Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Endpoint summary

Description of key information

Oral route: The LD50 value of the test item in Wistar rats was established to be within the range of 300 to 2000 mg/kg bw (OECD 423 and EU Method B.1 tris).

Dermal route: The LD50 value of the test item in the Wistar strain rat was found to be > 2000 mg/kg bw (OECD 402 and EU Method B.3)

Inhalation route: The substance is a low melting point solid with a vapour pressure of 0.078 Pa at 25 °C and it is expected that inhalation exposure from identified uses will be low. In addition, the most likely route of exposure for workers and consumers is the dermal route. However, classification for acute inhalation toxicity (category 4) has been adopted as a precautionary approach.

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:

23 September 2008 - 07 November 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study; well documented study report

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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 Deutschland, Sulzfeld, Germany.
-Age at study initiation: approx. 8~10 weeks
-Weight at study initiation: Body weight variation did notexceed +/-20% of the sex mean.
-Fasting period before study: Food was withheld overnight (for a maximum of 20 hours) prior to dosing until 3-4 hours after administration of the test substance.
-Housing: Group housing of 3 animals per cage in labeled Macrolon cages (MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom)..
-Diet: Free access to pelleted rodent diet (SM RIM-Z from SSNIFFB Spezialdiaten GmbH, Soest, Germany).
-Water: free access to tap-water.
-Acclimation period: Acclimatization period was at least 5 days before start of treatment under laboratory conditions.
ENVIRONMENTAL CONDITIONS
-Temperature (C): 19.5 -21.3 C
-Humidity (%): 30-70% (actual range: 37 -74%)
-Air changes: 15 air changes per hr
-Photoperiod: 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: Start: 23 September 2008; Completion: 07 November 2008.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 30, and 200 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw
- Justification for choice of vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.
- Lot/batch no. (if required): No data available.
- Purity: No data available.

MAXIMUM DOSE VOLUME APPLIED: 10mL/Kg
DOSAGE PREPARATION (if unusual): the formulations (w/w) were prepared within 4 hours prior to dosing. Homogeneity was accomplished to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. The concentration of the test substance in vehicle was varied to allow constant dosage volume in terms of mL/kg body weight.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: the toxicity of the test substance was assessed by stepwise treatment of groups of 3 females. The first group was treated at a dose level of 2000 mg/kg. The absence or presence of mortality of animals dosed at one step determined the next step, based on the test procedure defined in the guidelines. The onset, duration and severity of the signs of toxicity were taken into account for determination of the time interval between the dose groups.

Doses:

300 and 2000 mg/kg bw

No. of animals per sex per dose:

12 Females, each dose group consisted of 3 animals.

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing:

-Motility/viability: twice daily. The time of death was recorded as precisely as possible.

-Body weights: Days 1 (pre-administration), 8 and 15 and at death.

-Clinical signs: At periodic intervals on the day of dosing (Day 1) and once daily thereafter, until Day 15.


- Necropsy of survivors performed: yes

- Other examinations performed: None.

Statistics:

No statistical analysis was performed.

Preliminary study:

Not applicable

Sex:

female

Dose descriptor:

LD50

Effect level:

500 mg/kg bw

Mortality:

Five females in the high dose group were found dead on Days 9. 12 or 13. No further mortality occurred.

Clinical signs:

Clinical signs observed during the study period were as follows:
2000 mg/kg group: lethargy, hunched posture, watery discharge of the eyes, uncoordinated movements, ptosis, piloerection, lean appearance and/or hypothermia were noted in all animals during the observation period.
300 mg/kg group: hunched posture and piloerection were noted in all animals between
Days 1 and 7.

Body weight:

The mean body weight gain shown by the animals at 300 mg/kg over the study period was considered to be similar to that expected of normal untreated animals of the same age and strain.

Gross pathology:

Emaciation, dark red discoloration of the thymus and reduced size of the thymus was noted in one surviving animal at 2000 mg/kg. One animal, which was found dead, showed irregular surface of the forestomach. All animals found dead showed beginning or advanced autolysis.
No abnormalities were found at macroscopic post mortem examination of the animals at 300 mg/kg.

Other findings:

- Organ weights: No data available.
- Histopathology: No data available.
- Potential target organs: No data available.
- Other observations: No data available.

Results are shown in the attachments.

Table 1: Mortality

Table 2: Clinical signs

Table 3: Body weights

Table 4: Macroscopic findings

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

Migrated information

Conclusions:

The oral LD50 value of the test substance in Wistar rats was established to be within the range of 300- 2000 mg/kg body weight. According to the OECD 423 test guideline the LD50 cut-off value was considered to be 500mg/kg body weight.

Executive summary:

In an acute oral toxicity study, the oral LD50value of the test substance in Wistar rats was established to be within the range of 300- 2000 mg/kg body weight. According to the OECD 423 test guideline the LD50 cut-off value was considered to be 500mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

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:

24 September 2014 to 16 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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:

ANIMALS AND ANIMAL HUSBANDRY
- Five male and five female Wistar (RccHan:WIST) strain rats were supplied by Harlan Laboratories UK Ltd, Oxon, UK.
- On receipt the animals were randomly allocated to cages.
- Female rats were nulliparous and non-pregnant.
- After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card.
- At the start of the study the animals weighed at least 200 g and were 8 to 12 weeks of age.
- The weight variation did not exceed ± 20 % of the mean weight for each sex.
- Animals were housed in suspended solid floor polypropylene cages furnished with woodflakes.
- The animals were housed individually during the 24-hour exposure period and in groups of five, by sex, for the remainder of the study.
- Free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories Ltd, Oxon, UK) was allowed throughout the study.
- Diet, drinking water and bedding were routinely analysed and were not considered to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Temperature and relative humidity were set to achieve limits of 19 to °C and 30 to 70 % respectively.
- Rate of air exchange was at least 15 changes per hour.
- Lighting was controlled by a time switch to give 12 hours continuous light (06:00 to 18:00) and 12 hours darkness.
- The animals were provided with environmental enrichment items which were not considered to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Type of coverage:

semiocclusive

Vehicle:

arachis oil

Details on dermal exposure:

TEST ITEM FORMULATION AND EXPERIMENTAL PREPARATION
- The test item was weighed out according to each animal's individual body weight and moistened with arachis oil BP prior to application.
- Absorption of the test item was not determined.

PROCEDURE
- On the day before treatment the back and flanks of each animal were clipped free of hair.
- Using available information on the toxicity of the test item, a single group of animals was treated with the test item at a dose level of 2000 mg/kg.
- The appropriate amount of test item, moistened with arachis oil, was applied as evenly as possible to an area of shorn skin (approximately 10 % of the total body surface area) using a graduated syringe.
- A piece of surgical gauze was placed over the treatment area and semi-occluded with a piece of self-adhesive bandage.
- Animals were caged individually for the 24-hour exposure period.
- Shortly after dosing the dressings were examined to ensure that they were securely in place.
- After the 24-hour contact period the bandage was carefully removed and the treated skin and surrounding hair were wiped with cotton wool moistened with arachis oil BP to remove any residual test item.
- As no mortalities were noted a further group of animals (four males and four females) was similarly treated with the test item at a dose level of 2000 mg/kg body weight to give a total of five males and five females. After the 24-Hour contact period the bandages were carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with arachis oil BP to remove any residual test item. These animals were returned to group housing for the remainder of the test period.
- The animals were observed for deaths or overt signs of toxicity 0.5, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.
- After removal of the dressings and subsequently once daily for fourteen days, the test sites were
examined for evidence of primary irritation and scored according to the scale shown below. Any other skin reactions were also recorded.
- Individual body weights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.
- At the end of the study the animals were killed by cervical dislocation.
- All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

Five males and five females

Control animals:

no

Statistics:

DATA EVALUATION
- Data evaluations included the relationship, if any, between the exposure of the animal to the test item and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, body weight changes, mortality and any other toxicological effects.
- Using the mortality data from the study, an estimate of the acute dermal median lethal dose (LD50) was made for the test item.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

- Individual mortality data are given in Table 1 (attached).
- No unplanned animal deaths took place during the study.

Clinical signs:

- Individual clinical observations are given in Table 1 (attached).
- No signs of systemic toxicity were noted during the observation period.

Body weight:

- Individual body weights and body weight changes are given in Tables 4 (attached).
- Animals showed expected gains in body weight, except for one female which showed body weight loss during the first week but expected gain in body weight during the second week.

Gross pathology:

- Individual necropsy findings are given in Table 5 (attached).
- No abnormalities were noted at necropsy.

Other findings:

- Individual dermal reactions are given in Tables 2 and 3 (attached).
- Very slight erythema and very slight edema were noted at the test sites of the initial two treated animals up to two days after dosing. There were no signs of dermal irritation noted at the test sites of the additional eight treated animals.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be > 2000 mg/kg bw.

Executive summary:

GUIDELINE

The study was performed to assess the acute dermal toxicity of the test item in the Wistar strain rat in compliance with the OECD Guideline for the Testing of Chemicals No 402 “Acute Dermal Toxicity” (adopted 24 February 1987) and Method B.3 Acute Toxicity (Dermal) of Commission Regulation (EC) No 440/2008.

 

METHODS

Initially, two animals (one male and one female) were given a single, 24 hour, semi-occluded dermal application of the test item to intact skin at a dose level of 2000 mg/kg body weight. Based on the results of the initial test, a further group of eight animals (four males and four females) was similarly treated. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

 

RESULTS

No animal deaths took place during the study and there were no signs of systemic toxicity. Very slight erythema and very slight edema were noted at the test sites of the initial two treated animals up to two days after dosing. There were no signs of dermal irritation noted at the test sites of the additional eight treated animals. Animals showed expected gains in body weight, except for one female which showed body weight loss during the first week but expected gain in body weight during the second week. No abnormalities were noted at necropsy.

 

CONCLUSION

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Oral

 

The key study assessed acute oral toxicity in the rat using the Acute Toxic Class Method (OECD 423 and EU Method B.1 tris).

Initially, test item was administered by oral gavage to three female Wistar rats at 2000 mg/kg bw. In a stepwise procedure, additional groups of females were dosed at 2000, 300 and 300 mg/kg bw. All animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15).

Five females were found dead on Days 9, 12 or 13. No further mortality occurred.

Clinical signs observed during the study period were lethargy, hunched posture, watery discharge of the eyes, uncoordinated movements, ptosis, piloerection, lean appearance and/or hypothermia in all animals in the 2000 mg/kg bw dose group. Clinical signs observed in animals receiving 300 mg/kg bw were hunched posture and piloerection between Days 1 and 7.

The mean body weight gain shown by the animals at 300 mg/kg bw over the study period was considered to be similar to that expected of normal, untreated, animals of the same age and strain.

Emaciation, dark red discolouration of the thymus and reduced size of the thymus was noted in one surviving animals at 2000 mg/kg bw. One animal, which was found dead, showed irregular surface of the forestomach. All animals found dead showed beginning or advanced autolysis. No abnormalities were found at macroscopic post-mortem examination of animals at 300 mg/kg bw.

The oral LD50 value of the test item in Wistar rats was established to be within the range of 300 to 2000 mg/kg bw. 

 

Inhalation

 

The substance is a low melting point solid with a vapour pressure of 0.078 Pa at 25 °C and it is expected that inhalation exposure from identified uses will be low. In addition, the most likely route of exposure for workers and consumers is the dermal route. However, classification for acute inhalation toxicity (category 4) has been adopted as a precautionary approach.

 

Dermal

 

The study was performed to assess the acute dermal toxicity of the test item in the Wistar strain rat in compliance with the OECD Guideline for the Testing of Chemicals No 402 “Acute Dermal Toxicity” (adopted 24 February 1987) and Method B.3 Acute Toxicity (Dermal) of Commission Regulation (EC) No 440/2008.

 

Initially, two animals (one male and one female) were given a single, 24 hour, semi-occluded dermal application of the test item to intact skin at a dose level of 2000 mg/kg body weight. Based on the results of the initial test, a further group of eight animals (four males and four females) was similarly treated. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

 

No animal deaths took place during the study and there were no signs of systemic toxicity. Very slight erythema and very slight edema were noted at the test sites of the initial two treated animals up to two days after dosing. There were no signs of dermal irritation noted at the test sites of the additional eight treated animals. Animals showed expected gains in body weight, except for one female which showed body weight loss during the first week but expected gain in body weight during the second week. No abnormalities were noted at necropsy.

 

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be > 2000 mg/kg bw.

Justification for classification or non-classification

Oral

The LD50 value reported following administration via the oral route was 300-2000 mg/kg bw in the rat. Classification for acute oral toxicity (category 4) is therefore applicable under the terms of Regulation (EC) 1272/2008.

 

Dermal

The LD50 value reported following administration via the dermal route was > 2000 mg/kg bw in the rat. Classification for acute dermal toxicity is therefore unnecessary under the terms of Regulation (EC) 1272/2008.

Inhalation route: The substance is a low melting point solid with a vapour pressure of 0.078 Pa at 25 °C and it is expected that inhalation exposure from identified uses will be low. In addition, the most likely route of exposure for workers and consumers is the dermal route. However, classification for acute inhalation toxicity (category 4) has been adopted as a precautionary approach.