Reaction products of m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2007-10-02 to 2007-11-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

2004/73/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

641-048-8

Cas Number:

110839-13-9

Molecular formula:

C37H48N4O4

IUPAC Name:

Reaction products of m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Europe) Laboratories Inc. TOXI COOP Ltd. 1103 Budapest, Cserkesz u. 90.
- Age at study initiation: Young adult rats, 9 -10 weeks old
- Weight at study initiation: 200 – 215 g
- Fasting period before study: 24 hrs
- Housing: Group caging (3 animals/cage)
- Diet: Animals received ssniff® R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum
- Water (e.g. ad libitum): Tap water from the municipal supply ad libitum
- Acclimation period: 22 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 34 - 69
- Air changes: 8 - 12 air exchanges/hour by central air-condition system
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: PEG 400

Details on oral exposure:

A single oral administration - followed by a fourteen days observation period - was performed by gavage. Animals were treated with the test item prepared freshly in the morning hours. A constant treatment volume of 10 mL/kg body weight was applied. The concentration was adjusted to ensure constant volumes at all dose levels.

Doses:

The acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg/kg bw as the starting dose in three female rats. As all the three animals were found dead in the first treatment group (2000 mg/kg bw), a second group of three animals was dosed at 300 mg/kg bw. A single mortality occurred in the second treatment group. A third treatment group of three animals was then dosed at 300 mg/kg body weight. No animals were found dead. Further testing was not required according to the test guidelines (OECD 423, Directive 2004/73/EC B.1. tris).

No. of animals per sex per dose:

2000 mg/kg bw: 3 females
300 mg/kg bw: 2 test groups 3 females each

Control animals:

no

Details on study design:

Observation
Animals were observed daily for 14 days after dosing.

Clinical Examination
Animals were observed individually 30 minutes, 1 h, 2 h, 3 h, 4 h, 6 h after the treatment and once each day for 14 days thereafter. Individual observations were performed on the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The time of onset and cessation of symptoms and death were recorded as precisely as possible.

Weight Assessment
The body weights were measured and recorded on day 0 (beginning of the experiment), on days 7 and 14 with a precision of 1 g.

Pathology
All animals including those that died during the test were subjected to gross pathology. All surviving animals were exsanguinated under pentobarbital (100 mg/kg bw Euthasol® 40 %; Lot No.: 07E29 8, Expiry Date: April 2009, Product of AST Beheer B.V. Oudewater, Netherlands) anaesthesia. After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed. All gross pathological changes were recorded for each animal on the post mortem record sheets.

Statistics:

Evaluation of Results
The method used is not intended to allow calculation of a precise LD50 value, but an LD50 value is assigned, according to the OECD Guideline No. 423 and Directive 2004/73/EC B.1 tris. Individual animal data are provided and all data are summarised in tabular form, showing for each dose group the number of animals used, the number of animals displaying signs of toxicity, the number of animals found dead during the test and the time of death of each individual animal. Toxic response data and reversibility were recorded for each dose level. Nature, severity and duration of clinical observations were described.

Results and discussion

Preliminary study:

No preliminary study

Effect levels

Effect levels 1

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

>= 300 - <= 2 000 mg/kg bw

Effect levels 2

Sex:

female

Dose descriptor:

LD50

Effect level:

1 000 mg/kg bw

Remarks on result:

other: cut-off value

Mortality:

The test item caused mortality at 2000 mg/kg bw and at 300 mg/kg bw dose levels in female CRL:(WI) BR rats as follows:
2/3 animals at 2000 mg/kg bw
0/3 animals at 300 mg/kg bw (group 1)
1/3 animals at 300 mg/kg bw (group 2)

Clinical signs:

other: 2000 mg/kg bw – Treatment group 1 Activity decrease (3/3), squatting position (3/3), hunched back (3/3), sanguineous nasal discharge (1/3), diarrhoea (3/3), piloerection (3/3), decreased righting reflex (1/3), decreased grip and limb tone (1/3), decreased

Gross pathology:

2000 mg/kg bw – Treatment group 1
In dead animals (No.: 7869, 7878), congestive liver and dilated, liquid filled stomach and intestines were observed.
In surviving animal (No.: 7855) pinprick-sized haemorrhages were found in the lungs.

300 mg/kg bw – Treatment group 2
All animals (No.: 7821, 7829, 7863) survived. Pinprick sized haemorrhages in the lungs (2/3) and hydrometra (1/3) were noted for this group.

300 mg/kg bw – Treatment group 3
In dead animal (No.: 7877), reddish mottled lungs, congestive liver, liquid filled stomach and intestines, and hydrometra were observed.
In one of the surviving animals (No.: 7838) pinprick-sized haemorrhages were found in the lungs.

In summary, macroscopic findings in the gastro-intestinal tract (large amount of liquid) were due to the local effect of the test item in dead animals dosed with 2000 mg/kg bw or 300 mg/kg bw. The pulmonary alterations (reddish colour) and congestive liver were signs of circulatory disturbance developed during the death.
The haemorrhages in the lungs were due to the method of anaesthesia and exsanguinations, which are also observable in untreated animals after anaesthesia. Hydrometra due to the sexual cycle of animals is a common alteration in experimental rats

Other findings:

No other findings

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The acute oral LD50 value of the test item was between 300 mg/kg bw and 2000 mg/kg bw in female CRL:(WI) BR rats and was ranked into Category 4 with a cut off value of 1000 mg/kg bw.

Executive summary:

The acute toxic class method according to OECD 423 and Directive 2004/73/EC B.1. tris was performed with the test item. Three groups of three female CRL:(WI) BR Wistar rats were treated with the test item by single oral gavage at dose levels of 2000 mg/kg bw (Treatment group 1) and 300 mg/kg bw (Treatment groups 2 and 3) in Polyethylene glycol 400 (PEG 400) in three independent experiments. Based on the results of the study and according to the criteria of the relevant test guidelines no further testing was performed. The concentrations of the formulations were adjusted to ensure the constant treatment volume of 10 mL/kg bw (200 mg/mL and 30 mg/mL, respectively).

The following mortality was found after a single oral administration of the test item at 2000 mg/kg bw and 300 mg/kg bw/day dose levels in female CRL:(WI) BR rats:

Treatment group:	1	2	3
Dose level (mg/kg bw):	2000	300	300
Number of animals treated:	3	3	3
Mortality:	2/3	0/3	1/3

Clinical signs:

Treatment group 1 – 2000 mg/kg bw

Two animals of the first treatment group were found dead on day 1 shortly after the daily observation. Severe activity decrease, squatting position, hunched back, cyanosis, diarrhoea, piloerection, convulsions, dyspnoea, decreased righting reflex, decreased grip-and limb tone and decreased body tone were noted for these animals. Surviving animal became symptom-free ten days after the treatment.

Treatment group 2 – 300 mg/kg bw

Decreased activity, squatting position, hunched back, diarrhoea and piloerection were noted two hours after the treatment. All animals were symptom-free on day five, and no clinical symptoms were noted subsequently.

Treatment group 3 – 300 mg/kg bw

One animal of the third treatment group was found dead one day after the treatment. Severe activity decrease, squatting position, hunched back, diarrhoea and piloerection were observed. The surviving animals became symptom free three days after the treatment.

A transiently reduced body weight gain was noted in surviving animals dosed with 2000 mg/kg bw or 300 mg/kg bw on week 1 or on week 2, respectively. The summarised body weight gain was considered to be normal.

Necropsy revealed distended, liquid filled intestines and stomach at 2000 mg/kg bw or 300 mg/kg bw due to the local effect of the test item resulting in death of these animals. There were no test item related necropsy findings in surviving animals. Under the conditions of the present study, a single oral administration of the test item caused death of female CRL:(WI)BR rats at 2000 mg/kg bw (2/3) and 300 mg/kg bw (1/6).

The acute oral LD50 value of the test item was between 300 mg/kg bw and 2000 mg/kg bw in female CRL:WI (BR) rats and it was ranked into Category 4.