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Description of key information

Repeated dose toxicity: oral: 
No adverse effects were observed in male and female Wistar rats in a combined repeated dose/reprotox study conducted according to OECD Guideline 422 (Martell, 2013). Under the experimental conditions of this study, the NOAEL of the test substance in Wistar rats was 300 mg/kg/day for males and females. This study was selected as key study.
In addition, no adverse effects were observed in male and female Crj:CD(SD) rats in a 28 days repeated dose test conducted according to OECD 407. The NOAEL in this study was derived to be 150 mg/kg bw/d for both males and females, based on the toxicological effects at the 600 mg/kg dose.
Repeated dose toxicity: dermal: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure .
Repeated dose toxicity: inhalation:
No study with the target substance is available. Data from supporting substance morpholine is used to cover this endpoint. In a two-year chronic inhalation study, performed according to a method equivalent to OECD Guideline 452 (HAZLETON INC, 1983), rats exposed by inhalation to the read-across substance morpholine at concentrations of 0, 36, 181 or 543 mg/m³, 6 hours/day, 5 days/week for 104 weeks showed normal growth, survival and haematology and clinical chemistry parameters. A systemic intoxication was not observed. At the highest dose, chronic nasal irritation and some ocular injury was observed. Owing to its corrosivity, morpholine exposure resulted in irritation and inflammation of the upper digestive tract on oral intake, irritation of the respiratory tract and eyes following inhalation and, in high concentrations, irritation on contact with the skin.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-15 to 2012-07-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented GLP study performed according to OECD Guideline 422. However, dose formulations were not analyzed.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
no analytical verification of the dose is performed
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: # 1D403

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from humidity and light

OTHER SPECIFICS:
- Name of test material (as cited in study report): Jeffcat DMDEE
- Physical state: Liquid
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BIOAGRI Laboratorios
- Age at study initiation: 10 - 11 weeks old
- Weight at study initiation: males: 324.4 - 341.5 grams; females: 204.3 - 217.3 grams; satellite males: 336.0 - 371.0 grams; satellite females: 207.4 - 208.0 grams
- Fasting period before study: no
- Housing: Each animal was housed individually, except during cohabitation. After acclimation, one male was placed into each female cage for pairing. After pairing, females that presented vaginal smears with the presence of sperm were considered mated and housed individually. The rats were housed in polypropylene cages (41x34x19 cm) with wire mesh tops and bedding material (wood shavings). Clean cages were provided twice weekly for all animals. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-22.6°C
- Humidity (%): 40.5-69.9%
- Air changes (per hr): 10-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For each dosage group, the appropriate amount of Jeffcat DMDEE was weighed into a pre-calibrated beaker. The vehicle (deionized water) was added in sufficient quantity to achieve the desired concentration. Each solution was stirred and dispensed into individual containers properly identified. A sufficient quantity of the vehicle was similarly dispended for administration to control animals. The prepared solutions were stored at room temperature.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Parental animals (males and females) were treated with a volume of 4 mL/kg bw, starting at 10-11 weeks old and ending when the animals were euthanized. Satellite animals (5 animals per sex of the control and 5 animals per sex of the high dose group) were kept for 14 days after the scheduled necropsy of parental animals without treatment, for observation of reversibility, persistence or delayed occurrence of toxic effects. Satellite animals were not mated and, consequently, were not used for assessment of reproduction/developmental toxicity.
Frequency of treatment:
7-day per-week-basis
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 in the main study
5 animals per sex of the control group and 5 animals per sex of the high dose group for the satellite group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dosage levels (in mg/kg body weight/day) were selected following consultation with the Sponsor.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
All animals underwent a daily clinical observation for overt signs of ill health. These include, but are not limited to, changes in skin and fur, eye and mucous membranes, respiratory, circulatory, autonomic and central nervous system, motor activity and behavioral patterns.

BODY WEIGHT: Yes
Males were weighed on the first day of dosing and weekly thereafter (including mating and post-mating periods). Females were weighed on first day of dosing and once a week during premating and mating periods, on days 0, 7, 14 and 20 of gestation, and during lactation on the same days as the weighing of litters (on days 0 and 4 postnatal).

FOOD CONSUMPTION
Food consumption was determined on the same day of body weight determination during premating and lactation periods, except on day 0. During the gestation period food consumption was determined on days 3, 6, 9, 12, 15, 18 and 20. After the mating period, food consumption of males was determined weekly. Food consumption was not determined for exposed animals during the mating period.

HAEMATOLOGY: Yes
The following parameters were examined:
red blood cell count, hemoglobin, hematocrit, platelets, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total white blood cell count, differential leukocyte count, band neutrophils, monocytes, segmented neutrophils, lymphocytes, eosinophils, basophils
Clotting parameters: prothrombin time, activated partial thromboplastin time.

Hematology determinations were performed on 5 parental animals/sex/group, randomly selected from each group and on satellite animals from control and high dose group. The animals were fasted overnight and anesthetized by CO2 prior to blood collection (cardiac puncture).

CLINICAL CHEMISTRY: Yes
Clinical chemistry parameters:
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total protein, albumin, glucose, total cholesterol, urea nitrogen, creatinine, sodium, potassium, calcium, globulin, albumin/globulin ratio.

Clinical chemistry determinations were performed on 5 parental animals/sex/group, randomly selected from each group and on satellite animals from control and high dose group. The animals were fasted overnight and anesthetized by CO2 prior to blood collection (cardiac puncture). The clinical chemistry determination was performed in an accredited laboratory - Sabin - DF.

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observational battery:
Sensory reactivity to stimuli and motor activity assessment were performed in 5 animals/sex/group. For males these evaluations were performed at the end of the dosing period before scheduled necropsy, and for females, these evaluations were performed during the lactation period. The following parameters were assessed:
A - Autonomic Functions: lacrimation, salivation, palpebral closure, prominence of the eye, piloerection, respiration, urination and defecation;
B - Reactivity and sensitivity: sensor motor responses to approach tactile and tail flick;
C - Excitability: reactions to handling and behavior in an open field;
D - Gait and sensor motor coordination: degree of mobility and gait pattern in an open field;
E - Abnormal clinical signs: including convulsions, tremors, unusual behavior and deposits around the eyes, nose or mouth

OTHER:
Organ weights: At scheduled necropsy, testes and epididymides of all males were weighed.
Organ weights were obtained for the following organs from 5 animals/sex/group:
liver, kidneys, adrenals, thymus, spleen, brain, heart
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At termination, all parental animals were examined macroscopically for any abnormalities or pathological changes. The animals were euthanized in a carbon dioxide chamber. The number of implantation sites and corpora lutea were recorded. The animals were disposed in biological garbage and then incinerated. All pups were grossly examined for abnormalities of the oral, thoracic and abdominal cavities.

HISTOPATHOLOGY: Yes
At the scheduled necropsy, the following organs of all animals were preserved:
testes, epididymides, ovaries, prostate, semincal vesicle and coagulating gland, bulbourethral gland, organs showing alterations

The following organs and tissues of 5 animals/sex/group were preserved:
adrenals (right and left), bone marrow (femur), brain (cerebrum, cerebellum and pons), esophagus, heart, intestine (duodenum, jejunum, ileum - including Peyer's patches, cecum, colon, rectum/anus), kidneys (right and left), liver (3 lobes), lungs, lymph nodes (mesenteric and submaxillary), pancreas, peripheral nerve (sciatic), spinal cord (cervical, midthoracic and lumbar sections), spleen, stomach (glandular and non-glandular), trachea, thymus, thyroid/parathyroid, urinary bladder, uterus, all gross lesions

Full histopathology of the preserved organs and tissues listed above were performed in highest dose and control animals. The examination of the thymus was extended to females of other dosage groups and satellite females, because changes were observed in the highest dose group.
Statistics:
Quantitative variables such as body weights, food consumption and organ weights were analyzed by One Way Analysis of Variance (ANOVA), followed by Dunnett's test if significance is detected, or by the non-parametric test of Kruskal-Willis, according to the results of tests for normaility and homogeneity of variance. For qualitative or non-parametric data such as clinical findings, macroscopic and microscopic findings and fetal findings, comparison between means were carried out using Fisher's Exact Test or the Chi-Square Test. The level of significance was set at 5%.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Two females and one male at 300 mg/kg/day presented clinical signs between treatment days 25 to 41. These clinical signs were variable in nature, transitory, occurred in only a few animals, and were not observed in the high dose group satellite animals. The significance of these findings is not clear, but they are unlikely to be specific to the test article.
Mortality:
no mortality observed
Description (incidence):
The test substance did not cause mortality during the study
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain were unaffected at all dose levels
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected at all dose levels.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean total protein, globulin and albumin in males exposed to 150 and 300 mg/kg/day were statistically significant lower with a dose related trend, while potassium in females at 300 mg/kg/day was also higher than control. However, these findings were not observed in any other high dose groups, either study or satellite and are unlikely to be specific to the test article. Decreased diffuse lymphocyte cellularity in thymus and thymus involution was observed in females exposed to 150 and 300 mg/kg/day, respectively. In both cases, these lesions were correlated with a lower thymus weight. These findings were not observed in treated and satellite males or satellite females. It is important to note that for all treated females, including the control group females, thymus weights were substantially decreased in comparison to the satellite control group. This is most likely due to the animals' physiological response to pregnancy, and not test article related.

Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
During the functional observational battery, hyperreflexia was observed in three females at the 300 mg/kg/day dose level. The incidence of this finding was not statistically significant when compared to controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Kidney weights in males at 300 mg/kg/day was statistically significantly higher (+21%) when compared to the control group. Relative to body weight, although the difference was higher (+31.2%), it was not statistically significant, although relative to brain weight, kidney weights were statistically significant again (+20.8%). These changes were not dose related and no macroscopic or microscopic lesions were found, this finding was not considered to be test item related.
A statistically significant increase was observed in liver, kidneys, spleen and thymus weights of treated satellite males when compared to the control group. Relative to body weight, only liver and kidney weights were statistically significant, but relative to brain weight, the difference in these four organs was statistically significant again. Despite differences, no supporting macroscopic or microscopic lesions were noted in these organs, and these findings were not considered to be treatment-related.
Mean thymus weight was statistically significant lower in females exposed to 300 mg/kg/day (-39.5%) compared to the control group. Also, a statistically significant decrease was observed in thymus weights relative to body weight (-33%) and relative to brain weight (-39.1%). It is important to note that for all treated females, including the control group females, terminal thymus weights were substantially decreased in comparison to the female satellite control group and the treated and satellite males. This observation is most likely due to the female animals' physiological response to, and following, pregnancy, and is not considered test article related.
Finally, the differences found in liver and kidney weights in treated satellite females were not considered test item related because they were moderate in magnitude and there were no supporting macroscopic or microscopic lesions observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings were observed upon necropsy in male or female rats
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Decreased diffuse lymphocyte cellularity in thymus was observed in five females exposed to 150 mg/kg/day (animals 71, 74, 76, 77 and 82), while in females at 300 mg/kg/day was noted a thymus involution in rats 95, 98, 102, 104 and 106. These findings were not observed in treated and satellite males or satellite females, terminal thymus weights were substantially decreased in comparison to the female satellite control group. This observation is most likelly due to the animals' physiological response to, and following, pregnancy, and is not considered test article related.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Critical effects observed:
not specified
Conclusions:
Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test item in Wistar rats was 300 mg/kg/day for males and females. The substance is therefore not classified as STOT RE.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read across based on a study performed with supporting substance Morpholine (CAS 110-91-8). The read across justification is attached in IUCLID Section 13.
Reason / purpose:
read-across source
Dose descriptor:
NOEC
Remarks:
Systemic
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest concentration tested in the study, no chronic systemic toxicity was observed.
Critical effects observed:
not specified
Conclusions:
No reliable study is available with the target substance. Data from the supporting substance morpholine is used in a read-across strategy. Under conditions of this bioassay, morpholine was not regarded as systemically toxic. Effects noted with respect to ocular and nasal changes are attributed to direct exposure to these tissues. Morpholine was not considered to be carcinogenic.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
181 mg/m³
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral:

A combined repeated dose/reprotox/developmental screening study is performed with the target substance, in which male and female rats were exposed to 0, 50, 150 and 300 mg/kg body weight/day via gavage according to OECD Guideline 422 (Martell, 2013). Under the experimental conditions of the study, the NOAEL of the target substance was 300 mg/kg/day for males and females.

In this study, several findings were observed in males and females at mid and high dose levels. Two females and one male at high dose presented clinical signs between treatment days 25 and 41. These clinical signs were variable in nature, transitory and occurred in only a few animals. These clinical signs were not observed in the high dose group satellite animals. During the functional observational battery, hyperreflexia was observed in three females at the 300 mg/kg/day dose level. The incidence of this findings was not statistically significant when compared to controls. The significance of these findings is not clear, but they are unlikely to be specific to the test article. In males exposed to mid and high dose, total protein, globulin and albumin were statistically significant lower with a dose related trend, while potassium in females at high dose level was higher than control. However, these findings were not observed in any other high dose group, either the satellite group.

Females at mid dose showed decreased diffuse lymphocyte cellularity in thymus and at high dose displayed a thymus involution. In both cases, these lesions were correlated with a lower thymus weight, although with statistical reach only at high dose level. These findings were not observed in treated and satellite males or satellite females. It is important to note that for all treated females, including the control group females, terminal thymus weights were substantially decreased in comparison to the female satellite control group. This observation is most likely due to the animals' physiological response to, and following, pregnancy, and is not considered test article related. This study was selected as key study.

In a subacute oral toxicity study (28 days) with the target substance performed according to OECD Guideline 407, no significant, toxicologically relevant adverse effects have been observed at 150 mg/kg bw/day (actual dose received) for male and female Cjr:CD(SD) rats (Imatanaka, 1994). Adverse effects have been observed at 600 mg/kg dose. As no dose was tested in between 150 and 600 mg/kg bw/day in this study and no adverse effects have been observed in the combined repeated dose/reprotox test, the NOAEL of 300 mg/kg bw/day will be used for DNEL derivation.

Repeated dose toxicity: dermal:

No repeated dose toxicity via dermal application is required as reliable studies via other routes of administration are available.

Repeated dose toxicity: inhalation:

No reliable study via inhalation route of administration with the target substance is available. Data from the supporting substance morpholine is used to cover this endpoint. In a two-year chronic inhalation study (Huntsman, 1983), male and female rats that inhaled the read-across substance morpholine at concentrations of 0, 10, 50, or 150 ppm (0, 36, 181 or 543 mg/m³), 6 hours/day, 5 days/week for 104 weeks showed normal growth, survival, hematology, and clinical chemistry. The incidence of neoplasia in morpholine-exposed rats was not altered significantly compared to the concurrent controls. Rats exposed at the 150 ppm concentration developed focal erosion and focal squamous metaplasia of the epithelium of the anterior nasal cavity. Obvious evidence of chronic nasal irritation and inflammation with neutrophilic infiltration was documented in these same tissues. Ocular injury, including retinal degeneration, corneal irritation, uveitis, and corneal damage, were demonstrated only in rats exposed at 150 ppm. The distribution of ocular changes recorded in the groups exposed at 10 or 50 ppm morpholine was similar to that seen in the controls. Chronic exposure of rats to morpholine for 2 years at concentrations of 150 ppm or less revealed no carcinogenic potential or chronic systemic toxicity. Consistent with its known irritating properties, morpholine produced only local irritation, which was limited almost exclusively to high-dose animals.

 

The 2-year chronic inhalation study is considered to be the best available base for setting of relevant toxicological parameters. Based on this study, a systemic NOEC of 181 mg/m³ (50 ppm) for repeated dose toxicity is derived. A local NOEC was derived of 36 mg/m³. As morpholine is classified as corrosive to the skin, while the target substance is not classified for skin irritation, this local NOAEC cannot be used to derive a DNEL long-term exposure (local effects).

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation, DMDEE should not be classified for STOT repeated exposure via the oral route or inhalation route.