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Description of key information

Acute toxicity - oral: A K1 acute oral toxicity test was performed in female Sprague Dawley rats according to

a guideline similar to OECD Guideline 425 and EPA OPPTS 870.1100 (Lemoncelli, 2012). The LD50 for the oral route was determined to be 550 mg/kg bw (88.94 - 2340 mg/kg, 95% CL).
Acute toxicity - inhalation: An acute inhalation study does not need to be conducted as the substance is classified as corrosive to the skin (according to REACH, Annex VIII section 8.5, column 2). In addition, acute oral and acute dermal toxicity studies are available.
Acute toxicity - dermal: A K1 acute dermal toxicity test was performed in male and female Sprague-Dawley rats according to a guideline similar to OECD Guideline 402 and EU Method B.3 (Lemoncelli, 2012). The LD50 for the dermal route was determined to be > 1000 and < 2000 mg/kg bw.

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:
2012-07-04 - 2012-08-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)
Deviations:
yes
Remarks:
Temperature and relative humidity in animal room deviated from the protocol-stated ranges; no impact on outcome or integrity of the study, no effect on health status of the animals
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
yes
Remarks:
Temperature and relative humidity in animal room deviated from the protocol-stated ranges; no impact on outcome or integrity of the study, no effect on health status of the animals
GLP compliance:
yes
Test type:
up-and-down procedure
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Jeffamine RFD-270 Amine
- Substance type: Colourless liquid
- Physical state: Liquid
- Lot/batch No.: 8802-8-9
- Analytical purity: 92%
- Composition of test material, percentage of components: 0.02 wt% water
- Purity test date: 2010-07-15
- Storage condition of test material: Sample stored in cool, well-ventilated storage area prior to testing
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: 9 weeks at start of dosing
- Weight at study initiation: 196 to 222 grams
- Fasting period before study: Animals were fasted overnight prior to dose administration
- Housing: Animals were group housed by sex upon receipt and individually housed upon assignment to study in compliance with the National Research Council "Guide for the Care and Use of Laboratory Animals". Calvert is a USDA registered and fully AAALAC accredited facility.
- Diet (e.g. ad libitum): ad libitum except during fasting
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Study animals were acclimated to their housing for a minimum of 5 days prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 22°C
- Humidity (%): 28 to 84%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on oral exposure:
DOSAGE PREPARATION:
The proper amount of the test article was measured according to volume since it was a liquid, and was allocated/dosed neat as received from the Sponsor at 2000 and 550 mg/kg with a pH=9. The stock bottle was inverted several times prior to dispensing. At 175 mg/kg the test article was dissolved in distilled water (175 mg brought to a volume of 5 mL). The resulting clear liquid had a pH equal to 12.
Doses:
175, 550 and 2000 mg/kg
No. of animals per sex per dose:
6 rats in total:
2 female rats in the 175 mg/kg dose group
3 female rats in the 550 mg/kg dose group
1 female rat in the 2000 mg/kg dose group
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Mortality checks were made at least once daily. Clinical observations were recorded prior to dosing, as well as at 30 minutes, 4 hours post-dose, and daily thereafter up to day 15. Body weights were recorded on the day of dosing (day 1), and on days 8 and 15, or upon death.
- Necropsy of survivors performed: yes, all surviving rats were euthanized by CO2 asphyxiation and necropsied on day 15.
Statistics:
The LD50 was calculated for the main test using the AOT425StatPgm developed by Westat May, 2001.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
550 mg/kg bw
Based on:
test mat.
95% CL:
88.94 - 2 430
Mortality:
175 mg/kg: no mortality observed
550 mg/kg: two of three animals receiving the test article at 550 mg/kg were found dead
2000 mg/kg: the single animal at 2000 mg/kg was as well found dead
Clinical signs:
other: No clinical signs were observed at 175 mg/kg or in the initial surviving animal at 550 mg/kg. Abnormal gait and stance, decreased activity, and decreased body tone were observed in the animal at 2000 mg/kg. In addition to these signs piloerection was obse
Gross pathology:
Terminal necropsy revealed no visible lesions in the surviving animals at 175 and 550 mg/kg. Necropsy of the animals found dead revealed lesions of the intestines and stomach including fluid-filled red, lining red and a dark red circular lesion.
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Based on the results of this study, the oral LD50 for the test substance in rats was estimated to be 550 mg/kg (95% PL Confidence interval of 88.94 to 2430 mg/kg).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
550 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is classified as corrosive to the skin
other:
Endpoint conclusion
Endpoint conclusion:
no study available

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
Study period:
2012-07-18 - 2012-08-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
yes
Remarks:
temperature and relative humidity ranges were broader than specified by the guideline, no impact on the outcome of the study; age range 7 to 9 weeks instead of 8-24 weeks, no impact on the outcome or integrity of the study
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
yes
Remarks:
temperature and relative humidity ranges were broader than specified by the guideline, no impact on the outcome of the study; age range 7 to 9 weeks instead of 8-24 weeks, no impact on the outcome or integrity of the study
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Jeffamine RFD-270 Amine
- Substance type: Colourless liquid
- Physical state: Liquid
- Lot/batch No.: 8802-8-9
- Analytical purity: 92%
- Composition of test material, percentage of components: 0.02 wt% water
- Purity test date: 2010-07-15
- Storage condition of test material: Sample stored in cool, well-ventilated storage area prior to testing
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: 7 to 10 weeks
- Weight at study initiation: 184 to 300 grams
- Fasting period before study: no data
- Housing: Animals were group housed by sex upon receipt and individually housed upon assignment to study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Study animals were acclimated to their housing for a minimum of five days prior to dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 25°C
- Humidity (%): 30 to 84%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Type of coverage:
occlusive
Vehicle:
water
Details on dermal exposure:
TEST SITE
- Area of exposure: dorsal area of the trunk
- % coverage: > = 10%
- Type of wrap if used: The test article was applied on an intact skin site for each animal, covered with a gauze patch/dental dam, wrapped with an elastic bandage and secured with non-irritating tape for 24 (± 0.5) hours.

REMOVAL OF TEST SUBSTANCE
Post-exposure, the site was unwrapped and wiped appropriately (with gauze and water) to remove residual test article.

TEST MATERIAL
Dosage levels were calculated on a mg/kg basis for each individual animal based on body weight. For the liquid test article, the dose was adjusted for test article relative density (1.04 g/mL).

Duration of exposure:
24 ± 0.5 hours
Doses:
One group of ten animals (5/sex) received the test article at 2000 mg/kg (2.1 mL/kg). A full definitive test was performed and three groups of ten (5/sex) received the test article at 50 (5 mL/kg), 200 (5 mL/kg), and 1000 mg/kg (1 mL/kg).
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/Morbidity: once daily
Clinical observations: Clinical observations were recorded immediately after unwrap and daily thereafter through day 15. At the time of clinical observations, the dose site was also evaluated for dermal irritation. When present, dermal irritation was scored and recorded using descriptive scoring.
Body weight: Animals were weighed prior to dosing on Day 1 and on days 8 and 15 or upon death.
- Necropsy of survivors performed: yes: all surviving animals were euthanized by CO2 asphyxiation following final observation.
The terminal necropsy included examination of:
- the external body surface
- all orifices
- the thoracic, abdominal and pelvic cavities and their contents
Statistics:
Body weights were summarized using descriptive statistics (mean and standard deviation).
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 1 000 - < 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed at 50 or 200 mg/kg. Nine of ten animals were found dead at 2000 mg/kg and one of ten were found dead at 1000 mg/kg.
Clinical signs:
other: All animals at 50 mg/kg were observed to be clinically normal without signs of dermal irritation throughout the study. At 2000 mg/kg, all ten animals exhibited signs of irritation and necrosis at the application site by day 2 with clinical observations. T
Gross pathology:
No visible lesions were observed in the animals at terminal necropsy. Necropsy of found dead animals revealed: fluid filled stomachs in four of eight animals found dead in the 2000 mg/kg group on day 3 as well as dark red intestines in one animal found dead on day 4. Necropsy of the found dead animal in the 1000 mg/kg group on day 3 revealed dark red intestines.
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Based upon the results of the acute dermal toxicity study in rats with the test substance, the estimated LD50 was considered to be greater than 1000 mg/kg but less than 2000 mg/kg based on the results of the preliminary test (9 of 10 rats were found dead). Therefore, based on the criteria of the CLP Regulation, the substance is considered to be a category 4 acute dermal toxicant.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50

Additional information

Acute toxicity: oral

Lemoncelli (2012) investigated the acute oral toxicity via gavage of 175, 550 and 2000 mg/kg bw test substance in female Sprague-Dawley rats (6 rats in total: 2 in the 175 mg/kg dose group, 3 in the 550 mg/kg dose group and 1 in the 2000 mg/kg dose group). At 175 mg/kg no mortality was observed; at 550 mg/kg two of three animals receiving the test article were found dead and at 2000 mg/kg the single animal was as well found dead. The acute oral LD50 for male and female rats treated with the test substance was determined to be 550 mg/kg with confidence limits of 88.94 to 2430 mg/kg. No clinical signs were observed at 175 mg/kg or in the initial surviving animal at 550 mg/kg. Abnormal gait and stance, decreased activity, and decreased body tone were observed in the animal at 2000 mg/kg. In addition to these signs piloerection was observed in one animal at 550 mg/kg prior to death. Terminal necropsy revealed no visible lesions in the surviving animals at 175 and 550 mg/kg. Necropsy of the animals found dead revealed lesions of the intestines and stomach including fluid-filled red, lining red and a dark red circular lesion.

Acute toxicity: inhalation

No reliable studies were available for the inhalation route. An acute inhalation study does not need to be conducted as the substance is classified as corrosive to the skin (Regulation (EC) No 1907/2006, Annex VIII section 8.5). In addition, acute oral and acute dermal toxicity studies are available.

Acute toxicity: dermal

Lemoncelli (2012) investigated acute dermal toxicity of the test substance in male and female Sprague-Dawley rats (5 animals per sex and per dose) after 24 hours of exposure to either 50, 200, and 1000 mg/kg bw in the full definitive test. One group of ten animals (5/sex) received the test article at 2000 mg/kg. After 14 days of observation, the dermal LD50 for the test substance was > 1000 mg/kg. No mortality was observed at 50 or 200 mg/kg. Nine of ten animals were found dead at 2000 mg/kg and one of ten were found dead at 1000 mg/kg.

All animals at 50 mg/kg were observed to be clinically normal without signs of dermal irritation throughout the study. At 2000 mg/kg, all ten animals exhibited signs of irritation and necrosis at the application site by day 2 with clinical observations. The one remaining animal in this group continued with irritation and necrosis and was clinically normal at day 14. At 200 mg/kg, irritation was observed in four animals starting on day 2 with full resolution by day 6. Clinical observations were present in one animal at 200 mg/kg on day 2 only. At 1000 mg/kg, irritation was observed starting on day 2 in eight animals with necrosis beginning in three of ten animals on day 4. Irritation almost completely resolved in the remaining animals by day 15. Clinical observations were present in five animals at 1000 mg/kg on day 2 only.

No visible lesions were observed in the animals at terminal necropsy. Necropsy of found dead animals revealed: fluid filled stomachs in four of eight animals found dead in the 2000 mg/kg group on day 3 as well as dark red intestines in one animal found dead on day 4. Necropsy of the found dead animal in the 1000 mg/kg group on day 3 revealed dark red intestines.

Justification for classification or non-classification

Based on the results of the acute oral and acute dermal toxicity study and according to the criteria of the CLP Regulation, the test substance should be classified as an acute oral and acute dermal toxicant category 4, H302 and H312 (CLP)