4,4'-Methylenedianiline, oligomeric reaction products with 1-chloro-2,3-epoxypropane

Endpoint summary

Description of key information

The test item 4,4’-methylenebis[N,N-bis(2,3-epoxypropyl)aniline], was administered daily to Sprague-Dawley rats, by oral gavage, at dose-levels of 10, 50 or 200 mg/kg/day for 13 weeks.
Under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 50 mg/kg/day (based on clinical signs, decreased mean body weight and, hematology and clinical biochemistry findings at 200 mg/kg/day). The toxic effects >=200 mg/kg are considered to be caused by local (irritative, corrosive) effects of the test material to the gastrointestinal tract.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

4,4'-methylenebis[N,N-bis(2,3-epoxypropyl)aniline] is methylenebisbenzenamine with four epoxy groups and is considered as a monoconstituent substance under REACH with a purity > 80% and containing the following impurities: triglycidyl-(2-hydroxy-3-chloropropyl)-methylenedianiline in a range of 3.5 to 4%, dimeric TGMDA in a range of 3 to 5%, triglycidyl-(2,3-dihydroxypropyl)-methylenedianiline in a range of 0.3 to 1%, triglycidyl-(2-hydroxypiperidine)-methylenediamine in a range of 0.5 to 1%, triglycidyl methylenedianiline in a range of 0.3 to 0.8%, (2-hydroxy-3-chloropropyl)-dimeric TGMDA in a range of 0.2 to 0.6%, and trimeric TGMDA in a range of 0.2 to 0.5%. The hypothesis is to read-across some data from the monoconstituent substance described above to the corresponding UVCB substance as described under REACH. The UVCB substance being chemically similar and just differ from the monoconstituent substance by the purity of the main constituent which is < 80% but still stay in the same order of magnitude. The impurities from the monoconstituent substance being also present in the same order of magnitude in the UVCB substance containing also two additional substances, 2-oxiranemethanamine, %{N}-[4-[[4-[[3-chloro-2-(oxiranylmethoxy)propyl](oxiranylmethyl)amino]phenyl]methyl]phenyl]-%{N}-(oxiranylmethyl)- in a range of 0.5 to 2 % and less than 1% 1-[(4-{4-[bis(oxiran-2-ylmethyl)amino]benzyl}phenyl)(oxiran-2-ylmethyl)amino]-3-[(4-{4-[(3-chloro-2-hydroxypropyl)(oxiran-2-ylmethyl)amino]benzyl}phenyl)(oxiran-2-ylmethyl)amino]propan-2-ol. The main assumption is that the minor differences in terms of percentage between the mono and UVCB substance are not significant in respect of all properties under consideration. The full report on read-across approach is attached in section 13 of the IUCLID file.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
-Number: 87 rats (43 males and 44 females).
-Strain and Sanitary status: Sprague-Dawley, Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier
Sustained-Virus Antibody Free (COBS-VAF®).
-Age/weight: On the first day of treatment, the animals were approximately 6 weeks old. The males
had a mean body weight of 229 g (range: 197 g to 379 g) and the females had a mean body weight
of 172 g (range: 147 g to 201 g).
-Acclimation: the animals were acclimated to the study conditions for a period of 9 days before the beginning of the treatment period.
Environmental conditions
. temperature : 22 ± 2°C,
. relative humidity : 50 ± 20%,
. light/dark cycle : 12h/12h,
. ventilation : approximately 12 cycles/hour of filtered, non-recycled air.
Housing
-The animals were housed in pairs, by sex and group, in polycarbonate cages with stainless steel
lids (Tecniplast 2000P, 2065cm²) containing autoclaved sawdust (SICSA, Alfortville, France).
Each cage contained an object for the environmental enrichment of the animals (rat hut).
The cages were placed in numerical order on the racks. Every 9 weeks, all the racks were moved
clockwise around the room, rack by rack. In this way, for each group, identical exposure to
environmental conditions was achieved.
Food and water
-All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch Nos. 9827489 and
2537604 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly. The diet
formula is presented in Appendix 3. The animals had free access to bottles containing tap water
(filtered with a 0.22 μm filter). During periods of fasting, food, but not water, was removed.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400 batchN°MKBF9334V and MKBG6036V.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Daily for a period of 13 weeks

VEHICLE
- Justification for use and choice of vehicle (if other than water):PEG 400
- Concentration in vehicle: 2mg/l, 10 mg/l, 40mg/l
- Amount of vehicle (if gavage): 5 ml/kg/day
- Lot/batch no. (if required): batchN°MKBF9334V and MKBG6036V.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test item in samples of each control and test item dose formulation prepared for use in week 1, 4, 8, and 12 was detremined.
Acceptance criterion:
-Measured concentration=nominal concentration +/- 15%

Duration of treatment / exposure:

The dose formulations were administred daily for a period of 13 weeks (i.e. 92 (males) or 90 (females and male A 20491) day according to the necropsy schedule). Day 1 corresponds to the first day of the treatment.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
10 mg/kg, 50 mg/kg and 200 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

- 10 animals per sex in the control.
- 10 animals per sex for each concentrations

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected in agreement with the sponsor, based on the resullts of a previous 4-week preliminary toxicity study by the oral route (gavage) in rats (CiToxLAB France/Study N° 38871 TSR).

Positive control:

No positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality & morbidity: twice daily
general clinical signs: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: weekly


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes



OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule and dose groups: prior to treatment - all animals
week 13 - control and high dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of teh treatment
- Anaesthetic used for blood collection: Yes (Light isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: All
- Parameters were examined: Erytthrocytes, mean cell volume, packed cell volume, hemoglobin, mean cell hemoglobin concentration, thrombocytes, leucocytes, differential white cell count with cell morphology (neutrphiles, eosinophile, basophils, lymphocytes and large unstained cells and monocytes)

BLOOD BIOCHEMISTRY: Yes
- Time schedule for collection of blood: End of the treatment
- Animals fasted: Yes
- How many animals: All
- Parameters checked: Sodium, Potassium, Chloride , Calcium, Inorganic phosphorus and Glucose.

URINALYSIS: Yes
- Time schedule for collection of urine: End of the treatment period
- Animals fasted: Yes
- Parameters checked:
-Quantitative parameters: Volume, pH and specific garvity).
.-Semi-quantitative parameters: Protein, glucose, ketones, biliburin, nitrites, blood, urobilinogen, cytology of sediment (leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals and epithelial cells).
-Qualitative parametres: Appearance and color.
REACTIVITY TO MANIPULATION OR TO DIFFERENT STIMULI EXAMINATION: Yes


- Battery of functions tested: motor activity
other: Touch response, forelimb grip strength, pupillary reflex, visual stimulus, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, and rectal temperature (at the end of teh observation period)

Detailed obsrvations:
-In the cage:"touch escape"
-In the hand: fur apprearance, salivation, lacrimation, piloerection, exophtalmos, reactivity to handling, pupil size
-In the standard arena: grooming, palpebral closure, defecation, urination, tremors, twitches, convulsions, gait, arousal (hypo and hyper-activity), posture, stereotypic bahavior, breathing, ataxia and hypotonia.

OTHER:SEMINOLOGY:
-Before sacrifice at the end of the treatment period, each male was anesthetized by an intraperitoneal injection of sodium pentobarbital.
Epididymal sperm:
-The left epididymis was removed, weighed and sperm from the cauda was sampled for motility
and morphology investigations. Animals were then sacrificed.
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at -20°C pending further investigation.

Sperm motility:
-The sperm was evaluated on a slide, after appropriate dilution. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40-fold magnification. Results are expressed as the proportion of motile and non-motile spermatozoa.

Sperm morphology:
-The morphology was determined from a sperm smear, after eosin staining and counting of
100 spermatozoa per slide. Results are expressed as the proportion of spermatozoa in each of the
following categories:
. normal,
. normally shaped head separated from flagellum,
. abnormal head separated from flagellum,
. abnormal head with normal flagellum,
. abnormal head with abnormal flagellum,
. abnormal flagellum with normal head.

Sperm count:
-After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber.
Results are expressed as the number of spermatozoa per cauda and per gram of cauda.

Testicular sperm head count:
-The left testis was weighed and ground. The resulting preparation was diluted and sperm heads
resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted in a Neubauer cell.
Results are expressed as the number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10).

Sacrifice and pathology:

Sacrifice:
-On completion of the treatment period, after 14 hours fasting, all animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrified by exsanguination.
Organ weight:
-The body weight of each animal was recorded before sacrifice at the end of the treatment period.

Macroscopic post-mortem examination:
-A complete macroscopic post-mortem examination was performed on all animals. This included
examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the
brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and
tissues and the neck with its associated organs and tissues.

Preservation of tissues:
-For all study animals, the tissues specified in the Tissue Procedures Table were preserved in
10% buffered formalin (except for the eyes and optic nerves and Harderian glands, and the testes
and epididymides which were fixed in Modified Davidson's Fixative).
A bone marrow smear for the determination of the bone marrow differential cell count (1)
(see § Bone marrow) was prepared from the femur of each animal sacrificed on completion of the
treatment period.

Preparation of histological slides
-All tissues required for microscopic examination were trimmed according to the RITA guidelines,
when applicable, embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with
hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS).
A toluidine blue special stain was performed for all mesenteric lymph nodes.
The tissue processing was performed at ProPath under the responsibility of CiToxLAB France.
Details concerning the conduct of the delegated phase are presented in Appendix 20.

Microscopic examination:
-A microscopic examination was performed on all tissues listed in the Tissue Procedure Table for
animals of the control and high-dose groups (groups 1 and 4).
In addition testicular staging was performed for control and high-dose males (groups 1 and 4).
A detailed examination of the testes was performed, using a thorough understanding of tubule
development through the different stages of the spermatogenic cycle. Transverse sections of the
testes were stained with hematoxylin-PAS in order to detect retained spermatids, missing germ
cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc..
Following microscopic examination of tissues from animals of the control and high-dose groups
(groups 1 and 4), a microscopic examination (hematoxylin-eosin stain) was also performed on:
. the liver from low- and intermediate-dose (groups 2 and 3) females,
. the mesenteric lymph node from low- and intermediate-dose (groups 2 and 3) males and
females,
. the stomach from low- and intermediate-dose (groups 2 and 3) males and females,
. the duodenum from low- and intermediate-dose (groups 2 and 3) males and females.
A special stain "Toluidin blue" was used for the mesenteric lymph nodes from all animals to allow
the characterization and a semi-quantification of the mast cells in this organ.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mortality: No, clinical signs: Yes.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality: No, clinical signs: Yes.

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

The test item concentrations in the administered dose formulations analyzed in weeks 1, 4, 8 and 12 remained within an acceptable range of variations when compared to the nominal values.

Mortality:
-There were no test item treatment-related deaths.

Clinical signs:
_In the animals treated at 200 mg/kg/day, hunched posture (from week 8 in males and on week 12 in one female), reflux at dosing (one male on week 4), piloerection (from week 6 in males) and loud breathing (one male on week 7) were considered to be test item treatment-related.

Functional Observation Battery:
-Detailed clinical examination: rectal temperature was slightly decreased at 200 mg/kg/day. With the exception of dose-related abnormal fur appearance in both sexes from 10 mg/kg/day, there were no treatment-related findings.

Reactivity to stimuli and reflexes: there were no abnormal responses or findings.

Motor activity: there were no toxicologically significant findings.

Mean body weights and mean body weight changes:
-When compared with controls, there were statistically significant lower mean body weights from 50 mg/kg/day in females and at 200 mg/kg/day in males. Taking into account the amplitude of the changes, these decreases were considered of toxicological significance in both sexes at
200 mg/kg/day.

Food consumption: There were no effects on mean food consumption.

Ophthalmology: There were no ophthalmological findings at the end of the treatment period.

Estrous cycle: There were no effects on estrous stages.

Hematology:
-When compared with controls, there were statistically significant lower mean increased Hemoglobin concentration, White blood cells, Basophils and Lymphocytes count in males given 200 mg/kg/day. Theses findings were considered to be related to the test-item
treatment.

Blood biochemistry:
-Taking into account the amplitude of the changes and/or the presence of similar finding in the opposite sex, the increased inorganic phosphorus level (both sexes), decreased creatinine, glucose (males), total protein and albumin levels (males), increased urea level (males), and increased total cholesterol levels (females) were considered to be of toxicological significance at 200 mg/kg/day.

Thyroid investigations: awaiting decision from the Sponsor in light of study results

Urinalysis: there were no effects on mean urinalysis parameters.

Pathology
Seminology: there were no effects on mean sperm cells parameters (count, motility and
morphology), testicular sperm heads count and daily testicular production rate.

Organ weights: the relative liver weights were significantly higher in females given 200 mg/kg/day.

Macroscopic post-mortem examination: the red discoloration observed in the mesenteric lymph node from most males and females treated at 200 mg/kg/day and the distented colon seen in occasional males and females treated at 50 or 200 mg/kg/day were considered to be related to the
test item administration.

Microscopic examination:
-At microscopic examination, test item-related non adverse microscopic findings were seen in the liver (hepatocellular hypertrophy, increased glycogen storage in females treated at 200 mg/kg/day), mesenteric lymph node (sinusal erythrocytes, pigmented macrophages and mast cells infiltrates in males and females treated at 50 or 200 mg/kg/day), stomach (decreased goblet cell numbers and gland elongation in males and females treated at
200 mg/kg/day) and duodenum (basophilia and atrophy of villi in males and females treated at 200 mg/kg/day).

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Based on clinical signs, decreased mean body weight and, hematology and clinical biochemistry indings at 200 mg/kg/day).

Critical effects observed:

not specified

Conclusions:

The test item 4,4’-methylenebis[N,N-bis(2,3-epoxypropyl)aniline], was administered daily to Sprague-Dawley rats, by oral gavage, at dose-levels of 10, 50 or 200 mg/kg/day for 13 weeks. Under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 50 mg/kg/day (based on clinical signs, decreased mean body weight and, hematology and clinical biochemistry indings at 200 mg/kg/day).

Executive summary:

The objective of this study, (According to the OECD 408), was to evaluate the potential toxicity of the test item, 4,4’-methylenebis[N,N-bis(2,3-epoxypropyl)aniline], following daily oral administration (gavage) to rats for 13 weeks.

4,4’-methylenebis[N,N-bis(2,3-epoxypropyl)aniline] (batch No. AAB0290400), at dose-levels of 10, 50 or 200 mg/kg/day. The test item was administered during a 13-week period by gavage under a constant dosage-volume of 5 mL/kg/day. In addition, one group of ten males and ten females received the vehicle, PEG 400, and acted as a control group.

The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed once before the beginning of the treatment period and then once a week. A Functional Observation Battery (FOB) was performed for all animals once at the end of the treatment period. Body weight and food consumption were recorded at least once a week during the study.

Ophthalmological examinations were performed on all animals before the beginning of the treatment period and in control and high-dose groups on completion of the treatment period. Hematology, blood biochemistry and urinalysis were performed on all animals at the end of the treatment period. Seminology investigations in all males were performed at the end of the treatment period. The estrous cycle stage was determined in all females daily for 5 consecutive days at the end of the treatment period. On completion of the treatment period, the animals were sacrificed and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from all animals from groups 1 and 4 and on all macroscopic lesions.

The test item concentrations in the administered dose formulations analyzed in weeks 1, 4, 8 and 12 remained within an acceptable range of variations when compared to the nominal values.

Mortality: There were no test item treatment-related deaths.

Clinical signs: In the animals treated at 200 mg/kg/day, hunched posture (from week 8 in males and on week 12 in one female), reflux at dosing (one male on week 4), piloerection (from week 6 in males) and loud breathing (one male on week 7) were considered to be test item treatment-related.

Functional Observation Battery: Detailed clinical examination: rectal temperature was slightly decreased at 200 mg/kg/day. With

the exception of dose-related abnormal fur appearance in both sexes from 10 mg/kg/day, there were no treatment-related findings.

Reactivity to stimuli and reflexes: there were no abnormal responses or findings.

Motor activity: there were no toxicologically significant findings.

Mean body weights and mean body weight changes: When compared with controls, there were statistically significant lower mean body weights from 50 mg/kg/day in females and at 200 mg/kg/day in males. Taking into account the amplitude of the changes, these decreases were considered of toxicological significance in both sexes at 200 mg/kg/day.

Food consumption:There were no effects on mean food consumption.

Ophthalmology: There were no ophthalmological findings at the end of the treatment period.

Estrous cycle:There were no effects on estrous stages.

Hematology: when compared with controls, there were statistically significant lower mean increased Hemoglobin concentration, White blood cells, Basophils and Lymphocytes count in males given 200 mg/kg/day. Theses findings were considered to be related to the test-item treatment.

Blood biochemistry: taking into account the amplitude of the changes and/or the presence of similar finding in the opposite sex, the increased inorganic phosphorus level (both sexes), decreased creatinine, glucose (males), total protein and albumin levels (males), increased urea level (males), and increased total cholesterol levels (females) were considered to be of toxicological significance at 200 mg/kg/day.

Thyroid investigations: awaiting decision from the Sponsor in light of study results

Urinalysis: there were no effects on mean urinalysis parameters.

Seminology: there were no effects on mean sperm cells parameters (count, motility and morphology), testicular sperm heads count and daily testicular production rate.

Organ weights: the relative liver weights were significantly higher in females given200 mg/kg/day.

Macroscopic post-mortem examination: the red discoloration observed in the mesenteric lymph node from most males and females treated at 200 mg/kg/day and the distented colon seen in occasional males and females treated at 50 or 200 mg/kg/day were considered to be related to the test item administration.

Microscopic examination: at microscopic examination, test item-related non adverse microscopic findings were seen in the liver (hepatocellular hypertrophy, increased glycogen storage in females treated at 200 mg/kg/day), mesenteric lymph node (sinusal erythrocytes, pigmented macrophages and mast cells infiltrates in males and females treated at 50 or 200 mg/kg/day), stomach

(decreased goblet cell numbers and gland elongation in males and females treated at 200 mg/kg/day) and duodenum (basophilia and atrophy of villi in males and females treated at 200 mg/kg/day).

Conclusion

The test item 4,4’-methylenebis[N,N-bis(2,3-epoxypropyl)aniline], was administered daily toSprague-Dawley rats, by oral gavage, at dose-levels of 10, 50 or 200 mg/kg/day for 13 weeks.Under the experimental conditions of this study, the No Observable Adverse Effect Level(NOAEL) was considered to be 50 mg/kg/day (based on clinical signs, decreased mean body weight and, hematology and clinical biochemistry indings at 200 mg/kg/day).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the above assement on oral repeated dose toxicity, the substance does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council) as implementation of UN-GHS in the EU.