Reaction mass of 3-[(2-aminoethyl)ammonio]propane-1-sulfonate and 3,3'-(ethane-1,2-diyldiammonio)dipropane-1-sulfonate and ethane-1,2-diamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2008 - February 2009

Reliability:

1 (reliable without restriction)

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2009

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)

Test material

Test material information

Constituent 1

Reference substance name:

3-[(2-aminoethyl)amino]propane-1-sulfonic acid

Cas Number:

14235-54-2

Molecular formula:

C5H14N2O3S

IUPAC Name:

3-[(2-aminoethyl)amino]propane-1-sulfonic acid

Details on test material:

- Name of test material (as cited in study report): 3-[(2-aminoethyl)ammonio]propane-1-sulfonate
- Molecular formula (if other than submission substance): C5H14N2O3S
- Molecular weight (if other than submission substance): 182.24
- Substance type: liquid
- Physical state: clear yellowish-brown liquid
- Lot/batch No.: 06060423/101
- Expiration date of the lot/batch: not available
- Stability under test conditions: stable
- Storage condition of test material: room temperature, in the dark
- Composition of test material, percentage of components: Actually the concentration was 48.5 % w/w (of the test substance in water).
- Other Name:EPS

Test animals

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, 97633 Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 169.5g; females: 125.0 g. All animals were within plus/minus 20 % of the mean weight per sex at the time of the
allocation to the groups.
- Fasting period before study: no
- Housing: Makrolon cages Type IV (33 cm x 55 cm area, 20 cm height).
Group caging (2 or 3 animals of one group and the same sex per cage).
The first 3 animals of a group and the second 2 animals of a group were housed together.
- Diet: ad libitum; Ssniff R/M-H maintenance diet for rats and mice (item V1534-3), supplied by Ssniff Spezialdiäten GmbH,
59494 Soest, Germany.
Exception: Feed was withdrawn on days prior to blood sampling at 5:00 p.m., only from the animals, where blood was to be taken, and was re-offered
immediately after the blood sampling.
Random samples of the feed are analysed for contaminants by the supplier. One sample is analysed also for contaminants in addition by an independent external laboratory (once/year). The limits of tolerance are derived from the "Deutsche Futtermittelverordnung" (German feed regulation).
- Water:Tap water, acidified with HCl to pH greater than or equal to 3, from an automatic watering system, ad libitum. Random samples of the water are analysed by "AGES", 1226 Vienna, Austria, to check, if the water fulfils the requirements for drinking water for humans (exception: the pH).
- Acclimation period: 6 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.9°C
- Humidity (%): 48%
- Air changes (per hr): 12 per hour
- Photoperiod (hrs dark / hrs light): artificial light from 6 a.m.to 6.p.m. (12 hrs light/12hrs dark).


IN-LIFE DATES: From:31 March 2008 To:14 May 2008

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

An oral administration was performed by stomach intubation using a metal gavage once a day in the morning on 7 days per week.
The individual dose volumes were calculated using the last determined body weights.
The dose volume was uniformly 10 mL per kg body weight for all groups.

Groups, doses (main study):

• K (negative control group) - vehicle only,
• KS (negative control satellite group) - vehicle only,
• A (low dose group) - 100 mg "3-[(2-aminoethyl)ammonio]propane-1-sulfonate" per kg body weight,
•B (mid dose group) - 316 mg "3-[(2-aminoethyl)ammonio]propane-1-sulfonate" per kg body weight,
•C (high dose group) - 1000 mg "3-[(2-aminoethyl)ammonio]propane-1-sulfonate" per kg body weight,
•CS (high dose satellite group) - 1000 mg "3-[(2-aminoethyl)ammonio]propane-1-sulfonate" per kg body weight,

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determinations of the test substance were performed in some selected preparations for concentration.
The following analyses were performed:
Actual concentration:
2 samples of 2.0 mL each of the preparations for groups A, B and C+CS were taken using the syringe plus probe by the technician at the beginning, in the middle and at the end of the dosing procedure for the group concerned.
The test samples were stored deep frozen (-80°C) and then thawed at room temperature.
A deviation of the individual samples from the mean of at most ± 10 % was tolerated.
A test for stability was not determined, as the test substance was found to be stable in the hydrolysis test (see separate report ARC--L-3190 for details).
See table 18.

Duration of treatment / exposure:

The animals of all groups were treated with the test substance solutions or with the vehicle once a day on 28 consecutive days.
Animals of the satellite groups KS and CS were kept after cessation of dosing without a further administration for 15 additional days.

Frequency of treatment:

An oral administration was performed once a day in the morning on 7 days per week for 28 consecutive days.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 316, 1000 mg/kg bw/day (m/f)
Basis:
nominal in water

No. of animals per sex per dose:

5 animals per sex and dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses chosen are derived from and based on the results of the Dose Range Finding.
Summarized results of the dose range finding study: 20 male and 20 female rats (same strain, same conditions as in the main study) were used.
Groups, doses (Dose Range Finding):
•K-Df (negative control group) - vehicle only,
•A-Df (low dose group) - 100 mg "3-[(2-aminoethyl)ammonio]propane-1-sulfonate" per kg body weight,
•B-Df (mid dose group) - 316 mg "3-[(2-aminoethyl)ammonio]propane-1-sulfonate" per kg body weight,
•C-Df (high dose group) - 1000 mg "3-[(2-aminoethyl)ammonio]propane-1-sulfonate" per kg body weight,

All animals survived until the scheduled terminal sacrifice. No toxic signs were observed up to 1000 mg per kg b.w..
1000 mg per kg b.w. is the highest feasible dose for subacute toxicity study. The low dose shall induce no toxic effect and was set at 10 % of the high dose. The mid dose was interpolated geometrically.

Dose selection rationale for the dose range finding study was based on the results of an acute toxicity test. The LD50, rat, oral is > 5000 mg/kg b.w. (information obtained from the sponsor).


- Rationale for animal assignment: Random.
- Rationale for selecting satellite groups: According to the guidelines: additional five animals per sex in the control and in the high dosed group are kept for observation of reversibility, persistence or delayed occurrence of toxic effectst.
- Post-exposure recovery period in satellite groups: 14 days post treatment
- Section schedule rationale:Random.

Positive control:

None.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were carefully observed for general signs and the health status once a day and additionally checked once a day for
viability.
- Cage side observations were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days 0, 7, 15, 21, 28: all animals of all groups.
Days 34, 42: all animals of the satellite groups.
Special emphasis was put on skin, fur, eyes, visible mucous membranes, incisors, secretion and excretion, body odour, autonomous activities
(e.g. lacrimation, piloerection, pupillar size, abnormal breathing, and body surface temperature), vocalisation, abnormal locomotion, movements and posture, presence of convulsions or paralysis, stereotypes, bizarre behaviour, visible or palpable tissue masses.



BODY WEIGHT: Yes
- Time schedule for examinations:
The individual body weights were determined:
Day 1 to 28: Once a week, all animals.
Days 34 and 42: Satellite groups.
The body weight gain was calculated.



FOOD CONSUMPTION :
- Food consumption: Determined per cage in weekly intervals in all animals.



HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken on Day 29 from all animals of groups K, A, B and C and of all animals of groups KS and CS
on Day 43.
- Anaesthetic used for blood collection: Yes. Ether
- Animals fasted: Yes
- How many animals: all animals
-The following parameters were examined.
• Red blood cell count (RBC)
• Haemoglobin concentration (HGB)
• Haematocrit (HCT)
• Mean corpuscular haemoglobin (MCH)
• Mean corpuscular haemoglobin concentration (MCHC)
• White blood cell count (WBC)
• Mean cell volume (MCV)
• Platelet count (PLT)
• Differential white blood cell count (% of the different cell species)
• Prothrombin time (Quick) as an indicator of blood clotting capacity.



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:Blood was taken on Day 29 from all animals of groups K, A, B and C and of all animals of groups KS and CS
on Day 43.
- Animals fasted: Yes
- How many animals:all animals
-The following parameters were examined.
• alanin aminotransferase (ALT, GPT)
• albumin (ALB)
• alkaline phosphatase (AP)
• aspartate aminotransferase (AST, GOT)
• cholesterol (CHOL)
• creatinine (CREA)
• gamma-glutamyltransferase (GGT)
• glucose (GLU)
• potassium (K)
• sodium (Na)
• total protein (TP)
• urea (UREA)




NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined::
On Day 28: all animals of all groups.
On Day 42: all animals of satellite groups.

- Battery of functions tested: sensory activity / grip strength / motor activity / other:
The eyelid and auricular reflexes (tactile and proprioceptive) were tested by slightly touching the cornea and the interior of a pinna with a nylon cord of approximately 0.6 mm diameter. Shaking of the head was considered as positive response.
Acoustic reactivity was tested by response to a moderate sound (clapping of the hands). Twitching of the body was considered as positive.
Visual reactivity was derived from the reaction to a dark sheet of paper, brought near to the animal without any physical contact. Turning towards the paper was considered as positive.
For the testing of the righting reflex, the animals were held between the front- and hind legs, turned on their backs and dropped from a height of 30 cm onto the bedding material of the standard arena. A landing on all four legs is considered to be a normal reaction.
The measurement of the forelimb grip strength was determined using a (Digital Force Gauge DFIS 100 from Chatillon, Greensboro, North Carolina,
USA).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

None.

Statistics:

Statistical method used for:
1.all data with means and standard deviations determined, comparison of more than two groups: Analysis of variance followed by the Scheffé-test.
2.all data with means and standard deviations determined, for comparison of two groups only: t-test
3.counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled:H-test of Kruskal and Wallis followed
by the test of Nemenyi
4.counted events: Chi2-test
5.counted events, if the Chi2-Test was not applicable:Fisher's exact test

Results were analysed separately for males and females. P = 0.05 was chosen in each test. Two tailed test were used. Groups K and KS and C and CS
were treated separately for statistical analysis.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

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 examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No abnormal findings were made.
See table 7.

BODY WEIGHT AND WEIGHT GAIN
There was no statistically significant difference or dose related trend noted in the body weights of both sexes.
In the body weight gain, the high dosed group females had a lower body weight gain, when compared with the corresponding controls, in the first
week of dosing. This may be due to unpleasant sensations or due to a poor taste of the test substance, but is not given toxicological relevance due to single occurrence.
See table 10.

FOOD CONSUMPTION
There were no noteworthy differences or dose related trends noted in the feed consumption of both sexes. The differences between the groups are
too small to give an indication for a toxic effect.
See table 12.

HAEMATOLOGY
Significant group differences at the end of the recovery period stand alone without corresponding changes at the end of the dosing period and are
therefore considered as incidental. A possible interpretation may be the relative low mean of the actual control group.
See table 13.


CLINICAL CHEMISTRY
Only group differences in alanin aminotransferase are attributed to the test substance. The alanin aminotransferase was elevated in the high dosed
groups of both sexes, but gain statistical significance only in the males at the end of the dosing period.

In addition, alanin aminotransferase was higher in the high dosed females at the end of the recovery period. The elevated alanin aminotransferase
may be associated with mild hepatic toxicity.
Elevated gamma glutamyl transferase at the end of the dosing period lacks statistical significance and also lacks dose related increase and is
therefore not considered as toxicological relevant. Significant increase of gamma glutamyl transferase at the end of the recovery period adds to the indications for mild hepatic changes, derived from elevated alanin aminotransferase.
The higher urea is not considered to represent adverse effects as no dose response relationship was noted.
Only the aberrant alanin aminotransferase gives a hint on a target organ or a mode of action of the test substance. The others are regarded as
unspecific alterations.
See table 14.


NEUROBEHAVIOUR
No test substance related findings were made nor were there any significant group differences at the functional observations at the end of the dosing period.
In grip strength there were no significant differences between a dosed group and a control group.
The significant differences in "straightening up" in the males at the end of the recovery period is not regarded as toxicological relevant, as in general
the "additional observations" indicated a perfect condition of the animals.
See tables 8 and 9.

ORGAN WEIGHTS
Lowered spleen weights in high dosed females after recovery period lack a dose response and are not attributed to the test substance. Thus they are considered as incidental significances without a toxicological relevance.
See table 16.

GROSS PATHOLOGY
All animals were grossly normal at necropsy.
See table 15.

HISTOPATHOLOGY: NON-NEOPLASTIC
Some isolated findings in histopathology are regarded as part of the background pathology, inconspicuous in type and incidence. There were no test substance related findings made histopathologically.
See table 17.

Effect levels

Dose descriptor:

NOEL

Effect level:

>= 316 mg/kg bw (total dose)

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The only test substance related effect - elevated alanin aminotransferase - may be due to a very mild hepatic toxicity, what was noted in both sexes and persistent until the end of the recovery period.
The No-observed-effect-level of "3-[(2-aminoethyl)ammonio]propane-1-sulfonate" was at 316 mg per kg body weight in the males and in the females.

Executive summary:

This study was performed to evaluate the toxicity after a repeated oral administration of "3-[(2-aminoethyl)ammonio]propane-1-sulfonate" to rats, according to the EC-Guideline 96/54, method B.7., Official Journal of the European Community and to the OECD-Guideline 407, "Repeated Dose 28-day Oral Toxicity Study in Rodents".

According to EU-Directive 2001/59/EC, the application of "R48" is not considered as necessary for "3-[(2-aminoethyl)ammonio]propane-1-sulfonate", as no severe toxic effects were noted at doses of up to 1000 mg/kg.