Bis(2-hydroxyethyl)ammonium acetate

Endpoint summary

Description of key information

No relevant data are available on DEA acetate, but reliable 28-day oral gavage studies have been carried out on structurally-related read-across compounds. NOAELs of 150 and 1000 mg/kg bw/day were reported for RA1 and RA2, respectively.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1999-08-11 to 1999-09-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is the result of a structural analogue substance used as read-across substance. Study is conducted according to Guidelines in a GLP certified laboratory.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division CH-4414 Fullinsdorf/Switzerland
- Age at study initiation: 7 Weeks (6 weeks old on delivery, 1 week of Acclimatization)
- Weight at study initiation: At Acclimatization (Males = 122 - 158 grams (Mean 137 grams)) (Females = 102 - 127 grams (Mean 114 grams))
- Fasting period before study: None
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardised softwood bedding.
- Diet: Pelleted standard Provimi Kliba 3433 rat maintenance diet was available ad libitum. The feed batch was analysed for contaminants
- Water: Community tap water from Itingen was avaible ad libitum in water bottles
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light / 12 hours dark


IN-LIFE DATES: From: 11th August 1999 To: 15 September 1999

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

The test article formulations were prepared weekly.

RA1 was weighed into a tared glass beaker on a Mettler balance and the vehicle assed (weight:volume). The mixtures were then prepared using a magnetic stirrer and stored at room temperature (17 – 23ºC).

Homogeneity of the test article in the vehicle was maintained during the daily administration period using a magnetic stirrer.


DIET PREPARATION
- Rate of preparation of diet: Prepared weekly
- Mixing appropriate amounts with: Pelleted standard Provimi Kliba 3433 rat maintenance diet
- Storage temperature of food: Room temperature 17 - 23ºC


VEHICLE
- Concentration in vehicle: The test substance was administered to groups 1-4 at dose levels of 0, 30, 150 and 750 mg/kg body weight, respectively.
- Amount of vehicle (if gavage): 10 ml/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken during acclimatisation. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed using a GC method.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Dose was administered every day

Remarks:

Doses / Concentrations:
0, 30, 150 and 750 mg/kg/day
Basis:
nominal in water

No. of animals per sex per dose:

5 males and 5 females were in each group

Group 1 (Control): 0 mg/kg/day
Group 2: 30 mg/kg/day
Group 3: 150 mg/kg/day
Group 4: 750 mg/kg/day

Control animals:

yes

Details on study design:

The purpose of this oral toxicity study was to assess the cumulative toxicity of RA1 when administered daily to rats by gavage for a period of 28 days. This study should provide a rational basis for toxicological risk assessment in man. The results of this study should indicate potential target organs and should identify chemicals with neurotoxic potential.

Dose selection rationale:
Basd upon the results of a non-GLP 5 day dose range-finding study in which RA1 was administered by gavage to 2 rats per group and sex. Animals showed marked organ weight changes (spleen) at 600 mg/kg bw/day (males only) and 1000 mg/kg bw/day (both sexes).

Positive control:

Not required.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed for mortality/morbidity twice daily. The animals were observed for clinical signs once before commencement of administration, twice daily on days 1-3 (ca. 1 and 3 hours after administration), as well as once daily on days 4-28 (ca. 1 hour after administration).


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly thereafter.


BODY WEIGHT: Body weights were recorded weekly during pretest, treatment and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer. Please see Table 6 and 7
- Time schedule for examinations: Weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg bw/day: The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to a computer. Please see Tables 4 and 5
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube
- Anaesthetic used for blood collection: Yes Blood samples for hematology were collected from all animals under light ether anesthesia.
- Animals fasted: Yes the animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All 20 animals
- Parameters checked in table [No.?] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube
- Animals fasted: Yes the animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All 20 animals
- Parameters checked in table [No.?] were examined.


URINALYSIS: No .


FUNCTIONAL OBSERVATIONAL BATTERY:
During week 4, relevant parameters from a modified Irwin screen test were evalueated in all animals. Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge. The animals were placed with the forepaws inside a triangular grsaping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times.

Locomotor activity was measured quantitatively with Activity Monitor AM 1052 system. Animals were randomised and monitored during the fourth treatment week for a 60 minute period and the total activity of this time period was recorded.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes After 4 weeks all animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist. All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguinations. Samples of the following tissues and organs were collected at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution: adrenal glands, aorta, bone (sternum, femur), bone marrow (femur), brain (cerebrum, cerebellum, pons), cecum, colon, duodenum, epididymides, eyes with optic nerve, Harderian gland, heart, ileum (with Peyer's patches), jejunum with Peyer's patches, kidneys, larynx, lacrimal gland (exorbital), liver, lungs, lymph nodes (mesenteric, mandibular), mammary gland are, nasal cavity, oesophagus, ovaries, pancreas, pituitary, prostate gland, rectum, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin spinal cord, spleen, stomach, testes, thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina and gross lesions.

HISTOPATHOLOGY: Slides of the following organs and tissues were processed, embedded and slides at 2 to 4 micrometers, stained with H&E, and were examined by a pathologist: adrenal glands, bone marrow (femur), brain (cerebrum, cerebellum, pons), cecum, colon, duodenum, epididymides, heart, ileum (with Peyer's patches), jejunum with Peyer's patches, kidneys, liver, lungs, lymph nodes (mesenteric, mandibular), ovaries, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus, vagina and gross lesions.

OTHER: The following organ weights were recorded on the scheduled date of necropsy: brain, heart, liver, thymus, kidneys, adrenals, spleen, testes, epididymides. The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

Statistics:

The following statistical methods were used to analyse the cody weights, organs, and all ratios as well as clinical laboratory data:

If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one-t-test) based on a pool variance estimate was applied for the comparison between the treated groups and the control groups for each sex.

The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.

The Fisher's exact test was applied to macroscopial findings.

T-test was applied to grip strength and locomotor activity

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The total cholesterol and phospholipid levels were marginally lower in males and females treated at 750 mg/kg bw/day when compared with those of the controls. Plasma sodium and chloride levels were slightly raised in females in the 750 mg/kg bw/day group

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Locomotor Activity: A test article-related reduction in mean locomotor activity was noted in males (-9.3 %) and females (-30.7 %) treated with 750 mg/kg/day when compared with that of the control females.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Marginally higher relative kidney weights were noted in females treated at 750 mg/kg bw/day

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
No effects (All animals survived the study period. There were no clinical signs in any dose group.)

BODY WEIGHT AND WEIGHT GAIN:
No effects (body weight was unaffected by treatment with the test article)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
No effects

FOOD EFFICIENCY
Not examined

HAEMATOLOGY:
No effects

CLINICAL CHEMISTRY:
The total cholesterol and phospholipids levels were marginally lower in the males and females treated at 750 mg/kg/day when compared with those of the controls. These findings were considered to be indicative of minor changes in lipid metabolism.

The plasma sodium and chloride levels were slightly elevated in females treated with 750 mg/kg/day when compared with the control females. Although these findings were not seen in males, a relationship with treatment could not be excluded.

All other clinical biochemistry parameters of test article-treated animals were similar to those of the controls.


URINALYSIS
Not examined

FUNCTIONAL OBSERVATIONAL BATTERY:
No clinical signs or abnormal behaviour were evident in any animals during treatment week 4.

Grip Strength: The mean grip strength of the test article-treated animals compared favourably with those of the control animals.

Locomotor Activity: A test article-related reduction in mean locomotor activity was noted in males (-9.3 %) and females (-30.7 %) treated with 750 mg/kg/day when compared with that of the control females. Marginally, lower locomotor activity (-4.6 %) was noted in the females treated with 150 mg/kg/day, but these differences were considered to be within the range of normal variation and unrelated to the treatment with the test article.


ORGAN WEIGHTS:
The slightly higher (and statistically significant when compared to control females, p<0.05) kidney to body weight ratio of females treated at 750 mg/kg/day was considered to be a test article-related change.

The remaining organ weights were considered to be unaffected by treatment when compared with the control values.


GROSS PATHOLOGY:
There were no macroscopic findings which were considered to distinguish treated rats from control rats.

HISTOPATHOLOGY: NON-NEOPLASTIC:
A number of findings were noted in the organs/tissues examined. They were comparable to those occurring spontaneously in rats of the same strain and age, and their incidence, severity and morphologic appearance did not distinguish treated rats from control rats.

Dose descriptor:

NOAEL

Effect level:

ca. 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Table 3: Organ Weights (gram) in Males

		Group 1	Group 2	Group 3	Group 4
		0 mg/kg	30 mg/kg	150 mg/kg	750 mg/kg
Body Weight	Mean	263.402	268.082	255.076	278.118
	St. Dev.	35.699	25.657	21.763	35.638
Brain	Mean	1.90	1.91	1.88	1.92
	St. Dev.	0.15	0.09	0.05	0.08
Heart	Mean	0.920	0.887	0.861	0.949
	St. Dev.	0.163	0.056	0.086	0.114
Liver	Mean	7.87	7.38	7.28	7.79
	St. Dev.	1.85	1.15	1.17	1.21
Thymus	Mean	0.41	0.42	0.37	0.43
	St. Dev.	0.10	0.06	0.11	0.12
Kidneys	Mean	1.91	1.97	1.87	2.14
	St. Dev.	0.27	0.31	0.22	0.20
Adrenals	Mean	0.057	0.064	0.058	0.061
	St. Dev.	0.008	0.010	0.006	0.009
Spleen	Mean	0.662	0.663	0.603	0.723
	St. Dev.	0.134	0.140	0.080	0.126
Testes	Mean	3.41	3.31	3.16	3.11
	St. Dev.	0.40	0.20	0.20	0.21
Epididymides	Mean	1.008	0.996	0.935	0.953
	St. Dev.	0.172	0.104	0.029	0.116

 

 

Table 4: Organ Weights (gram) in Females

		Group 1	Group 2	Group 3	Group 4
		0 mg/kg	30 mg/kg	150 mg/kg	750 mg/kg
Body Weight	Mean	180.652	169.402	178.032	137.322
	St. Dev.	15.551	9.966	13.310	16.676
Brain	Mean	1.78	1.80	1.86	1.86
	St. Dev.	0.05	0.09	0.09	0.04
Heart	Mean	0.695	0.695	0.684	0.643
	St. Dev.	0.036	0.090	0.059	0.065
Liver	Mean	5.36	5.25	5.45	5.67
	St. Dev.	0.40	0.36	0.57	0.65
Thymus	Mean	0.47	0.35	0.41	0.40
	St. Dev.	0.07	0.07	0.17	0.07
Kidneys	Mean	1.38	1.35	1.44	1.50
	St. Dev.	0.07	0.11	0.14	0.15
Adrenals	Mean	0.079	0.079	0.085	0.084
	St. Dev.	0.009	0.014	0.017	0.011
Spleen	Mean	0.497	0.455	0.484	0.517
	St. Dev.	0.137	0.071	0.54	0.023

 

Table 5: Summary of male and female food consumption (g/animal/day)

Day of study	Group 1 control		Group 2 30 mg/kg		Group 3 150 mg/kg		Group 4 750 mg/kg
	Male	Female	Male	Female	Male	Female	Male	Female
1-8	21.9	14.7	20.9	14.1	22.3	15.1	21.9	15.4
8-15	22.7	17.1	21.6	15.8	22.9	16.5	23.1	16.3
15-22	22.7	17.8	22.1	15.9	22.5	17.2	24.1	16.4
22-28	23.2	18.2	23.3	17.5	24.6	17.5	24.3	17.0

 

 Table 7: Summary of haematology

Parameter	Group 1 control		Group 2 30 mg/kg		Group 3 150 mg/kg		Group 4 750 mg/kg
	Male	Female	Male	Female	Male	Female	Male	Female
RBC (T/L)	8.83	8.36	8.99	8.30	9.12	8.53	8.82	8.11
Haemoglobin (mmol/L)	9.97	9.58	10.12	9.51	10.15	9.73	10.10	9.22
Haematocrit (L/L)	0.48	0.46	0.49	0.46	0.49	0.47	0.48	0.45
MCV (fL)	54.6	55.5	54.2	55.1	53.3	54.9	54.5	55.9
MCH (fmol)	1.13	1.15	1.13	1.15	1.11	1.14	1.15	1.14
MCHC (mmol/L)	20.8	20.7	20.8	20.9	20.9	20.8	21.1	20.4
Platelets (g/L)	885	1018	981	972	1016	1019	887	969
Reticulocytes (%)	2.75	2.54	2.62	3.08	2.80	2.46	2.81	2.74
Reticulocytes (T/L)	0.2376	0.2093	0.2298	0.2504	0.2421	0.2004	0.2397	0.2135
Heinz bodies	0	0	0	0	0	0	0	0
Methaemoglobin (%)	0.8	0.9	0.8	0.9	0.7	0.7	0.9	0.9
WBC (g/L)	7.1	6.6	6.4	5.8	7.6	6.0	8.1	6.3

 

Table 8: Summary of clinical biochemistry

Parameter	Group 1 control		Group 2 30 mg/kg		Group 3 150 mg/kg		Group 4 750 mg/kg
	Male	Female	Male	Female	Male	Female	Male	Female
Glucose (mmol/L)	5.54	4.59	5.35	4.83	5.63	4.67	5.58	4.45
Urea (mmol/L)	6.38	7.20	6.15	7.69	5.40	6.56	6.93	7.10
Creatinine (µmol/L)	47.5	53.8	47.4	54.9	47.9	49.7	48.6	54.0
Uric acid (µmol/L)	37.8	50.2	28.4	35.6	45.6	37.0	41.6	50.6
Bilirubin total (µmol/L)	2.12	2.30	2.16	2.03	1.95	1.99	2.41	2.31
Cholesterol total (mmol/L)	1.95	1.60	1.96	1.52	1.88	1.57	1.52	1.15
Triglycerides (mmol/L)	0.65	0.36	0.83	0.34	0.92	0.39	0.75	0.33
Phospholipids (mmol/L)	1.76	1.58	1.80	1.61	1.76	1.68	1.43	1.33
ASAT (µkat/L)	1.20	1.17	1.18	1.20	1.30	1.13	1.20	1.26
ALAT (µkat/L)	0.53	0.46	0.61	0.44	0.56	0.42	0.53	0.38
Creatine kinase (µkat/L)	4.01	3.72	3.21	3.95	4.42	3.01	3.53	4.85
Calcium (mmol/L)	2.73	2.71	2.72	2.59	2.72	2.72	2.71	2.78
Phosphorous (mmol/L)	2.41	2.12	2.18	1.93	2.17	1.97	2.42	1.95
Sodium (mmol/L)	144.4	144.8	145.4	145.9	145.4	145.2	145.4	146.8*
Chloride (mmol/L)	100.6	101.6	100.9	102.9	100.8	101.6	100.4	103.8**
Albumin (g/L)	31.7	34.0	32.0	34.2	32.5	34.7	31.8	36.1
Total protein (g/L)	67.5	68.1	67.6	67.8	68.6	69.5	66.5	71.7

*/** Dunnett t-test based on pooled variance significant at 5 % or 1 % level, respectively.

Conclusions:

Based on the results of this 28-day gavage study in rats, 150 mg/kg bw/day of RA1 was established as the study no-observed-adverse-effect level (NOAEL).

Executive summary:

In this sub-acute toxicity study, RA1 was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 30, 150 and 750 mg/kg bw/day for a period of 28 days. A control group was treated concurrently with the vehicle only.

 

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment.

 

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pre-test and treatment periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

 

At the end of the dosing period, blood samples were withdrawn for haematology and plasma chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues (including those of the reproductive system) from all control and high dose animals, and all gross lesions from all animals.

The results are summarised as follows:

Mortality

All animals survived the study period

Clinical signs

No clinical signs were observed

Food consumption

There were no effects of the test substance on food consumption

Organ weights, gross pathology and histopathology

No treatment-reated effects observed

Clinical laboratory investigations

There were no effects of the test substance on the haematology parameters measured.

In a clinical chemistry examination, the total cholesterol and phospholipids levels were found to be marginally lower in the males and females treated at 750 mg/kg bw/day when compared with those of the controls. These findings were considered to be indicative of minor changes in lipid metabolism. The plasma sodium and chloride levels were slightly elevated in females treated with 750 mg/kg/day when compared with the control females. Although these findings were not seen in males, a relationship with treatment could not be excluded.

All other clinical biochemistry parameters of test article-treated animals were similar to those of the controls.

Functional observations

A test article-related reduction in mean locomotor activity was noted in males (-9.3 %) and females (-30.7 %) treated with 750 mg/kg bw/day when compared with that of the control females. Marginally, lower locomotor activity (-4.6 %) was noted in the females treated with 150 mg/kg bw/day, but these differences were considered to be within the range of normal variation and unrelated to the treatment with the test article.

Based on the results of this study, 150 mg/kg bw/day of RA1 was etablished as the NOAEL.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

No repeated dose studies were available on DEA acetate, but two good-quality, reliable, GLP-compliant, OECD-guideline studies on the structurally-related compounds RA1 (Braun et al. 2000) and RA2 (Allard & Becker, 1997) meet the REACH tonnage-driven data requirements.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No studies are available on the repeated dose toxicity of DEA acetate, but oral studies have been conducted on structurally-related read-across compounds.

 

In a 28-day study, RA1 was administered via daily oral gavage to SPF-bred Wistar rats (5/sex) at up to 750 mg/kg bw/day. Slightly elevated plasma sodium and chloride levels were seen in high-dose females. High-dose males and females demonstrated significant treatment-related reductions in locomotor activity. The no-observed-adverse-effect level (NOAEL) was determined to be 150 mg/kg bw/day (Braun et al. 2000). A 28-day study on Sprague-Dawley rats (5/sex) given RA2 by daily oral gavage at up to 1000 mg/kg bw/day. No treatment-related significant adverse effects were seen in treated animals (NOAEL 1000 mg/kg bw/day), although no neurobehaviour assessment was conducted (Allard and Becker, 1997).

As neurobehavioural effects (reductions in locomotor activity) were seen with RA1 in high dose animals, and such affects were not assessed in detail in the RA2 study, a health-precautionary NOAEL of 150 mg/kg bw/day is considered critical.

According to Annex VIII of Regulation (EC) No 1907/2006 REACH Regulation, repeated dose toxicity studies only need to be conducted on one species taking into consideration the most appropriate route of administration regarding human exposure. As the oral route of exposure is most appropriate for DEA acetate, repeated dose toxicity studies were not carried out for the dermal or inhalation routes.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP, OECD guideline study (reliability 2).

Repeated dose toxicity: via oral route - systemic effects (target organ) neurologic: behaviour

Justification for classification or non-classification

Oral repeated dose toxicity studies in rats with RA2 and RA1 yielded NOAELs of 1000 and 150 mg/kg bw/day respectively (Allard & Becker, 1997; Braun et al. 2000). According to Regulation (EC) No. 1272/2008, classification is applicable when significant toxic effects are seen to occur at 300 mg/kg bw/day or less for an oral 28-day rat study. As such, the results of these studies indicate that classification of DEA acetate is not required.