Registration Dossier

Administrative data

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:
experimental study
Adequacy of study:
key study
Study period:
12 September 2012 - 04 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
neurotoxicological investigations were based on the following guidelines:
. Commission Regulation (EC) No. 440/2008, B.43, 30 May 2008,
. USA EPA Health Effects Test Guideline OPPTS 870.3050, July 2000
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: approximately 6 weeks old on the day of treatment
- Mean body weight at study initiation: 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)
- Fasting period before study: no
- Housing: the animals were housed in pairs, by sex and group, in polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 9 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 20 September 2012 to 07 January 2013.
Route of administration:
oral: gavage
Vehicle:
other: PEG 400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was mixed with the required quantity of vehicle according to the process described in a previous homogeneity/stability study.

VEHICLE
- Justification for use and choice of vehicle:
- Concentration in vehicle: 2, 10 and 40 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: assessed to homogeneity study (satisfactory results)
Stability: assessed in stability study.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
10, 50 and 200 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, based on the results of a previous 4-week preliminary toxicity study by the oral route (gavage) in rats.

In this preliminary toxicity study, 12 males and 12 females Sprague-Dawley rats were allocated to 4 groups and received the test item or the vehicle (PEG 400) by oral route (gavage) once daily for a period of 28 days (with the exception of high-dose group where administrations were stopped on study day 15 in order to allow evaluation of recovery in two out of four surviving animals), at the following dosages: 100, 300 or 1000/750/400 mg/kg/day. A constant dosage-volume of 5 mL/kg/day was used, with the exception of occasional changes in the volume administered in the high-dose group (3.75 mL on study day 10 and 2.67 mL/kg on study day 13 to allow use of dose formulation already prepared on this day). Control animals (group 1) received the vehicle only.

The findings were the following:
Mortality: in the high-dose group, one male was found dead on day 15 and one female was prematurely sacrificed on day 11. Prior to their death, the following clinical signs were observed: ptyalism, hunched posture, piloerection, dyspnea, thin appearance, soiled head and forelimbs, and/or hypoactivity, pallor of eyes, half-closed eyes, and decreased grasping reflex.

Clinical signs: in the high-dose group, ptyalism, piloerection, hunched posture and thin appearance were observed in all males and females. Hypoactivity and half-closed eyes were also noted in all females. In the mid-dose group, all animals had piloerection and ptyalism. Hunched posture was also observed in all females. In the low-dose group, both males and females had piloerection and ptyalism was noted in all males.

Mean body weights and body weight changes: males from the high-dose group had a statistically significant lower mean body weight change for the period of days 1-8 ( 15 g versus +61 g in control animals). From day 22 (i.e. recovery period), both males and females gained weight (+50 g in males versus +28 g in controls and +47 g in females versus +10 g in controls). At 300 mg/kg/day, males had a statistically significant lower mean body weight change for the period of days 8-15 (-72% compared with control values), days 22-28 (-79% compared with control values) and for the whole study period, i.e. days 1-28 (-64% compared with control values). At 100 mg/kg/day, body weight was unaffected by the test item treatment.

Mean food consumption: in the high-dose group, lower mean food intake, which correlated with the effect observed on body weight, was noted in both sexes for the period of days 1-7 (-40% and 38% in males and females respectively, compared with control group) and days 8-14 (-69% and 72% in males and females respectively, compared with control group). From days 15 to 21 (i.e. recovery period), food consumption returned to normal for both males and females. At 300 mg/kg/day, slight lower food intake, which correlated with the effect observed on body weight, was recorded in males throughout the study period. In females, food consumption was not affected by the test item treatment. At 100 mg/kg/day, food consumption was not affected by the test item treatment.

Hematology: in males, from 300 mg/kg/day and in the surviving female in the 1000/750/400 mg/kg/day group, there were decreases in leucocytes, reticulocytes count, neutrophils percentage, eosinophils, basophils and lymphocytes counts. When compared with controls, all differences were not statistically significant but considered to be related to treatment with the test item.

Pathology: following daily oral administration of the test item at the dose-levels of 100 or 300 mg/kg/day to the rat, minimal to slight hepatocytic hypertrophy was seen in the liver which correlated with the higher weight noted at necropsy. At 300 mg/kg/day, single cell necrosis and hypertrophy of venous endothelial cells in portal tracts were seen in one male and glycogen in both sexes. At 1000 mg/kg/day minimal hepatocytic hypertrophy was seen in the liver of the male. In both animals, there was minimal hypertrophy of bile duct cells and of venous endothelial cells in portal tracts. In the male, minimal fusion of villi with increased number of nuclei was seen in the duodenum and eosinophilic material (minimal), consistent with fibrin was seen in the villi of the duodenum and jejunum. In the female, minimal superficial erosion was seen in the stomach and edema in the pancreas. Both these changes correlated with macroscopic changes seen at necropsy. Lymphoid atrophy, possibly in relationship with the poor status of the animals was seen in the thymus and the spleen of the female. This correlated with changes seen at necropsy.

Overall, 1000/750/400 mg/kg/day and 300 mg/kg/day were considered to be excessive dose levels, based on in-life and pathology data. Therefore, 200 mg/kg/day was selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing a 4- to 5 fold interval (i.e. 10 and 50 mg/kg/day).

- Animal assignment: computerized stratification procedure.
Positive control:
no (not required).
Observations and examinations performed and frequency:
MORTALITY/MORBIDITY:
- Time schedule: once a day during the acclimation period and at least twice a day during the treatment period.

CLINICAL SIGNS:
- Time schedule: each animal was observed at least once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT:
- Time schedule: once before the beginning of the treatment period, on the first day of treatment and then at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: once a week, over a 7-day period, during the study.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule: before the beginning of the treatment period (all animals) and on control and high-dose animals on one occasion at the end of the treatment period.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: once in week 11 (for all animals) or week 12 (for one male).

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS:
- Time schedule: at the end of the treatment period.
Sacrifice and pathology:
ORGAN WEIGHTS: see table below

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination of all study animals.

HISTOPATHOLOGY:
- on all tissues listed in the table below for the control and high dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- on all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period,
- 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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
ptyalism and hunched posture
Mortality:
mortality observed, treatment-related
Description (incidence):
ptyalism and hunched posture
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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):
effects observed, treatment-related
Description (incidence and severity):
treatment-related but of no toxicological importance
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY:
There were no test item treatment-related deaths.
On day 19 (week 3), one animal given 200 mg/kg/day (group 4M) was prematurely sacrificed because of an error of sexing. A male replaced this female animal from day 19.

CLINICAL SIGNS:
Ptyalism was considered to be related to the treatment with the test item, but of minor toxicological significance.

In the 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.
The others clinical signs are findings commonly observed in this species and strain.

BODY WEIGHT:
When compared with controls, there were statistically significant lower mean body weights and/or mean body weight changes 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.

FUNCTIONAL OBSERVATION BATTERY
Detailed clinical examination
All control and treated animals had normal touch escape response, no lacrimation, normal pupil size, no tremors, no twitches, no clonic/tonic convulsions, no hyperactivity, no ataxia, no hypotonia, normal gait, normal posture, no stereotypy, no abnormal behavior and normal breathing.
Rectal temperature was slightly decreased at 200 mg/kg/day (37.5°C in males vs. 38.5°C in controls and 38.7°C in females vs. 39.0°C in controls).

Reactivity to stimuli and reflexes
Visual stimulus response, pupillary reflex, auditory startle reflex, tail pinch response, righting reflex and landing foot splay were normal.

There were a few abnormal responses (touch response at 50 mg/kg/day in 2/10 males or forelimb strength at 200 mg/kg/day in 1/10 males). In the absence of any obvious dose-level relationship and of similar findings in the opposite sex, a test item treatment-related effect was considered unlikely.

Motor activity
The decreases in mean number of horizontal movement at 200 mg/kg/day (males and females) and in mean number of rearing (males) were considered to be treatment-related but of no toxicological importance.

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

MONITORING OF ESTROUS CYCLE
There were no obvious effects on estrous stages.

HAEMATOLOGY:
When compared with controls, there were statistically significant lower mean white blood cells count, hemoglobin concentration, basophils and lymphocytes counts in males given 200 mg/kg/day. These findings were considered to be of toxicological significance.

CLINICAL CHEMISTRY:
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 glucose, total protein and albumin levels (males), and increased total cholesterol (females) levels were considered to be of toxicological significance at 200 mg/kg/day.

URINALYSIS:
When compared with controls, there were no significant differences in urinalysis parameters.

ORGAN WEIGHTS:
The mean relative liver weights were statistically significantly higher in females given 200 mg/kg/day (+27%, p<0.01). This difference correlated to the microscopic hepatocellular hypertrophy in this organ and was related to the test item administration. There was a similar trend in males at 200 mg/kg/day (relative weight: +20%). However, in the absence of microscopic findings, this was considered to be mainly due to the lower body weight.

Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates and/or were not consistent for the sexes.
The statistically significant by higher relative epididymides and testes weights and the higher relative kidney weights in males treated at 200 mg/kg/day were considered to be due to the lower body weight at this dose-level.

GROSS PATHOLOGY:
The red discoloration observed in the mesenteric lymph nodes from 8/10 males and 6/10 females treated at 200 mg/kg/day was related to the test item administration. This correlated microscopically with the sinusal erythrocytes in these lymph nodes.
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. This correlated with dilated lumen at microscopic examination.

The other macroscopic findings had no histological correlates or correlated with common histological findings in control rats, and were considered to be incidental.
Specifically the yellow, brown or black discoloration of the lungs in some control or treated males correlated to microscopic pigment or hemorrhage. These findings were considered to be of no toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Test item-related non adverse microscopic findings were seen in the liver, mesenteric lymph node, stomach and duodenum.

. Liver
Minimal non adverse hepatocellular hypertrophy was seen in 6/10 females treated at 200 mg/kg/day.
It was accompanied by an increase in glycogen.
These lesions could correlate to the increase in cholesterol levels in high dose females (also seen in males).

. Mesenteric lymph node
There was an increase in mast cell and pigmented macrophages in the medullary sinuses in males and females treated at 50 or 200 mg/kg/day. The presence of these cells was recorded with the toluidine blue special stainings.
Sinusal red blood cells were also recorded at 50 or 200 mg/kg/day and correlated to the red discoloration seen macroscopically, together with the pigment.
These findings were considered not to be adverse.

. Stomach
Dose-related, minimal to slight decreased goblet cells and elongation of glands were seen in males and females treated at 200 mg/kg/day.
This was considered not to be adverse in view of the low magnitude of this change.
A focal degeneration/necrosis was noted in the stomach (pylorus) from one male treated at 50 mg/kg/day. The relationship to test item treatment was considered to be unclear in view of the low incidence of this change.

. Duodenum
There was a villous basophilia in the duodenum, together with atrophy (shortening of the villi length) mostly in males treated at 200 mg/kg/day.
These findings were considered not to be adverse in view of the low magnitude of these changes.

The colon dilated lumen observed in three high-dose males and females was considered to be probably related to test item administration and correlated to the macroscopic distension with feces.
In addition the glands from the cecum of one high-dose male were atrophic (grade 2, slight). A relationship to test item administration was not excluded.

Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, and/or are common background findings for the Sprague-Dawley rat.
Among these were the disruption wall of the aorta seen in one control male and one high dose male. It was accompanied by inflammation and hemorrhage in the adjacent tissue. This lesion was considered to be related to this strain of rats and was not considered as drug related. Pigment was seen in the lungs from some control and high-dose males. This could be related to a previous hemorrhage in this organ. It was associated with inflammation (in pleura or in alveoli). These changes were also considered to be spontaneous findings in this strain of rats.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on clinical signs, decreased mean body weight and, hematology and clinical biology findings at 200 mg/kg/day.
Critical effects observed:
not specified
Conclusions:
The test item 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 biology findings at 200 mg/kg/day).
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 13 weeks.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item at dose-levels of10, 50 or 200 mg/kg/day. The test item was administered during a 13-week period by gavage under a constant dosage-volume of5 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 forall animalsonce 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 maleswere 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.

 

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 weekmales and on weekone female), reflux at dosing (one male on week 4), piloerection (from weekmales) and loud breathing (one male on week 7) were considered to be test item treatment-related.

 

Functional Observation

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 notoxicologically 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.

 

Laboratory investigations

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 glucose (males), total protein and albumin levels (males), and increased total cholesterol levels (females) were considered to be of toxicological significance at 200 mg/kg/day.

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).

 

Conclusion

The test item was administered daily to Sprague-Dawley rats, by oral gavage, at dose-levels of10, 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

Additional 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 indings at 200 mg/kg/day). The effects at higher doses (dose-range finder study) included mortality after a few days of exposure. The assumption is that the primary effect causing mortality was caused by severe irritation in the gastro-intestinal tract.

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.