Reaction products of 1,4-cyclohexanedimethanol with propylene oxide and ammonia

Repeated dose toxicity: oral

Administrative data

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:

2012-07-19 - 2012-09-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Dose formulations were not analyzed.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 1,4-cyclohexanedimethanol, propylene oxide and ammonia

Cas Number:

1220986-58-2

Molecular formula:

C11H24N2O

IUPAC Name:

Reaction products of 1,4-cyclohexanedimethanol, propylene oxide and ammonia

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Jeffamine RFD-270 Amine
- Substance type: Colourless liquid
- Physical state: Liquid
- Lot/batch No.: 8802-8-9
- Analytical purity: 92%
- Composition of test material, percentage of components: 0.02 wt% water
- Purity test date: 2010-07-15
- Storage condition of test material: Sample stored in cool, well-ventilated storage area prior to testing

Specific details on test material used for the study:

Name of test material (as cited in study report): Jeffamine RFD-270 Amine
- Substance type: Colourless liquid
- Physical state: Liquid
- Lot/batch No.: 8802-8-9
- Analytical purity: 92%
- Composition of test material, percentage of components: 0.02 wt% water
- Purity test date: 2010-07-15
- Storage condition of test material: Sample stored in cool, well-ventilated storage area prior to testing

Test animals

Species:

rat

Strain:

other: Wistar Hannover

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BIOAGRI Laboratorios
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 304-446 g (males) ; 179 to 267 g (females)
- Fasting period before study: not applicable
- Housing: Each animal was housed individually, except during cohabitation. After acclimation, one male was placed into females cages for pairing (up to 2 females: 1 male). 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 a week 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: A larger number of animals than necessary were ordered in order to permit the selection and/or replacement of individual animals before the start of treatment. Accordingly, a total of 70 males and 70 females were received for acclimation and were 10-11 weeks of age. Upon receipt from the supplier, animals were placed into cages (1 animal/cage/sex) examined and acclimated for 8 days. All animals were observed daily for morbidity and mortality and the estrous cycle was checked. At the end of this period, the animals were weighed and a detailed clinical examination was performed. Animals showing abnormal signs or irregular estrous cycle were not used in the study. Only animals with weights within ± 20 % from mean body weight were used in this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 - 24.5 °C
- Humidity (%): 40.5-69.9 %
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: For each dosage group, the appropriate amount of Jeffamine RFD 270 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 identitified. A sufficient quantity of the vehicle was similarly dispensed for administration to control animals. The prepared solutions were stored at room temperature.
Test solutions were prepared daily at the testing facility and were administered within 2 hours after preparation. The test solutions were stirred continuously during the administration to maintain the homogeneity.

The volume administered each day was 4 mL/kg body weight.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Parental animals (males and females) were treated, starting at 11-12 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 3

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 4

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

actual ingested

No. of animals per sex per dose:

12 animals 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 groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage levels were selected in agreement with the sponsor.
10 mg/kg bw/day as the expected dose which causes no signs;
75 mg/kg bw/day as the intermediate dose level;
150 mg/kg bw/day as the expected dose which causes signs of systemic toxicity, but no death or severe suffering.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/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 behavioural 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 post natal).

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

CAGE SIDE OBSERVATIONS/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 lactation. The following parameters were assessed:
A - Autonomic Functions: lacrimation, salivation, palpebral closure, prominence of the eye, piloerection, respiration;
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. Animals found dead or euthanized moribund during the study were evaluated by gross examination as soon as possible after death. 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), peripheral nerve (sciatic), salivary gland, 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. TThe examination of the lung, liver, brain and kidneys was extended to animals of other dosage groups.

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-Wallis, 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%.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Most of male and female rats exposed to 150 mg/kg bw/day presented piloerection, apathy, respiratory sounds, dyspnea and rinocromodacryorrhea periodically from day 8 to day 44 of the test item administration. Males No 86, 93 and 108 at 150 mg/kg bw/day were found dead on days 12, 19 and 19 of treatment, respectively; while males rats No 84, 88, 89, 90 and 109 were sacrificed by animal welfare on days 12 and 13, after severe episodes of limited mobility and prostration. Females N° 96, 99, 101, 104, 105 and 115, at the same dose level were found dead on days 38, 12, 17, 25, 41 and 28 of treatment, respectively. These effects were dose related and could be attributed to treatment with the test item.

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Marked effects (decreases) on body weight, body weight gain and food consumption were observed in males and females in the high dose groups and in the high dose satellite male and female groups (150 mg/kg/day). Furthermore, even during the recovery period, a clear recovery in body weights was not observed in the satellite animals. Accordingly, changes observed in body weight, body weight gain and food consumption of males and females at 150 mg/kg/day, were considered to be treatment-related with toxicological difference.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Marked effects (decreases) on body weight, body weight gain and food consumption were observed in males and females in the high dose groups and in the high dose satellite male and female groups (150 mg/kg/day). Furthermore, even during the recovery period, a clear recovery in body weights was not observed in the satellite animals. Accordingly, changes observed in body weight, body weight gain and food consumption of males and females at 150 mg/kg/day, were considered to be treatment-related with toxicological difference.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some changes in mean red blood cell count and mean corpuscular hemoglobin concentration, or mean platelets were statistically significant when compared to the control groups (males or females, different concentrations). However, these changes were not considered to be test item related because they were isolated findings or normal biological variations.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Markedly higher mean aspartate aminotransferase levels were observed in male and females rats exposed to 75 and 150 mg/kg bw/day. No other alterations were found in the hemogram, leukogram and clotting parameters. However, these chemistry effects in the mid and high dose males and females, while not statistically significant, were observed in treated satellite males and females. Furthermore, these findings were supported by other observations from macroscopic and microscopic pathology examinations and are considered to be biologically relevant. Moreover, male rats at high dose level had a decrease in cholesterol levels, which were outside the historical control range and are considered to be dose related. Also this finding was observed in treated satellite males, confirming it as a treatment related effect.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No important alterations in organ weights were observed in treated male and female rats compared to the control groups. Only numerical differences were found and they were considered as normal biological variations.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Most of males exposed at 150 mg/kg bw/day presented slight congestion in brain, liver and lung; while the most frequent macroscopic findings in females at the same dose level were moderate congestion in liver, kidneys and brain. These findings were dose related and although they were not statistically significant, should be attributed to treatment with the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mild congestion in brain, liver, lung and kidney were the most common histopathological findings in both male and female rats exposed to 150 mg/kg bw/day, being statistically significant to the control group. These effects were dose related and should be considered treatment related.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

gross pathology

histopathology: non-neoplastic

mortality

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the NOAEL of the test substance in Wistar rats was 10 mg/kg bw/day for male systemic toxicity and maternal systemic toxicity.