Tris(2-hydroxyethyl)ammonium acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

1997-06-19 to 1997-07-24

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

427-360-5

EC Name:

-

IUPAC Name:

427-360-5

Details on test material:

- Description: Light Yellow Solid
- Stability of test compound: Stable in bi-distilled water for at least 48 hours at 20 deg C

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Total Number of Animals per Group: 5 males, 5 females.
Total Number of Animals: 20 males, 20 females.
Age at delivery: 6 weeks.
Bodyweight and range at acclimatization/pretest: Males = 197-230g (mean 213 grams), Females = 164-200g (mean 181 grams).
Acclimatization: 7 days test conditions, following a health examination. Only animals without any visible signs of illness were used for the study.
Accomodation: Groups of 5 in Makrolon type-4 cages with standard softwood bedding.
Diet: Pelleted standard Kliba 343 ad libitum.
Water: Community tap water ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Method: Oral by gavage.

Frequency: Once daily, 7 days per weeks for a total of 28 days.

Dose Levels:
Group1 (control): 0mg/kg per treatment day.
Group 2: 50mg/kg per treatment day.
Group 3: 200mg/kg per treatment day.
Group 4: 1000mg/kg per treatment day.

Dose Volume: 10ml/kg body weight per treatment day.

Duration of acclimatization: 7 days.

Duration of treatment: 28 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples taken from each concentration group were dissolved with about 70 ml of bi-distilled water by means of an ultrasonic bath. The, the 100 ml volumetric flasks were filled to the mark with bi-distilled water. Depending on the dose group, the latter sample solutions were further diluted with bi-distilled water to yield concentrations within the calibration range. A 2ul aliquot was then quantified using GC.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days per week for a total of 28 days.

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 200 and 1000 gm/kg
Basis:
nominal in water

No. of animals per sex per dose:

Concentration Number of Animals
Male Female
Group 1 (Control) 0 gm/kg per treatment day 5 5
Group 2 50 gm/kg per treatment day 5 5
Group 3 200 gm/kg per treatment day 5 5
Group 4 1000 gm/kg per treatment day 5 5

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this oral toxicity study was to assess the cumulative toxicity of Read Across substance 2 when administered by oral gavage to rats daily for a period of 28 days.
This study should provide a rational basis for the toxicological risk assessment in man. The results of this study should indicate potential target organs.

Dose selection rationale:
Based upon data from an acute toxicity study performed by the sponser and the results of a 5-day dose-range-finding study in which RA2 was administered by gavage to 3 rats per group and sex.
Treatment with test article at 0, 200 and 1000mg/kg resuled in no test article-related findings, except reduced body weight in females at 1000mg/kg.

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

Asmples of major organs from all animals of group 1 (0 mg/kg) and group 4 (1000 mg/kg), as well as gross lesions from all animals were processed as H&E stained slides and examined by light microscopy.

Observations for mortality / viability were recorded twice daily and clinical signs of toxicity were recorded at least once daily.

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 the RCC computer.

Body weights were recorded at least weekly during pretest and treatment period.

The ophthalmoscopic examinations of both eyes of all animals were performed after the application of a mydriatic solution using a Microflex 2 Ophthalmoscope. A description of any abnormality was recorded.

Blood samples for haematology and clinical biochemistry were collected from all animals after 4 weeks of treatment under light ether anaesthesia. The animals were fasted in metabolism cages for approximately 18 hours prior to blood sampling but allowed access to water ad libitum. Urine was collected during the 18 hour fasting period into a specimen vial.

The parameters measured for haematology were as follows: erythrocyte count, haemoglobin, haematocrit, MCV, MCH, MCHC, platelet count, reticulocyte count, nucleated erythrocytes, Heinz bodies, methaemoglobin, total leukocyte count, differential leukocyte count, red cell morphology, and coagulation factors.

The parameters measured for clinical biochemistry were as follows: glucose, urea, creatinine, uric acid, bilirubin, cholesterol, triglycerides, phospholipids, ASAT/GOT, ALAT/GPT, lactate dehydrogenase, creatine kinase, alkaline phosphatase, calcium, phosphorous, sodium, potassium, chloride, albumin, total protein, globulin.

The parameters measured for urinalysis were as follows: osmolarity, color, appearance, pH, protein, glucose, ketone, bilirubin, blood, nitrite, urobilinogen, urine sediment.

Sacrifice and pathology:

All animals were weighed and necropsied and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist. At the end of the treatment period the animals were anestheized by intraperitoneal injection of sodium pentobarbitone and sacrificed by exsanguination.

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in phosphate buffered neutral 4% formaldehyde solution: Adrenal glands , aorta, bone - sternum and femur; bone marrow - femoral and sternal ; brain, epididymides , esophagus , eyes with optic nerve and Harderian gland, heart , joint - femorotibial , kidneys, large intestine - cecum, colon and rectum; lacrimal glands - exorbital, larynx, liver, lungs , lymph nodes - mesenteric and mandibular, mammary gland area, nasal cavity, ovaries , pancreas , pituitary gland, prostate gland, salivary glands - mandibular and sublingual ; sciatic nerve, seminal vesicles , skeletal muscle, skin , small intestine - duodenum, jejunum and ileum; spinal cord - cervical , midthoracic and lumbar segments ; spleen , stomach, testes , thymus, thyroid glands with parathyroid glands, tongue , trachea, urinary bladder, uterus, vagina and all gross lesions.

The following organ weights were recorded on the date of necropsy: Adrenals, brain , heart, kidneys, liver, ovaries, pituitary, spleen , testes , thyroid including parathyroid gland. Slides of adrenals, heart , kidneys, liver, lungs, spleen , stomach and testes collected at terminal sacrifice from the animals of group I (control) and group 4 (high-dose), as well as gross lesions from rats of all groups were examined by a pathologist.

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 the ophthalmoscopy data and macroscopial findings.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

All animals survived their asigned study period. There were no clinical signs in any dose group.

Mortality:

no mortality observed

Description (incidence):

All animals survived their asigned study period. There were no clinical signs in any dose group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight was unaffected by treatment with the test article.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect on food consumption. The slightly higher food consumption in test-article treated males was already noted during pretest.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmologic findings were noted in a small proportion of animals from all groups. They included vitreous flaoters and corneal opacity. These findings occurred at similar incidences in the control and treated groups at the end of the treatment period. T

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on heamatology.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effecteds on clinical chemistry.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on urinalysis.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute and relative organ weights did not distinguish treatment groups from controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

A number of gross lesions were observed. The type and incidence of these gross lesions did not differ between treated rats and controls.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

A number of microscopic lesions were observed. The type, incidence and severit of these lesions was not different between treated rats and controls.

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY
All animals survived their assigned study period.
 
CLINICAL SIGNS
There were no clinical signs in any dose groups
 
FOOD CONSUMPTION/RELATIVE FOOD CONSUMPTION
There was no effect on food consumption. The slightly higher consumption in test-article treated males was already noted during pretest.
 
BODY WEIGHTS
Body weight was unaffected by treatment
 
OPHTHALMOSCOPIC EXAMINATIONS
Ophthalmologic findings were noted in a small proportion of animals from all groups. They included vitrous floaters and corneal opacity. These findings occurred at similar incidences in the control and treated groups at the end of the treatment period. Therefore, they were considered to be unrelated to treatment with test the test article.
 
CLINICAL LABORATORY INVESTIGATIONS 
There were no treatment-related effects on heamtology, clinical biochemistry and urinalysis data at termination of the treatment which could be considered of toxicological significance. The only change of note, however, was a slight decreased glucose and slightly increased urea level in females (p<0.01 – p<0.05) of group 4.
 
These findings, which were of minor degrees and within the range of the common variation of these parameters, were considered to be a reflection of metabolic adaptions, and therefore of no toxicological relevance.
 
All other statistical differences in the results of the clinical laboratory data were considered to be incidental and spontaneous in nature, and of normal biological variation for rats of this strain and age.
 
PATHOLOGY 
Organ weights and organ weight ratios
The absolute and relative weights were not different between treated groups and controls
 
MACROSCOPIC FINDINGS
A number of gross lesions were observed. The type and incidence of these gross lesions were not different between treated rats and controls.

MICROSCOPIC FINDINGS
A number of microscopic lesions were observed. The type, incidence and severity of these lesions was not different between treated rats and controls.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1:               Summary of male and female body weights (g)

 

 

Day of study	Group 1 Control		Group 2 50 mg/kg		Group 3 200 mg/kg		Group 4 1000 mg/kg
	Male	Female	Male	Female	Male	Female	Male	Female
1	261 ± 14.7	207 ± 16.3	272 ± 21.3	200 ± 6.6	264 ± 11.8	197 ± 11.5	267 ± 13.7	201 ± 11.8
8	313 ± 13.3	238 ± 14.9	328 ± 27.0	226 ± 9.8	321 ± 16.1	219 ± 15.4	323 ± 16.6	221 ± 10.6
15	351 ± 15.9	248 ± 9.4	372 ± 36.0	239 ± 9.4	360 ± 15.2	232 ± 15.0	360 ± 22.2	234 ± 15.4
22	380 ± 16.5	261 ± 12.0	404 ± 42.7	251 ± 12.0	396 ± 20.9	239 ± 16.2	403 ± 25.1	244 ± 20.3
28	397 ± 14.8	265 ± 12.4	422 ± 42.8	256 ± 12.7	414 ± 24.8	248 ± 13.8	418 ± 29.4	251 ± 13.9

¹Significantly different from the control group; p<0.05

²Significantly different from the control group; p<0.01

 

 

 

Table 2:               Summary of male and female food consumption (g/animal/day)

 

 

Day of study	Group 1 Control		Group 2 50 mg/kg		Group 3 200 mg/kg		Group 4 1000 mg/kg
	Male	Female	Male	Female	Male	Female	Male	Female
1-8	26.9	19.5	28.2	18.7	27.4	17.7	26.9	16.2
8-15	27.9	20.4	30.0	19.7	28.9	18.8	29.3	18.5
15-22	26.5	19.6	28.9	19.4	28.2	18.4	30.1	18.3
22-28	27.7	20.3	29.5	20.1	28.3	19.4	29.4	19.2

¹Significantly different from the control group; p<0.05

²Significantly different from the control group; p<0.01

Table 3:               Summary of male and female relative food consumption (g/kg bw/day)

 

 

Day of study	Group 1 Control		Group 2 50 mg/kg		Group 3 200 mg/kg		Group 4 1000 mg/kg
	Male	Female	Male	Female	Male	Female	Male	Female
1-8	85.9	81.6	85.9	82.6	85.3	80.6	83.5	73.3
8-15	79.6	82.0	80.8	82.6	80.3	81.6	81.5	79.0
15-22	69.8	75.1	71.6	77.2	1.1	77.0	74.8	75.0
22-28	69.7	76.5	69.9	78.5	68.4	78.5	70.4	76.4

¹Significantly different from the control group; p<0.05

²Significantly different from the control group; p<0.01

 

 

Table 4:               Summary of male and female ophthalmoscopic examinations

 

 

	Group 1 Control		Group 2 50 mg/kg		Group 3 200 mg/kg		Group 4 1000 mg/kg
	Male	Female	Male	Female	Male	Female	Male	Female
No. of abnormalities detected
Pretest	3/5	3/5	4/5	3/5	5/5	4/5	2/5	5/5
At week 4	3/5	3/5	2/5	3/5	3/5	3/5	3/5	5/5
Corneal opacity
Pretest	2/5	1/5	1/5	2/5	0/5	1/5	3/5	0/5
At week 4	2/5	2/5	3/5	2/5	2/5	2/5	2/5	0/5

¹Significantly different from the control group; p<0.05

²Significantly different from the control group; p<0.01

Table 5:               Summary of haematology

 

 

Parameter	Group 1 Control		Group 2 50 mg/kg		Group 3 200 mg/kg		Group 4 1000 mg/kg
	Male	Female	Male	Female	Male	Female	Male	Female
RBC (T/L)	9.72	8.57	9.17	8.40	8.94	8.69	8.95	8.65
Haemoglobin (mmol/L)	11.1	10.0	10.6	9.7	10.4**	10.0	10.4**	10.0
Haematocrit (L/L)	0.53	0.47	0.50	0.47	0.49	0.47	0.49	0.48
MCV (fL)	54.8	55.3	54.8	55.4	55.2	54.3	55.2	55.4
MCH (fmol)	1.15	1.17	1.16	1.16	1.17	1.15	1.16	1.15
MCHC (mmmol/L)	20.9	21.0	21.2	20.8	21.2	21.1	21.0	20.8
Platelets (g/L)	984	857	1036	958	963	847	1001	918
Reticulocytes (%)	2.57	2.95	2.53	2.99	2.66	2.70	2.50	2.69
Reticulocytes (T/L)	0.2438	0.2517	0.2274	0.2460	0.2381	0.2342	0.2176	0.2269
HFR (%)	6.3	6.6	7.2	9.8	7.8	8.8	9.8	6.3
MFR (%)	32.6	33.2	36.9	34.3	34.4	36.6	37.1	34.5
LFR (%)	61.2	60.1	53.9	55.9	57.8	54.6	53.1	59.2
Heinz bodies	0	0	0	0	0	0	0	0
Methaemoglobin (%)	0.6	0.6	0.6	0.6	0.7	0.7	0.6	0.8
WBC (g/L)	10.8	5.3	11.3	5.9	10.9	5.9	9.5	4.9

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

⁺Steel test significant at 5 % level

 

 

 

 

Table 5:               Summary of clinical biochemistry

 

 

Parameter	Group 1 Control		Group 2 50 mg/kg		Group 3 200 mg/kg		Group 4 1000 mg/kg
	Male	Female	Male	Female	Male	Female	Male	Female
Glucose (mmol/L)	5.53	6.79	6.39	6.66	6.51	6.95	5.73	5.60*
Urea (mmol/L)	4.84	5.68	5.42	5.73	5.50	5.90	5.23	7.59*
Creatinine (µmol/l)	31.3	37.4	29.9	38.2	30.9	37.3	33.0	38.1
Uric Acid (µmol/L)	17.7	31.7	22.9	26.3	20.7	24.6	24.3	42.0
Bilirubin total (µmol/L)	3.42	4.49	3.22	3.70	3.36	3.85	3.03	4.61
Cholesterol total (mmol/L)	2.27	2.86	2.03	2.73	2.60	2.52	2.32	2.78
Triglycerides (mmol/L)	0.55	0.38	0.65	0.34	0.62	0.29	0.68	0.46
Phospholipids (mmol/L)	1.65	2.29	1.57	2.21	1.85	2.23	1.72	2.41
ASAT (µkat/L)	1.43	1.13	1.17*	1.22	1.29	1.27	1.31	1.30
ALAT (µkat/L)	0.68	0.39	0.43*	0.43	0.45*	0.44	0.44*	0.44
Creatine kinase (µkat/L)	3.12	1.79	2.32*	2.15	2.19**	2.16	2.30	2.53*
Alkaline phosphatase (µkat/L)	5.97	3.16	5.08	3.83	6.55	4.05	5.61	3.84
Calcium (mmol/L)	2.94	2.87	2.95	2.84	2.90	2.83	2.99	2.86
Phosphorous (mmol/L)	2.38	1.93	2.43	1.95	2.40	2.09	2.57	2.12
Sodium (mmol/L)	142.0	139.9	142.7	141.3	141.2	140.8	142.5	140.0
Potassium (mmol/L)	3.27	3.59	3.54*	3.35	3.60*	3.43	3.38	3.55
Chloride (mmol/L)	113.0	113.6	112.6	114.6	112.6	113.8	111.4	112.8
Albumin (g/L)	33.8	34.4	32.7	34.5	32.9	33.8	31.4**	33.5
Total protein (g/L)	75.2	73.1	72.8	72.5	72.6	72.2	71.2*	71.2

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

⁺Steel test significant at 5 % level

Applicant's summary and conclusion

Conclusions:

The oral administration of RA2 to Wistar rats at doses of 0, 50, 200 and 1000 mg/kg/day, for 28 days, resulted in no deaths and no effects on clinical signs, food consumption, body weight, ophthalmoscopic findings, organ weights, macroscopical or microscopical findings.
 
In group 4 (1000 mg/kg/day) a slight decreased glucose and slightly increased urea level in females, and slightly decreased albumin and total protein level in males was recorded and considered to be a reflection of metabolic adaptations. No toxicological relevance was attributed to this finding.
 
Based on the results of this study, 1000 mg/kg body weight/day of RA2 was established as the no-observed-adverse-effect-level (NOAEL) and 200 mg/kg as the no-observed-effect-level (NOEL).

Executive summary:

In this subacute toxicity study, RA2 was adminstered daily by oral gavage to SPF-bred Sprague Dawley rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days.

 

Each group comprised 5 animals per sex which were sacrificed after 28 days of treatment. Clinical signs, food consumption and body weights were recorded periodically during the acclimatization and treatment period. Ophthalmosopic examinations were performed during acclimatization and at the end of the treatment period.

 

At the end of the dosing period, blood samples were withdrawn for haematology and plasma chemistry analyses, and urine samples were collected for urinalysis. All animals were killed, necropsied and examined post mortem. Samples of major organs from animals of group 1(0 mg/kg) and group 4 (1000 mg/kg), as well gross lesions from all animals were processed as hematoxylin and eosin stained slides and examined by light microscopy.

 

The results are summarized as follows:

 

Mortality

All animals survived their assigned study period

 

Clinical signs

There was no effect on body weight.

 

Food consumption/relative food consumption

There was no effects on food consumption or relative food consumption

 

Ophthalmoscopic Examinations

There were no test article-related ophthalmological findings.

 

Clinical Laboratory investigations

 

There were no treatment-related effects on heamatology, clinical biochemistry and urinalysis data at termination of the treatment which could be considered of toxicological significance. The only change of note, however, was a slight decreased glucose and slightly increased urea level in females (p<0.01 – p<0.05) of group 4.

 

These findings, which were of minor degrees and within the range of the common variation of these parameters, were considered to be a reflection of metabolic adaptions, and therefore of no toxicological relevance.

 

All other statistical differences in the results of the clinical laboratory data were considered to be incidental and spontaneous in nature, and of normal biological variation for rats of this strain and age.

 

Based on the results of this study, 1000 mg/kg body weight/day of RA2 was established as the no-observed-adverse-effect-level (NOAEL) and 200 mg/kg as the no-observed-effect-level (NOEL).