 0.5% solution in methylcellulose. Since no relevant toxicity data were available for the estimation of a lethal dose-level and any existing data were taken into account, the starting dose-level was 300 mg/kg bw for ethical reasons. After the first assay, the next higher dose-level of 2000 mg/kg bw was tested. Since no toxicity was observed at this higher dose-level, the results were confirmed in other females.

Each animal was observed at least once a day for mortality and clinical signs for 15 days. The bodyweight was recorded before treatment then on days 1, 8 and 15. All surviving animals were necropsied at the end of the observation period.

No unscheduled deaths occurred during the study and no clinical signs were observed in any animals. A trend towards a slightly lower mean bodyweight gain was noted in females given the test substance at 2,000 mg/kg, when compared to historical control data. There were no macroscopic post-mortem findings.

The acute oral LD50 was found greater than 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:

Study complete and sufficient to fulfill the endpoint requirements.

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

Study period:

From 04 Sept 2012 to 18 Sept 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: Young adult rats, 10-11 weeks old
- Weight at study initiation: 244 to 428 g (males: 405-428 g; females: 244-259g)
- Housing: Standard housing conditions
- Bedding: Lignocel bedding for laboratory animals was available to animals during the study
- Diet (e.g. ad libitum): Ssniff SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance" ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 26 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3 °C
- Humidity: 30–70 %
- Air changes: 15-20 air exchanges/h
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Deviation: The temperature (22±3ºC) and the humidity (30–70%) values deviated from the required range during the acclimation period in the animal room. However, these deviations had no effect on the purpose and integrity of the study.

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

3.62 µm

Geometric standard deviation (GSD):

4.49

Remark on MMAD/GSD:

The Geometric standard deviation of the generated aerosol during the animal exposure was greater than 4 µm, it came from the characteristics of the test item. However, it had no effect on the purpose and integrity of the study. The proportion of aerosol less than 4µm was 52.6%.

Details on inhalation exposure:

Atmosphere generation:
The test substance formulation was aerosolised using a rotating brush powder disperser (Palas GmbH, Karlsruhe, Germany) located at the top of the exposure chamber and compressed air. The compressed air was passed through a respiratory quality filter train and condensate separator prior to use. The dust aerosol produced was then ducted to the exposure system using suitable tubing and various devices may have been included between the generation and exposure systems in order to increase the proportion of the aerosol in the target particle size range.

Animal exposure system
The animals were exposed, nose-only, to an atmosphere of the test substance using a TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany). This system comprises of two, concentric anodised aluminium chambers and a computer control system incorporating pressure detectors and mass flow controllers.
Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where, under positive pressure, it was distributed to the individual exposure ports. The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animal’s nares to enter the exposure port. After passing through the animal’s breathing zone, used aerosol entered the outer cylinder from where it was exhausted through a suitable filter system. Atmosphere generation was therefore dynamic.
Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). The flow of air through each port was at least 0.7 L/min. This flow rate was considered adequate to minimize re-breathing of the test atmosphere as it is about twice the respiratory minute volume of a rat.
The homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was not specifically determined during this study.However, chambers of this design have been fully validated and have been shown to produce evenly distributed atmospheres in the animals’ breathing zones (Pauluhn, 1994).

Exposure procedure:
Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber. Only the nose of each animal was exposed to the test atmosphere.
Following an equilibration period of at least the theoretical chamber equilibration time (T99) (Silver, 1946) (actual equilibration time = 17 minutes), a group of six rats (three male and three female) was exposed to an atmosphere of the test material for a period of 4 hours. A target concentration of 5 mg/L was used for the exposure. Since no deaths occurred and the mean achieved concentration was 101 % of the target, no further data were required.


Exposure Monitoring:

Test atmosphere concentrations:
The test atmosphere was sampled at regular intervals during the exposure period. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone) by pulling a suitable, known volume of test atmosphere through weighed GF10 glass fibre filters (Whatman GmbH, Hahnestraße 3 – D-37586 Dassel, Germany). The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.
The nominal concentration was calculated by dividing the mass of test substance disseminated into the chamber by the total volume of air that went through the chamber during the same period.
The mean achieved test atmosphere concentration was 5.05 +/- 0.19 mg/L and the nominal chamber concentration was 6.67 mg/L.

Particle size analysis:
The particle size of the test atmosphere was determined three times during the exposure period using a 7-stage impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 0.55, 0.96, 1.55, 2.11, 3.56, 6.66 and 10.55 µm was calculated.
From these data, using software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated. In addition, the proportion (%) of aerosol less than 4µm (considered to be the inhalable portion) was determined.
The mean MMAD was 3.62 µm and the geometric standard deviation (GSD) was 4.49. The Geometric standard deviation of the generated aerosol during the animal exposure was greater than 4 µm, it came from the characteristics of the test item. However, it had no effect on the purpose and integrity of the study. The proportion of aerosol less than 4µm was 52.6%.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Target concentration: 5.0 mg/L
Mean achieved atmosphere concentration: 5.05 mg/L ; Standard deviation: 0.19

No. of animals per sex per dose:

Three/sex/dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 d

OBSERVATIONS:
- Morbidity/Mortality: Animals were checked hourly during exposure, 1 h after exposure and twice daily (early and late in the working day) during the 14-d observation period for morbidity and/or mortality.

- Clinical Signs: All animals were observed for clinical signs at hourly intervals during exposure, as soon as practically possible following removal from restraint at the end of exposure, 1 h after exposure and subsequently once daily for 14 d.

- Bodyweight: Individual bodyweights were recorded prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.

- Necropsy: At the end of the 14-d observation period, the animals were euthanised by exsanguination under anaesthesia (intra-peritoneal injection of pentobarbital solution) and gross macroscopic examination was performed. All animals were subject to a gross necropsy which included a detailed examination of the abdominal and thoracic cavities. Special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity.

Statistics:

None

Key result

Sex:

male/female

Dose descriptor:

LC0

Effect level:

>= 5.05 mg/L air

Based on:

other: mean achieved concentration

Exp. duration:

4 h

Remarks on result:

other: (i.e., >5,050 mg/m3)

Mortality:

No mortality was observed in the group of six rats.

Clinical signs:

other: Wet fur and fur staining were commonly recorded on the day of exposure. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be biologically significant. Laboured and noisy re

Body weight:

Normal bodyweight gain was noted during the observation period for all animals.

Gross pathology:

None of the six animals were associated with any macroscopic findings.

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the study conditions, the 4 hour inhalation LC0 value of the test substance was equal or higher to 5050 mg/m3.

Executive summary:

The substance was tested for acute inhalation toxicity according to the OECD 436 guideline and in compliance with Good Laboratory Practice.

A group of three male and three female Wistar rats were exposed, nose-only, to an atmosphere of the test item using a flow-past exposure system. The animals were exposed for four hours to a target concentration of 5 mg/L followed by a fourteen day observation period.

Each animal was observed for mortality and clinical signs at hourly intervals during exposure, then one hour after exposure and at least once a day during the 14 -day observation period. Bodyweights were recorded before treatment then on the day of exposure (day 0) and on days 1, 3,7 and 14. All surviving animals were necropsied at the end of the observation period.

The mean achieved atmosphere concentration was 5.05 mg/L and the mean mass median aerodynamic diameter was 3.62 µm. No deaths occurred during the study. Laboured and noisy respiration and increased respiratory rates were noted for the exposed animals on the day of exposure and during several days after exposure. No abnormalities were detected in any animal from day 4 of the observation period. There were no effects on bodyweight gain and no macroscopic post-mortem findings.

The 4 -hour inhalation LC50 was found to be greater than 5.05 mg/L (ie 5050 mg/m3).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

5 050 mg/m³ air

Quality of whole database:

Study complete and sufficient to fulfill the endpoint requirements.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral route:

In an acute oral toxicity study performed according to the OECD 423 guideline and in compliance with Good Laboratory Practice, groups of 3 female rats received by gavage a single dose of 2000 or 300 mg/kg bw of the test substance. The starting dose-level was 300 mg/kg bw for ethical reasons. After the first assay, the next higher dose-level of 2000 mg/kg bw was tested. Since no toxicity was observed at this higher dose-level, the results were confirmed in other females. Animals were then observed for 14 days following exposure.

No unscheduled deaths occurred during the study and no clinical signs were observed in any animals. A trend towards a slightly lower mean bodyweight gain was noted in females given the test substance at 2,000 mg/kg, when compared to historical control data. There were no macroscopic post-mortem findings.

The acute oral LD50 was found to be greater than 2000 mg/kg bw (Duclos, 2012).

Inhalation:

In an acute inhalation toxicity study performed according to the OECD 436 guideline and in compliance with Good Laboratory Practice, a group of three male and three female Wistar rats were exposed, nose-only, to an atmosphere of the test item using a flow-past exposure system. The animals were exposed for four hours to a target concentration of 5 mg/L followed by a 14- day observation period.

Each animal was observed for mortality and clinical signs at hourly intervals during exposure, then one hour after exposure and at least once a day during the 14 day observation period. Bodyweights were recorded before treatment, on the day of exposure (day 0) and on days 1, 3,7 and 14. All the surviving animals were necropsied at the end of the observation period.

The mean achieved atmosphere concentration was 5.05 mg/L and the mean mass median aerodynamic diameter was 3.62 µm. No deaths occurred during the study. Laboured and noisy respiration, and increased respiratory rates were noted for the exposed animals on the day of exposure and several days after exposure. No abnormalities were detected in any animal from day 4 of the observation period. There were no effects on bodyweight gain and no macroscopic post-mortem findings.

The 4 -hour inhalation LC50 was found to be greater than 5.05 mg/L (ie 5050 mg/m3).

Justification for classification or non-classification

No mortalities were noted in rats treated either by the oral route with a single dose of 2000 mg/kg body weight or those exposed by inhalation for 4 hours to 5.05 mg/L.

On the basis of these studies and according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures, no classification is warranted with respect to acute oral and inhalation toxicities.