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Description of key information

In an acute oral toxicity test performed according to OECD guideline 423 and GLP principles, the oral LD50 of 4,4’-methylenebis-N-sec-butylaniline was found to be > 300 mg/kg bodyweight and < 2000 mg/kg bodyweight. An acute dermal study was performed according to OECD guideline 402 and GLP principles, the dermal LD50 value of 4,4’-methylenebis-N-sec-butylaniline in Wistar rats was determined to exceed 2000 mg/kg bodyweight. Both studies have Klimisch reliability 1.

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:
13 February 2018 - 20 March 2018
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:
December 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
December 2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.
Version / remarks:
November 2000
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Specific details on test material used for the study:
Adjustment was made for specific gravity of the test item.
Specific density: 0.99 g/cm3
Species:
rat
Strain:
other: Crl:WI (Han)
Sex:
female
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Young adult animals (approx. 8-10 weeks old)
- Weight at study initiation: 139 to 187 g.
- Fasting period before study: yes
- Housing: Group housing of 3 animals per cage in labeled polycarbonate Macrolon cages containing sterilized sawdust as bedding material. For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 21
- Humidity (%): 26 - 51%
- Air changes (per hr): ten or more
- Photoperiod (hrs dark / hrs light): 12/12

Deviations from the minimum level of daily mean target humidity occurred (lowest value 26%). This study plan deviation is considered not to have affected the integrity of the study because it did not noticeably affect the clinical condition of the animals or the outcome of the study.

IN-LIFE DATES: From: 13 february 2018 to 20 March 2018
Route of administration:
oral: gavage
Details on oral exposure:
GAVAGE METHOD: syringe with a plastic gavage cannula

Frequency: single dosage, on day 1.

MAXIMUM DOSE VOLUME APPLIED:
2000 mg/kg body weight.

- Rationale for the selection of the starting dose: maximum recommended dose according to OECD 423.


Doses:
2000 mg/kg body weight
300 mg/kg body weight

No. of animals per sex per dose:
2000 mg/kg: 6 (2 groups of three females in a stepwise manner)
300 mg/kg: 6 (2 groups of three females)
Control animals:
no
Details on study design:
The toxicity of the test item was assessed by stepwise treatment of groups of 3 females. 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. The first group was treated at a dose level of 2000 mg/kg. Based on the results, three additional groups were dosed at 2000, 300 and 300 mg/kg.

Animals were deprived of food overnight prior to dosing and until 3-4 hours after administration of the test substance. Water was available ad libitum.

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Postdose observations were performed at periodic intervals on the day of dosing (at least three times) and once daily thereafter.

Mortality/Viability: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

Body weights: Animals were weighed individually on day 1 (predose), 8 and 15.

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

- Necropsy of survivors performed: All moribund animals and animals surviving to the end of the observation period were sacrificed by oxygen/carbon dioxide procedure. All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded.

- Other examinations performed: none.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - < 2 000 mg/kg bw
Based on:
test mat.
Mortality:
At 2000 mg/kg, all animals were sacrificed for humane reasons between days 8 and 11.
At 300 mg/kg, no mortality occurred.
Clinical signs:
At 2000 mg/kg, in the first dose group, hunched posture and piloerection were noted on day 1 and ventro-lateral recumbency, hunched posture, decreased locomotor activity, piloerection, lean appearance and/or ptosis were noted from day 5 onwards.
At 2000 mg/kg, in the second dose group, hunched posture, uncoordinated movements, decreased locomotor activity and/or piloerection were noted from day 1 onwards.
At 300 mg/kg, hunched posture, uncoordinated movements and/or piloerection were noted for the animals between days 1 and 2. In addition, quick breathing and chromodacryorrhoea (nose) were noted for one animal on Day 1.
Body weight:
No abnormalities
Gross pathology:
At 2000 mg/kg, abnormalities of the thymus (reduced in size), liver (discolouration, pale) and stomach (irregular surface of the forestomach) and general emaciation were found for the animals that were sacrificed for humane reasons during the study, at macroscopic examination.

At 300 mg/kg abnormalities of the thymus (many tan foci) were noted for one animal. Macroscopic post mortem examination of the other animals did not reveal any abnormalities.

According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 500 mg/kg body weight.

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
In an acute oral toxicity test, the oral LD50 of 4,4’-methylenebis-N-sec-butylaniline was found to be > 300 mg/kg bodyweight and < 2000 mg/kg bodyweight.
Executive summary:

An acute oral toxicity test was performed according to OECD guideline 423 and GLP principles. Three female Wistar rats were exposed to 2000 mg/kg body weight 4,4’-methylenebis-N-sec-butylaniline.  In a stepwise procedure three additional groups of three females were dosed at 2000, 300 and 300 mg/kg body weight. At 2000 mg/kg, in the first dose group, hunched posture and piloerection were noted on day 1 and ventro-lateral recumbency, hunched posture, decreased locomotor activity, piloerection, lean appearance and/or ptosis were noted from day 5 onwards. At 2000 mg/kg, in the second dose group, hunched posture, uncoordinated movements, decreased locomotor activity and/or piloerection were noted from day 1 onwards. At 300 mg/kg, hunched posture, uncoordinated movements and/or piloerection were noted for the animals between days 1 and 2. In addition, quick breathing and chromodacryorrhoea (nose) were noted for one animal on day 1. At 2000 mg/kg, all animals were sacrificed for humane reasons between days 8 and 11. At 300 mg/kg, no mortality occurred. No abnormalities in weight were observed in all dose groups. Necroscopy performed on 2000 mg/kg dose groups showed abnormalities at the thymus (reduced in size), liver (discolouration, pale) and stomach (irregular surface of the forestomach) and general emaciation were found for the animals that were sacrificed for humane reasons during the study, at macroscopic examination. At 300 mg/kg, abnormalities of the thymus (many tan foci) were noted for one animal. Macroscopic post mortem examination of the other animals did not reveal any abnormalities.

Based on these results, the oral LD50 of 4,4’-methylenebis-N-sec-butylaniline was found to be > 300 mg/kg bodyweight and therefore  according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and  according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), 4,4’-methylenebis-N-sec-butylaniline should be classified as Category 4 and should be labeled as H302: Harmful if swallowed.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
300 mg/kg bw
Quality of whole database:
The study was performed according to OECD/EC guidelines and GLP principles.

Acute toxicity: via inhalation route

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:
22 March 2018 - 23 April 2018
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:
2017
Deviations:
no
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
yes
Specific details on test material used for the study:
Adjustment was made for specific gravity of the test item.
Species:
rat
Strain:
other: Crl: WI(Han)
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant:yes
- Age at study initiation: approx. 10 - 12 weeks old
- Weight at study initiation: Females: 191 - 226 g.
- Housing: During the acclimatization period the animals were group housed in Makrolon cages (MIV type, height 18 cm) containing sterilized sawdust as bedding material and paper as cage-enrichment. During the study the animals were individually housed in Makrolon cages (MIII type, height 18 cm.).
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

IN-LIFE DATES: From: 26 March 2018 To: 23 April 2018

Periodic analysis of the water was performed and feed was analyzed by the supplier for nutritional components and environmental contaminants.
It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 43 - 51
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: 18 cm^2
- % coverage: approx. 10% of the total body surface
- Type of wrap if used: a surgical gauze patch (Surgy 1D), successively covered with Coban elastic bandage. A piece of Micropore tape was additionally used for fixation of the bandages

REMOVAL OF TEST SUBSTANCE
- Washing: tap water
- Time after start of exposure: 24 hours


Duration of exposure:
24 hours
Doses:
2000 mg/kg bodyweight
No. of animals per sex per dose:
Main study: 2 females at 2000 mg/kg bodyweight
Range finding study: 1 female at 1000 mg/kg bodyweight and 1 female at 2000 mg/kg bodyweight
Control animals:
not required
Details on study design:
RANGEFINDING STUDY:
A range finding study was performed in order to select the dose causing no mortality or significant toxicity to be used in the main study. Initially, one animal was dosed at 1000 mg/kg bodyweight. A period of at least 48 hours was allowed between the dosing of each animal. Based on the results one additional animal was dosed at 2000 mg/kg.

MAIN STUDY:
- Duration of observation period following administration: 14 days
- Frequency of observations:
- Mortality: twice daily
- Clinical observations: at periodic intervals on the day of dosing and once daily thereafter.
- Body weights: on day 1 (pre-administration), day 8 and day 15.
- Irritation: The skin reactions were assessed approximately 24, 48 and 72 hours after the removal of the dressing and test item. Adjacent areas of untreated skin of each animal served as controls.
- Necropsy of survivors performed: yes


Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred.
Clinical signs:
No clinical signs were noted in any of the animals.
Body weight:
The mean body weight gain shown by the animals during the observation period was within the range expected for rats used in this type of study.
Gross pathology:
No abnormalities were seen at necropsy.
Other findings:
Irritation effects: General erythema of the right flank was noted for one animal on day 4.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of an acute dermal study, performed according to OECD guideline 402 and GLP principles, the dermal LD50 value of 4,4’-methylenebis-N-sec-butylaniline in Wistar rats was determined to exceed 2000 mg/kg bodyweight. As a consequence, 4,4’-methylenebis-N-sec-butylaniline is not classified according to CLP criteria.
Executive summary:

An acute dermal study was performed according to OECD guideline 402 and GLP principles. two female rats were exposed at test item concentration of 2000 mg/kg bodyweight and observed for 14 days. No mortality occurred. General erythema of the right flank was noted for one animal on day 4 only. No unexpected changes in body weight gain occurred, no clinical signs were observed and no abnormalities were seen at necropsy. These results demonstrate that the dermal LD50 value of 4,4’-methylenebis-N-sec-butylaniline in Wistar rats exceeds 2000 mg/kg body weight. As a consequence, 4,4’-methylenebis-N-sec-butylaniline is not classified according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
The study was performed according to OECD/EC guidelines and GLP principles.

Additional information

An acute oral toxicity test was performed according to OECD guideline 423 and GLP principles. Three female Wistar rats were exposed to 2000 mg/kg body weight 4,4’-methylenebis-N-sec-butylaniline. In a stepwise procedure three additional groups of three females were dosed at 2000, 300 and 300 mg/kg body weight. At 2000 mg/kg, in the first dose group, hunched posture and piloerection were noted on day 1 and ventro-lateral recumbency, hunched posture, decreased locomotor activity, piloerection, lean appearance and/or ptosis were noted from day 5 onwards. At 2000 mg/kg, in the second dose group, hunched posture, uncoordinated movements, decreased locomotor activity and/or piloerection were noted from day 1 onwards. At 300 mg/kg, hunched posture, uncoordinated movements and/or piloerection were noted for the animals between days 1 and 2. In addition, quick breathing and chromodacryorrhoea (nose) were noted for one animal on day 1. At 2000 mg/kg, all animals were sacrificed for humane reasons between days 8 and 11. At 300 mg/kg, no mortality occurred. No abnormalities in weight were observed in all dose groups. Necroscopy performed on 2000 mg/kg dose groups showed abnormalities at the thymus (reduced in size), liver (discolouration, pale) and stomach (irregular surface of the forestomach) and general emaciation were found for the animals that were sacrificed for humane reasons during the study, at macroscopic examination. At 300 mg/kg, abnormalities of the thymus (many tan foci) were noted for one animal. Macroscopic post mortem examination of the other animals did not reveal any abnormalities. Based on these results, the oral LD50 of 4,4’-methylenebis-N-sec-butylaniline was found to be > 300 mg/kg bodyweight.

An acute dermal study was performed according to OECD guideline 402 and GLP principles. A range finding study was performed in order to select the dose causing no mortality or significant toxicity to be used in the main study. In the main study, two female rats were exposed at test item concentration of 2000 mg/kg bodyweight and observed for 14 days. No mortality occurred. General erythema of the right flank was noted for one animal on day 4 only. No unexpected changes in body weight gain occurred, no clinical signs were observed and no abnormalities were seen at necropsy. These results demonstrate that the dermal LD50 value of 4,4’-methylenebis-N-sec-butylaniline in Wistar rats exceeds 2000 mg/kg body weight.

Justification for classification or non-classification

Based on these results, the oral LD50 of 4,4’-methylenebis-N-sec-butylaniline was found to be > 300 mg/kg bodyweight and therefore according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and  according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), 4,4’-methylenebis-N-sec-butylaniline is classified for oral toxicity in Category 4 and should be labeled as H302: Harmful if swallowed.

The dermal LD50 of 4,4’-methylenebis-N-sec-butylaniline was found to be > 2000 mg/kg bodyweight, therefore 4,4’-methylenebis-N-sec-butylaniline is not classified for dermal toxicity according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

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