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

Endpoint summary

Description of key information

Repeated dose toxicity - oral

Griffiths D. (2017) performed a 90 -day repeated dose toxicity study in rats according to OECD guideline 408 (GLP). A NOAEL of 10 mg/kg bw/day was derived for male and female systemic toxicity.

Betancourt Martell A. (2014) performed a repeated dose toxicity study in rats according to OECD guideline 422 (GLP). A NOAEL of 10 mg/kg bw/day was derived for male and female systemic toxicity.

Repeated dose toxicity - inhalation:

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 inhalation route of exposure.

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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

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

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

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

actual ingested

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

actual ingested

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

actual ingested

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.

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%.

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

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

Key result

Critical effects observed:

no

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2016-05-10 to 2016-12-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 21 September 1998

Deviations:

yes

Remarks:

Please refer to field "Any other information on materials and methods incl. tables.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 4M604
- Physical state/appearance: Clear colorless liquid
- Expiration date of the lot/batch: not indicated
- Purity: 100%
- Purity test date: not indicated


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark
- Stability under storage conditions: not indicated
- Stability under test conditions: The formulations were administered within two hours of preparation it was considered that the formulations were stable for this duration.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: not indicated


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was prepared at the appropriate concentrations as a solution in distilled water on a daily basis throughout the duration of the study.

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST strain

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: males weighed 167 to 189g, the females weighed 131 to 156g
- Number of animals in total: A total of 80 animals (40 males and 40 females) were accepted into the study.
- Fasting period before study: No
- Housing: The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): Yes free access
- Water (e.g. ad libitum): Yes free access. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Environmental enrichment: It was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Acclimation period: The animals were acclimatized for nine days during which time their health status was assessed.



DETAILS OF FOOD AND WATER QUALITY: A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used.
The diet, drinking water bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility.

IN-LIFE DATES: From 20 May 2016 (first day of treatment) to 19 August 2016 (final day of necropsy).

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of distilled water. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Vehicle:

water

Remarks:

Distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item preparation and Analysis:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in distilled water.

Homogeneity: The homogeneity of the test item formulations was determined by Envigo Research Limited, Shardlow, UK, Analytical Services. with respect to the concentration of test item in water formulations at nominal concentrations of 2 mg/mL and 30 mg/mL.

Stability: As the analytical method used was non stability indicating, the formulations were made up on a daily basis throughout the duration of the study. The formulations were administered within two hours of preparation it was considered that the formulations were stable for this duration.

See details on the method in section Details on analytical verification of doses or concentrations

DIET PREPARATION
not applicable

VEHICLE
Distilled water as the test item is stable in water.

Analytical verification of doses or concentrations:

yes

Remarks:

The test item concentration in the test samples was determined spectrophotometrically using an external standard technique.

Details on analytical verification of doses or concentrations:

Samples of test item formulation were taken on four occasions and analyzed for concentration of JEFFAMINE RFD 270 at Envigo Research Limited, Shardlow, UK, Analytical Services. The results i ndicate that the prepared formulations were within ± 10% of the nominal concentration and were all within acceptable ranges for the purpose of this study.

Apparatus:
Analytical and top-pan balances
General laboratory apparatus and glassware.

REAGENTS:
Control vehicle: Distilled water
Water: Reverse osmosis
Dilution solvent: Water

PREPARATION OF CALIBRATION STANDARDS
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 gr of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration range of 0.2 mg/mL.

On each occasion standard solutions derived from two stock standard solution were used for calculation.

Calibration solutions were injected onto instrument, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the instrument.
To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solution of 1.045 mg/mL by serial dilution covering the concentration range 0.1045 mg/mL to 0.2613 mg/mL.

PREPARATION OF TEST SAMPLES:
The formulations received were diluted with dilution solvent. An aliquot of the test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent this was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve to working concentration.

PREPARATION OF ACCURACY AND PRECISISON SAMPLES.
Sample of Distilled water were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These sample were then prepared for analysis as the test samples.


INSTRUMENTATION PARAMETERS:
- Spectrophotometer: Perkin-Elmer Lambda 20
- Cell path length: lambda max at ca. 192 nm
- Cell path length: 10 nm
- Reference medium: Water.


DATA EVALUATION AND CALCULATIONS:
The peak area response for the test item in each calibration standard chromatogramm was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for test item in sample and procedural recovery chromtograms was measured. The concentratio of test item was determined using the following equations:


X= Y-a / m

Where:
x=C° of the test item in the injected sample (mg/mL)
y= mean absorbance response of the tets item in the sample
m= slope of the calibration plot (counts per mg/mL)
a= y-axis intercept (counts)


The concentration of the tes item in the test sample was calculated using the following equation:

C=(x/mas)xFxSG

Where:
x=C° of the test item in the injected sample (mg/mL)
y= mean absorbance response of the tets item in the sample
F= sample dilution factor
SG= Specific gravity (where applicable) (g/mL)
Mass= Mass taken (g)

The recovery rate (R) of the spiked recovery sample was calculated using the following equation:
R= c/cfort)x100

Where:
R= Recovery rate (%)
C= Determined concentration of the test item in the spiked recovery sample (mg/mL)
Cfrot= Fortified concentration of the tets item in the spiked recovery sample (mg/mL)

VALIDATION OF THE ANALYTICAL PROCEDURE:
The analytical procedure was validated by determining the following parameters:
- The specificity of the chromatographic analysis in control sample chromatograms.
- The linearity of detector response over the calibration standard concentration range.
- The method accuracy (recovery) and precision, by analyzing five recovery samples at nominal concentrations of 2.5 mg/mL and 250 mg/mL
- The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.

HOMOGENEITY AND STABILITY IN VEHICLE FORMULATIONS
The homogeneity and stability of the test item in water formulations was assessed at nominal concentrations of 2.0mg/mL and 30 mg/mL. a

CONCENTRATION OF DOSE FORMULATIONS:
For each analysis occasion, freshly prepared test formulations were analyzed. Duplicate samples were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory result were achieved

MAJOR COMPUTERIZED SYSTEMS:
The computerized systems that have been used in this project are:
Perkin Elmer UV WinLab Enhances Security Version 6.0.3.0730
Delta BMS


RESULTS:
METHOD VALIDATION:
The analytical procedure was successfully validated for test item in water with respect to the specificity of analysis, the linearity of detector response, method accuracy and precision
The specificity of the analytical method was demonstrated by the absence of a peak at the characteristic retention time for test item in the control sample chromatogram.

The data was found to have a linear correlation within the calibration range of 0.1045 mg/mL to 0.2613 mg/mL. The R2 fit of the calibration curve to the data was considered to be acceptable.


A mean recovery value of 103% (CV=1.75%, n=5) was obtained for 2.0 mg/mL and 106% (CV= 0.728%, n=5) was obtained for 30 mg/mL.

The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.


HOMOGENEITY AND STABILITY OF DOSE FORMULATIONS:
The homogeneity and stability of test item in water formulations was assessed with respect to the level of concentration at nominal concentrations of 2.0 mg/mL and 30 mg/mL.
Homogeneity was confirmed at the initial stability time point. The mean analyzed concentration for the nine samples remained within 10% of the initial time zero value and the variation was less than 10%.

CONCENTRATION OF DOSE FORMULATIONS:
The mean concentrations were within applied limits +/- 10%, confirming accurate formulation.

CONCLUSION:
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.

The homogeneity and satbilty was confirmed for test item in water formulations at nominal concentrations of 2.0 mg/mL and 30 mg/mL when refrigerated for 12 days (low dose) and 21 days high dose)

The mean concentrations of test item in test formulations analyzed for the study were within +/- 10% of nominal concentrations, confirming accurate formulations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Low

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Medium

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

High

No. of animals per sex per dose:

- 10 males and 10 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen in collaboration with the Sponsor and were based on available toxicity data. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

- Rationale for animal assignment (if not random): Not applicable
- Rationale for selecting satellite groups: Not applcable
- Post-exposure recovery period in satellite groups: Not applicable

Concentrations and volumes:
Treatment Group
Control
concentration: 0 mg/mL
volume: 0 mL/kg
Low
concentration: 2 mg/mL
volume: 5 mL/kg
Medium
concentration: 6 mg/mL
volume: 5 mL/kg
High
concentration: 15 mg/mL
volume: 5 mL/kg

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
Food consumption was recorded for each cage group at weekly intervals throughout the study. Weekly food efficiency (body weight gain/food intake) was then calculated retrospectively.

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: No

WATER CONSUMPTION : Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all control and treated animals were examined pre-treatment with all control and high dose animals’ examined again before termination of treatment (during Week 12).
Examinations included observation of the anterior structures of the eye and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using an ophthalmoscope was performed.

IN-LIFE SAMPLING AND ANALYSIS:
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein.
Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.

HAEMATOLOGY: Yes
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.
- Parameters checked:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct):
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)

Total leukocyte count (WBC)
Differential leukocyte count:
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

BLOOD CHEMISTRY: Yes
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)

IMMUNOLOGY: No


URINALYSIS: No


FUNCTIONAL OBSERVATIONS
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli. These were performed at least one hour after daily dosing had been completed.

BEHAVIOURAL EXAMINATION: Yes
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988).The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity.
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength.
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

SENSORY REACTIVITY:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Sacrifice and pathology:

NECROPSY
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.


GROSS PATHOLOGY: Yes
Microscopic examination was conducted by the Study Pathologist (Kai Chiu Liu at Envigo CRS Limited, Eye Research Centre, Eye, Suffolk, IP23 7PX). A histopathology peer review was conducted at the test facility.


HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:

Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint)• Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and pons) Rectum
Caecum Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymides ♦ Skin
Esophagus Spinal cord (cervical, mid-thoracic
Eyes* and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum (including Peyer’s patches) Testes ♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Tongue•
Lungs (with bronchi) # Trachea
Lymph nodes (mandibular and mesenteric) Urinary bladder
Mammary glands Uterus (with cervix)
Muscle (skeletal)• Vagina


All tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). All tissues from control and 75 mg/kg bw/day dose group animals and Livers from 10 and 30 mg/kg bw/day dose group were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of adrenals, brain, epididymides, lungs, pancreas and thyroid from animals in the low and intermediate groups.

• Retained only and not processed
* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative
• Retained only and not processed

Other examinations:

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals Ovaries
Brain Spleen
Epididymides Testes
Heart Thymus
Kidneys Uterus
Liver

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group.

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Sporadic instances of increased salivation were evident in nine male animals and eight females treated with 75 mg/kg bw/day from Week 7. Intermittent instances of noisy respiration were also noted in four male animals and six females from Week 2. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are generally considered to be of no toxicological importance. No such effects were detected in animals of either sex treated with 30 mg/kg bw/day or 10 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male animals treated with 75 mg/kg bw/day showed a reduction in body weight gain during the first two weeks of treatment achieving statistical significance during week 1 (p<0.01).
Thereafter, slightly lower body weight gains were noted on some occasions during the treatment period which culminated in an actual body weight loss (p<0.05) during the final week of treatment. Overall body weight gain for this treatment group was found to be approximately 12 % lower than controls. Female animals treated with 75 mg/kg bw/day and 30 mg/kg bw/day showed slightly lower body weight gains in some instances with statistical significance being noted in the 75 mg/kg bw/day treatment group during week 8 (p<0.05) and week 12 (p<0.01). Overall body weight gains for these two treatment groups were approximately 10 % and 13 % lower than controls (for females treated with 30 mg/kg bw/day and 75 mg/kg bw/day respectively). No such effects were detected in male animals treated with 30 mg/kg bw/day or in animals of either sex treated with 10 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male animals treated with 75 mg/kg bw/day showed a marginal reduction in food consumption throughout the treatment period resulting in an overall reduction in food consumption for these animals (28%). No such effects were detected in the female animals
treated with 75 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day or 10 mg/kg bw/day.

Food efficiency:

no effects observed

Description (incidence and severity):

Any minor differences in food conversion efficiencies were considered likely to be due to differences in body weight gains and/or food intake.

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

no effects observed

Description (incidence and severity):

No adverse effect was detected on water consumption.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Opthalmoscopic examination of animals of both sexes from the control and 75 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At week 13 assessments, male animals treated with 75 mg/kg bw/day showed statistically significant increases in total Leukocyte count and Lymphocytes (p<0.05). Approximately half of the animals treated with 75 mg/kg bw/day showed values that exceeded the normal range values for each of these parameters, however, values noted for 4/10 control animals were below the normal range values. No clear dose-relationships were apparent. Females treated with 75 mg/kg bw/day showed a statistically significant reduction in mean corpuscular hemoglobin concentration (p<0.01) with no dose-relationship. All values for the test item treated females were within the historical control range, however, 3/10 females from the control group exceeded the normal range values. These changes were seen in the absence of any associated histopathological correlates and were considered not to be of any toxicological significance.

No toxicologically significant effects were noted in females treated with 75 mg/kg bw/day or in animals of either sex treated with 30 or 10 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At Week 13 assessments, males treated with 75 mg/kg bw/day showed a statistically significant reduction in chloride concentration (p<0.01) with a dose-relationship being apparent between 30 and 75 mg/kg bw/day. A statistically significant increase in Bile Acids (p<0.05) was also noted, however, a dose relationship was not apparent. All values were within the normal control range for chloride concentration for both treated and control animals. The majority of treated male animals showed bile acid values that exceeded the normal ranges, however, 4/10 control animals exhibited values that were lower than the normal ranges. Male animals treated with 75 mg/kg bw/day and 30 mg/kg bw/day also showed statistically significant increases in phosphorous, aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) (p<0.01 and p<0.05 for the high and intermediate dose groups respectively) with dose-relationships being apparent. Normal control ranges were exceeded in 4/10, 10/10 and 5/10 animals for each of these parameters respectively for animals treated with 75 mg/kg bw/day. For animals treated with 30 mg/kg bw/day normal historical range values were exceeded in 9/10 animals in respect to ASAT, whilst most
animals showed individual Phosphorous and ALAT values within the historical control data range. Female animals from all treatment groups showed a statistically significant increase in albumin/globulin ratio (p<0.05) but without a dose-relationship, most individual values were within the normal historical control data range. Females treated with 75 mg/kg bw/day showed a statistically significant increase in aspartate aminotransferase (ASAT) (p<0.05) with all values exceeding the normal historical control ranges. A statistically significant decrease in Cholesterol (p<0.05) was also noted for female animals treated with 75 mg/kg bw/day with a dose-relationship apparent between 30 and 75 mg/kg bw/day dose groups, however, most individual values were within the historical control data ranges. All of these changes were seen in the absence of any associated histopathological correlates and were considered not to be of toxicological significance.

No such effects were detected in males treated with 10 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in the behavioural parameters measured.
One male animal treated with 75 mg/kg bw/day exhibited noisy respiration during the Week 11 assessments

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 75 mg/kg bw/day showed a statistically significant increase in absolute and relative kidney weight (p<0.01). Approximately 80% of animals from both the treated and control groups showed values that exceeded the normal control ranges. Female animals treated with 75 mg/kg bw/day showed a statistically significant reduction in absolute and relative thymus weight (p<0.05). The majority of values were within the normal control range for treated animals, however, three animals from the control group showed values that were higher than the normal control ranges. As there were no corresponding test item related microscopic findings seen in the kidney and thymus to account for the effects on the organ weights, these findings are considered to be of no toxicological significance.

Males treated with 75 mg/kg bw/day and 30 mg/kg bw/day showed a statistically significant increase in absolute and relative adrenal weights (p<0.01 and p<0.05) in a dose-related manner. Approximately 50% of animals treated with 75 mg/kg bw/day exceeded the normal range values with the majority of animals treated with 30 mg/kg bw/day exceeding them also. Control animals exhibited an increase over the normal range values in no more than 40% of cases. These changes were considered to be related to the microscopic findings seen in the adrenals of animals treated with 75 mg/kg bw/day and were considered to be a stress related response.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy
There were no treatment-related findings at necropsy.
One female treated with 10 mg/kg bw/day and one animal of either sex treated with 30 mg/kg bw/day had reddened lungs at necropsy. One male treated with 10 mg/kg bw/day and one male treated with 75 mg/kg bw/day had an increased pelvic space in the left kidneys.
These observations were considered to be incidental findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment related microscopic abnormalities were detected:

Adrenal
Diffuse cortical hypertrophy was seen in animals treated with 75 mg/kg bw/day.

Brain
Vacuolation/hypertrophy of the choroid plexus was seen in animals treated with 75 or 30 mg/kg bw/day. Oil Red O and AB (Alcian Blue)/PAS (Periodic acid-Schiff) stains were used to try to identify the type of vacuolation but these were inconclusive.

Epididymides
Epithelial vacuolation was seen in males treated with 75 or 30 mg/kg bw/day.

Lungs
Mucous metaplasia of the alveolar epithelium and bronchiolar epithelium were seen in animals treated with 75 or 30 mg/kg bw/day. Oil Red O and AB (Alcian Blue)/PAS (Periodic acid-Schiff) stains were used to try to identify the type of vacuolation but these were inconclusive.

Pancreas
Degranulation of the acinar cells was seen in animals treated with 75 mg/kg bw/day.

Thyroid
Increased incidence of vacuolation of the follicular epithelium at slight severity was seen in animals treated with 75 mg/kg bw/day.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Functional Performance Tests
There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.

Details on results:

DISCUSSION
The oral administration of JEFFAMINE RFD 270 to rats for a period of ninety consecutive days at dose levels of 10, 30 and 75 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 30 or 75 mg/kg bw/day.

Clinical signs were detected in animals of either sex treated with 75 mg/kg bw/day during the study. Increased salivation was reported between Days 43 to 87 of the study and intermittent instances of noisy respiration were also noted from Days 9 to 76. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are generally considered not to be of toxicological importance.

Adverse effects on body weight gains and food consumption were noted in male animals treated with 75 mg/kg bw/day, this resulted in an overall body weight gain which was approximately 12% less than controls. Female animals treated with 75 mg/kg bw/day and 30 mg/kg bw/day showed occasional reductions in body weight gain throughout the treatment period, however, no significant effects were noted in food consumption for these animals.
Overall body weight gain for these two female treatment groups were approximately 13 % and 10 % lower than controls respectively.

Histopathological examination of the brain (in animals of either sex treated with 75 or 30 mg/kg bw/day), epididymides (males treated with 75 or 30 mg/kg bw/day) and thyroid (in animals of either sex treated with 75 mg/kg bw/day) revealed vacuolation in these tissues. In addition, mucous metaplasia of the alveolar epithelium and bronchiolar epithelium were seen in the lungs of animals given the test item. Oil Red O and AB (Alcian Blue)/PAS (Periodic acid-Schiff) stains were used to try to identify the type of vacuolation but these were inconclusive. The cause and significance of these findings is not clear but the changes are
unusual and are therefore considered to be related to the administration of the test item.
Diffuse cortical hypertrophy was seen in the adrenals in animals of either sex treated with 75 mg/kg bw/day; this corresponded with an increase in organ weight, ultimately this was considered to be a stress related response.
Acinar degranulation of the pancreas was considered to be a physiological effect due to the reduction in food intake. Hematological and blood chemistry investigations in animals of either sex did not reveal any findings of any toxicological significance.

There were no toxicologically significant effects observed during the weekly open field arena observations.

Taking into consideration the overall results from this study, it is considered that a dose level of 10 mg/kg bw/day could be assigned a No Observed Effect Level (NOEL) for systemic toxicity within the confines of this type of study.

CONCLUSION
Administration of JEFFAMINE RFD 270 by oral (gavage) to Wistar Han rats for 90 days was associated with test item related effects in the adrenal, brain, lungs, pancreas, thyroid and epididymides, of rats treated with 75 or 30 mg/kg bw/day. There were no toxicologically significant observations at 10 mg/kg bw/day in animals of either sex and a dose level of 10 mg/kg bw/day is therefore considered to be the ‘No Observed Effect Level’ (NOEL) for systemic toxicity within the confines of this type of study.

Key result

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Conclusions:

Administration of JEFFAMINE RFD 270 by oral (gavage) to Wistar Han rats for 90 days was associated with test item related effects in the adrenal, brain, lungs, pancreas, thyroid and epididymides, of rats treated with 75 or 30 mg/kg bw/day.There were no toxicologically significant observations at 10 mg/kg bw/day in animals of either sex and a dose level of 10 mg/kg bw/day is therefore considered to be the ‘No Observed Effect Level’ (NOEL) for systemic toxicity within the confines of this type of study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

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: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

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 - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic 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

Link to relevant study records

Reference

Endpoint:

sub-chronic 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

Additional information

Repeated dose toxicity- oral:

• A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test is performed in rats. The substance was administered daily to 12 animals/sex/group and 5 animals/sex/satellite groups by gavage, on a 7 -day-week-basis (from 14 days before mating period to 4 days post-delivery). The test item was administered at doses of 0 (vehicle - deionized water), 10, 75 and 150 mg/kg bw/d via gavage (OECD 422, Martell A., 2014).

 

The substance caused clinical signs of toxicity from day 8 to 44 of the test item administration; in addition, a marker total mortality of 50% in males and 50% in females occurred in the high dose group (150 mg/kg bw/day) during the study. These effects were dose related and therefore, attributed to treatment with the test item.

Decreases on body weight, body weight gain and food consumption were observed in males and females in the high dose group and in the high dose satellite male and female groups (150 mg/kg bw/day). Furthermore, even during the recovery period, a complete recovery in the bodyweights 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 bw/day were considered to be treatment-related with toxicological significance.

Other than the cage side observations noted, there were no test article effects on reaction to stimuli and motor activity assessment in the functional observational battery study.

Markedly higher mean aspartate aminotransferase levels were observed in male and female 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 the treated satellite males and females. Furthermore, these findings are considered to be treatment related at the high dose level males and females and satellite males and females. Moreover, male rats at the high dose level and the satellite male groups had decreased cholesterol levels which fell outside of the laboratory historical control value range. This finding was considered to be a treatment related effect.

Under the experimental conditions of this study, the NOAEL of the test item in Wistar rats was 10 mg/kg bw/day.

 

• A 90 -day repeated dose toxicity was also conducted with the substance according to OECD guideline 408 (Griffiths D., 2017).

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 10, 30 and 75 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Distilled water).

 

Administration of the test substance by oral (gavage) to Wistar Han rats for 90 days was associated with test item related effects in the adrenal, brain, lungs, pancreas, thyroid and epididymides, of rats treated with 75 or 30 mg/kg bw/day.

Histopathological examination of the brain (in animals of either sex treated with 75 or 30 mg/kg bw/ day), epididymides (males treated with 75 or 30 mg/kg bw/day) and thyroid (in animals of either sex treated with 75 mg/kg bw/day) revealed vacuolation in these tissues. In addition, mucous metaplasia of the alveolar epithelium and bronchiolar epithelium were seen in the lungs of animals given the test item. Oil Red O and AB (Alcian Blue)/PAS (Periodic acid-Schiff) stains were used to try to identify the type of vacuolation but these were inconclusive. The cause and significance of these findings is not clear but the changes are unusual and are therefore considered to be related to the administration of the test item.

Diffuse cortical hypertrophy was seen in the adrenals in animals of either sex treated with 75 mg/kg bw/day; this corresponded with an increase in organ weight, ultimately this was considered to be a stress related response.

Acinar degranulation of the pancreas was considered to be a physiological effect due to the reduction in food intake. Hematological and blood chemistry investigations in animals of either sex did not reveal any findings of any toxicological significance.

There were no toxicologically significant observations at 10 mg/kg bw/day in animals of either sex and a dose level of 10 mg/kg bw/day.

Under the experimental conditions of this study, the No Observed Effect Level of the test item in Wistar rats was 10 mg/kg bw/day.

 

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

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 inhalation route of exposure.

Justification for classification or non-classification

Based on the available results and according to the criteria of the CLP Regulation, the substance is considered not classified as STOT RE. There are no observations in the key study that would trigger such a classification.