Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Endpoint summary

Description of key information

Oral

The test item was tested for oral toxicity in rats according to OECD Guideline 423. The test substance caused mortality at 2000 mg/kg bw (3/3) and at 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.

Dermal

The test item was tested for dermal toxicity in a 24-hrs single administration of 2000 mg/kg bw in male and in female CRL:(WI)BR rats according to OECD Guideline 402. The test material caused erosion, erythema and sloughing on the treated skin, which recovered within 2 - 13 days. Mortalities were not observed. The acute dermal LD50 value of the test item is greater than 2000 mg/kg bw in male and female CRL:(WI)BR rats. No classification and labelling for dermal toxicity is required according to Regulation 1272/2008/EC (CLP).

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:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2001

Deviations:

no

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

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: 205 – 221 g
- Housing: Group caging (3 animals/cage)
- Diet (e.g. ad libitum): 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 and tap water from the municipal supply, ad libitum.
- Water: ad libitum
- Acclimation period: 22 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24 °C
- Humidity (%): 38 - 58 %
- Air changes (per hr): 8 - 12 air exchanges/hour by central air-condition system.
- Photoperiod (hrs dark / hrs light): light 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

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.

Preliminary study:

no preliminary study

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

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

Sex:

female

Dose descriptor:

LD50

Effect level:

500 mg/kg bw

Remarks on result:

other: cut off value

Mortality:

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

Clinical signs:

other: 2000 mg/kg bw – Treatment group 1 Activity decrease (3/3), prone position (1/3), squatting position (2/3), hunched back (3/3), cyanotic skin (3/3), piloerection (2/3) and dyspnoea (1/3) were noted for this group on the day of the treatment. White mucous f

Gross pathology:

2000 mg/kg bw – Treatment group 1
All animals (No.: 7842, 7847, 7886) were found dead. Dark red lungs (2/3), congestive liver (3/3) and distended, liquid filled small intestines (3/3) were noted at the gross necropsy.

300 mg/kg bw – Treatment group 2
In dead animal (No.: 7862), reddish mottled lungs and congestive liver were observed. In one of the two surviving animals, pinprick-sized haemorrhages were found in the lungs (No.: 7875).

300 mg/kg bw – Treatment group 3
Pale raised areas (No.: 7831) and point like haemorrhages (No.: 7837) were recorded in the lungs of these animals.

In the dead animals, distended, liquid filled intestines were related to the local effect of the test item at 2000 mg/kg bw. The pulmonary alterations and congestive liver were signs of circulatory disturbance developed during the death.
No macroscopic alterations related to any toxic effect of the test item were found in surviving animals. The haemorrhages and pale raised areas in the lungs were due to the method of anaesthesia and exsanguinations, which are also observable in untreated animals after anaesthesia.

Other findings:

no other findings.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Under the conditions of the present study, a single oral administration of the test item caused mortality at 2000 mg/kg bw (3/3) and at 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 with a cut off value of 500 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) Polyethylene glycol 400 (PEG 400) in three independent experiments. 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).

Results

Mortality

The test item caused mortality at 2000 mg/kg bw and 300 mg/kg bw/day dose levels in female CRL:(WI) BR rats as follows:

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

Clinical signs

Treatment group 1 – 2000 mg/kg bw

All animals of the first treatment group were found dead one day after the treatment. Activity decrease, prone position, squatting position, hunched back, cyanotic skin, piloerection and dyspnoea were observed. Onset of signs was 2 hours and 6 hours.

Treatment group 2 – 300 mg/kg bw

Activity decrease, squatting position, hunched back, diarrhoea and piloerection were observed in this group. Onset of the first signs was 2 hours for animal, which was found dead next day and 6 hours for surviving animals.

Treatment group 3 – 300 mg/kg bw

No animal of the third treatment group died but activity decrease, hunched back, diarrhoea and piloerection were observed with an onset of 30 minutes of the first signs. All animals became symptom-free from day 2.

Body weight

The body weight development of each animal treated with 300 mg/kg bw was normal throughout the two weeks observation period, similar to untreated female animals of the same age and strain.

Necropsy

Distended, liquid filled small intestines were signs of the local effect of the test item at 2000 mg/kg bw. There were no test item related necropsy findings in dead animal or in surviving animals at 300 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

discriminating dose

Value:

300 mg/kg bw

Quality of whole database:

The study is regarded as reliable without restrictions because it was conducted in accordance with GLP regulation and guideline.

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 exposure of humans via inhalation is not likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size

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:

from 2007-10-03 to 2007-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Version / remarks:

92/69/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/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
- Weight at study initiation: Male: 252 - 283 g; Female: 216 - 239 g
- Fasting period before study: None
- Housing: Individual caging (1 animal/cage); Type II polypropylene/polycarbonate
- Diet: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water from the municipal supply, ad libitum.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 -24 °C
- Humidity (%): 38 - 58 %
- Air changes (per hr): 8 - 12 air exchanges/hour by central air-condition system
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Type of coverage:

semiocclusive

Vehicle:

other: Test item was pulverized and moistened with water

Details on dermal exposure:

TEST SITE
- Area of exposure: approximately 10 per cent of the total body surface
- % coverage: appr. 10
- Type of wrap if used: semi occlusive plastic wrap

REMOVAL OF TEST SUBSTANCE
- Washing: water at body temperature
- Time after start of exposure: 24 hrs

TEST MATERIAL
- Amount applied: single dose of 2000 mg/kg bw
- For solids, paste formed: yes, with water

Duration of exposure:

24 hrs

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

Males: 5; 2000 mg/kg bw;
Females: 5; 2000 mg/kg bw;

Control animals:

no

Details on study design:

Observations: Animals were observed for a 14-day post-treatment period. Clinical examinations, body weight assessment and gross necropsy were conducted.

Clinical Examination: A careful individual clinical examination was made on the day of treatment 1 h and 5 h after the administration of the test item, 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.

Weight Assessment: The body weight of each animal was recorded on day 0 (beginning of the experiment), on day 7 and on day 14 with a precision of 1 g.

Pathology: On day of the last clinical observation, all animals were exsanguinated under carbon dioxide anaesthesia and gross necropsy was performed. 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 post mortem record sheets.

Evaluation of Results: The method used allows assigning an LD50 value, according to the OECD Guidelines for Testing of Chemicals, No. 402, and B.3. 92/69/EEC.
Individual animal data were provided and summarised in tabular form, showing for each dose group the number of animals used.
Clinical observations were noted individually and in summary tables.
Body weight changes were summarised in tabular form.
Necropsy findings were described and summarised in tabular form.

Statistics:

No statistics performed

Preliminary study:

No preliminary study was performed

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Mortality:

Male Female
Dose level (mg/kg bw): 2000 2000
Number of animals treated: 5 5
Number of dead animals: 0 0

Clinical signs:

other: There were no clinical signs. The behaviour and physical condition of animals were considered to be normal. 2000 mg/kg bw of the test item caused slight to marked erythema (5/5 male, 5/5 female), erosion (1/5 female) and sloughing (3/5 male, 5/5 female) o

Gross pathology:

No macroscopic alterations due to the effects of the test item were found. Gross necropsy revealed pinprick-sized haemorrhages (5/5 male, 2/5 female) and reddish mottled colour (1/5 female) in the lungs due to the method of anaesthesia and exsanguinations, which are also observable in untreated animals after anaesthesia.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, a single 24-hour dermal administration of 2000 mg/kg bw of the test item, the test item caused erosion, erythema and sloughing on the treated skin, which recovered within 2 - 13 days. Mortalities were not observed, neither in male nor in female CRL:(WI)BR rats. The acute dermal LD50 value of the test item is greater than 2000 mg/kg bw in male and female CRL: (WI) BR rats.

Executive summary:

The acute dermal toxicity study (Limit Test) with the test item was performed in compliance with OECD guideline No.: 402 and method B.3. 92/69/EEC. A limit test was carried out at 2000 mg/kg bw dose level. Five male and five female CRL:(WI)BR rats were treated with a dose of 2000 mg/kg bw. The test item was applied once in its original form by dermal route and held in contact with the skin for a 24-hour period. The observation period after patch removal continued for 14 days. Clinical observations were made one and five hours after the treatment, then once a day. Body weight was measured weekly. Gross necropsy was performed in all animals at the termination of the experiment.

Mortality

No mortality was observed in male and female animals up to the limit dose of 2000 mg/kg bw.

Clinical Observations

There were no clinical signs. The behaviour and physical condition of animals were considered to be normal. 2000 mg/kg bw of the test item caused slight to marked erythema (5/5 male, 5/5 female), erosion (1/5 female) and sloughing (3/5 male, 5/5 female) on the skin after 24-hour exposure. Skin became normal between days 2 and 10 in male animals and between days 8 and 13 in female animals.

Body Weight

The mean body weight and the body weight gain of the male and female animals were considered to be normal during the two-week observation period., similar to the expected values in untreated animals of the same age and strain.

The following table contains the body weight and body weight gain values of control animals of same age and strain.

Male animals: 

	Body weight (g)			Body weight gain (g)
	Day 0	Day 7	Day 14	Day 0 - 7	Day 7 - 14	Day 0 - 14
Mean	264.5	316.6	366.4	52.0	49.8	101.9
Sd	15.0	14.1	15.1	4.9	7.6	8.7
n	21	21	21	21	21	21

Female animals: 

	Body weight (g)			Body weight gain (g)
	Day 0	Day 7	Day 14	Day 0 - 7	Day 7 - 14	Day 0 - 14
Mean	226.7	242.7	251.3	16.0	8.7	24.7
Sd	6.8	7.3	10.2	7.8	5.8	11.1
n	23	23	23	23	23	23

Necropsy

No macroscopic alterations due to the effects of the test item were found. Gross necropsy revealed pinprick-sized haemorrhages (5/5 male, 2/5 female) and reddish mottled colour (1/5 female) in the lungs due to the method of anaesthesia and exsanguinations, which are also observable in untreated animals after anaesthesia.

Under the conditions of the present study, a single 24 -hour dermal administration of 2000 mg/kg bw of test item, the test item caused erosion, erythema and sloughing on the treated skin, which recovered within 2 - 13 days. Mortalities were not observed, neitehr in male nor in female CRL:(WI)BR rats. The acute dermal LD50 value of the test item is greater than 2000 mg/kg bw in male and female CRL:(WI) BR rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

The study is regarded as reliable without restrictions because it was conducted in accordance with GLP regulation and guideline.

Additional information

Acute Oral

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) Polyethylene glycol 400 (PEG 400) in three independent experiments. 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).

Results

Mortality

The test item caused mortality at 2000 mg/kg bw and 300 mg/kg bw/day dose levels in female CRL:(WI) BR rats as follows:

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

 

Clinical signs

Treatment group 1 – 2000 mg/kg bw

All animals of the first treatment group were found dead one day after the treatment. Activity decrease, prone position, squatting position, hunched back, cyanotic skin, piloerection and dyspnoea were observed. Onset of signs was 2 hours and 6 hours.

Treatment group 2 – 300 mg/kg bw

Activity decrease, squatting position, hunched back, diarrhoea and piloerection were observed in this group. Onset of the first signs was 2 hours for animal, which was found dead next day and 6 hours for surviving animals.

Treatment group 3 – 300 mg/kg bw

No animal of the third treatment group died but activity decrease, hunched back, diarrhoea and piloerection were observed with an onset of 30 minutes of the first signs. All animals became symptom-free from day 2.

Body weight

The body weight development of each animal treated with 300 mg/kg bw was normal throughout the two weeks observation period, similar to untreated female animals of the same age and strain.

Necropsy

Distended, liquid filled small intestines were signs of the local effect of the test item at 2000 mg/kg bw. There were no test item related necropsy findings in dead animal or in surviving animals at 300 mg/kg bw.

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.

Acute Dermal

The acute dermal toxicity study (Limit Test) with the test item was performed in compliance with OECD guideline No.: 402 and Method B.3. 92/69/EEC. A limit test was carried out at 2000 mg/kg bw dose level. Five male and five female CRL:(WI)BR rats were treated with a dose of 2000 mg/kg bw. The test item was applied once in its original form by dermal route and held in contact with the skin for a 24-hour period. The observation period after patch removal continued for 14 days. Clinical observations were made one and five hours after the treatment, then once a day. Body weight was measured weekly. Gross necropsy was performed in all animals at the termination of the experiment.

No mortality was observed in male and female animals up to the limit dose of 2000 mg/kg bw.

There were no clinical signs. The behaviour and physical condition of animals were considered to be normal. 2000 mg/kg bw of the test item caused slight to marked erythema (5/5 male, 5/5 female), erosion (1/5 female) and sloughing (3/5 male, 5/5 female) on the skin after 24-hour exposure. Skin became normal between days 2 and 10 in male animals and between days 8 and 13 in female animals.

The mean body weight and the body weight gain of the male and female animals were considered to be normal during the two-week observation period., similar to the expected values in untreated animals of the same age and strain.

The following table contains the body weight and body weight gain values of control animals of same age and strain.

Male animals:

	Body weight (g)			Body weight gain (g)
	Day 0	Day 7	Day 14	Day 0 - 7	Day 7 - 14	Day 0 - 14
Mean	264.5	316.6	366.4	52.0	49.8	101.9
Sd	15.0	14.1	15.1	4.9	7.6	8.7
n	21	21	21	21	21	21

 

Female animals:

	Body weight (g)			Body weight gain (g)
	Day 0	Day 7	Day 14	Day 0 - 7	Day 7 - 14	Day 0 - 14
Mean	226.7	242.7	251.3	16.0	8.7	24.7
Sd	6.8	7.3	10.2	7.8	5.8	11.1
n	23	23	23	23	23	23

No macroscopic alterations due to the effects of the test item were found. Gross necropsy revealed pinprick-sized haemorrhages (5/5 male, 2/5 female) and reddish mottled colour (1/5 female) in the lungs due to the method of anaesthesia and exsanguinations, which are also observable in untreated animals after anaesthesia.

Under the conditions of the study, a single 24-hour dermal administration of 2000 mg/kg bw of test item, the test item caused erosion, erythema and sloughing on the treated skin, which recovered within 2 - 13 days. Mortalities were not observed, neither in male nor in female CRL:(WI)BR rats. The acute dermal LD50 value of the test item is greater than 2000 mg/kg bw in male and female CRL:(WI) BR rats.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered to be classified as acute oral toxicity Cat. 4, H302: Harmful if swallowed for acute oral toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.