1-Octadecanamine, reaction products with 1,1'-methylenebis[4-isocyanatobenzene]

Endpoint summary

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2015 to 07 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Age at study initiation: six to eight weeks old
- Weight at study initiation: At the start of treatment the males weighed 180 to 202g and the females weighed 161 to 184g
- Housing: The animals were housed in groups of five 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): A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK) was used.
- Water (e.g. ad libitum): Mains drinking water ad libitum was supplied from polycarbonate bottles attached to the cage
- Acclimation period: 5 days

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

IN-LIFE DATES: From: 15 September 2015 to 07 March 2016:

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

The test substance was administered to rats by daily oral gavage for a period of 28 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. The homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Due to the complex nature of the test item and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of test item in the formulations was determined using a gravimetric technique and the stability of the test item in the formulations was not performed. Formulations were therefore prepared and dosed daily

Duration of treatment / exposure:

Test duration: 28 days.

Frequency of treatment:

Onced daily for twenty-eight consecutive days.

No. of animals per sex per dose:

Animals were allocated to treatment groups as follows:
Control: 0mg/kg bw/day: 5 male animals and 5 female animals
Low:: 30mg/kg bw/day: 5 male animals and 5 female animals
Intermediate: 300mg/kg bw/day: 5 male animals and 5 female animals
High: 1000mg/kg bw/day: 5 male animals and 5 female animals

Details on study design:

DOSE SELECTION:

Range-Finding Study:
The test item was administered by gavage to three groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for seven consecutive days, at dose levels of 250, 500 and 1000 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Polyethylene glycol 400). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to gross necropsy examination.

Results:
Mortality: There were no unscheduled deaths
Clinical observations: There were no clinical signs detected
Bodyweight: Males treated with 1000 mg/kg bw/day and females from all treatment groups showed a slight reduction in body weight gain between Days 1 and 3. Recovery in either sex was evident thereafter. No such effects were detected in males treated with the 500 or 250mg/kg bw/day
Food consumption: No adverse effect on overall food consumption or food conversion efficiency was detected.
Water consumption: No adverse effect on water consumption was detected.
Necropsy: No macroscopic abnormalities were detected.

Conclusion;
There were no adverse effects detected in the 7-Day Range-Finding Study in the Rat to exclude 1000mg/kg bw/day for use in future studies. Dose levels of 0 (control), 30, 300 and 1000mg/kg bw/day were selected for the 28-Day study

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
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
Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill.

FOOD CONSUMPTION: Yes
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION: Yes
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically.

HAEMATOLOGY: Yes
Hematological investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling. The following parameters were measured;

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)
Reticulocyte count (Retic)
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).

CLINICAL CHEMISTRY: Yes
Blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling. The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant;

Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)
Bile acids
Triglycerides (Trigs/Tri)

Functional Observations:-
Prior to the start of treatment and on Days 7, 14, 21 and 25, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion as follows;

Behavioral Assessment:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed;
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behaviour
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper/Hyopthermia
Skin Colour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation.

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
Vocalization
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Blink reflex
Startle reflex

Sacrifice and pathology:

On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.

Organ weights:
The following organs were dissected free from fat and weighed before fixation:

Adrenals
Brain
Epididymides
Heart
Kidneys
Piuitary (post-fixation)
Prostrate and Seminal Vesicles (with coagulating glands and fluids)
Liver
Ovaries
Spleen
Testes
Thymus
Thyroid/Parathyroid (post fixation)
Uterus and Cervix

Histopathology:
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Colon
Duodenum
Epididymides♦
Esophagus
Eyes *
Gross lesions
Heart
Ileum (including peyer’s patch) Testes ♦
Jejunum
Kidneys
Liver
Lungs (with bronchi)#
Lymph nodes (mandibular and mesenteric)
Mammary gland
Muscle (skeletal)
Ovaries
Pancreas
Pituitary
Prostrate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles (with coagulating glands and fluids)
Skin
Spinal cord (cervical, mid thoracic and lumbar)
Spleen
Stomach
Testes♦
Thymus
Thyroid/Parathyroid
Trachea
Urinary bladder
Uterus and Cervix
Vagina

* 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

Other examinations:

Thyroid Hormone Assessment:
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -20 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

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. Where the data were unsuitable for these analyses, pair-wise tests were performed using the Student ttest (parametric) or the Mann-Whitney U test (non-parametric).

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Details on results:

Mortality:
There were no unscheduled deaths.

Clinical Observations:
There were no clinical signs detected in any animal.

Functional Observations:-
Behavioral assessments:
There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests:
There were no treatment-related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.

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

Body Weight:
Females treated with 1000 mg/kg bw/day showed a statistically significant reduction (p<0.05) in body weight gain during the first week of treatment. Recovery was however evident thereafter. Males from this treatment group showed a statistically significant reduction (p<0.01) in body weight gain during the final week of treatment. Consequently these animalsshowed a reduction in overall body weight gain (14% for females and 11% for males) when compared to controls. No such effects were detected in animals of either sex treated with 300 and 30 mg/kg bw/day.

Food Consumption:
There were no treatment-related effects on ood consumption or food conversion efficiency. A slight reduction in food conversion efficiency was evident in females treated with 1000mg/kg bw/day during the first week of treatment and in males from this treatment group during the final week of treatment. These intergroup differences were considered to be the result of body weight fluctuations seen in these animals during these periods.

Water Consumption:
There were no treatment-related effects on water consumption.

In-Life Sampling and Analysis:-
Hematology:
There were no toxicologically significant effects detected in the hematological parameters examined. Females treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in total leukocyte and lymphocyte count. The majority of individual values were within historical control ranges therefore in the absence of any associated histopathology correlates these intergroup differences were considered not to be of toxicological importance.

Blood Chemistry:
There were no treatment-related effects detected in the blood chemical parameters measured. Statistical analysis of the data did not reveal any significant intergroup differences.

Terminal Investigations:-
Necropsy:
There were no treatment-related macroscopic abnormalities detected. One female treated with 30 mg/kg bw/day had enlarged adrenals at necropsy. In the absence of a similar effect at 300 or 1000 mg//kg bw/day or any associated histopathological correlates the macroscopic findings was considered to be incidental and unrelated to treatment.

Organ Weights:
There were no toxicologically significant effects detected in the organ weights measured. Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in liver weight both absolute and relative to terminal body weight. All of the individual relative values and all but one absolute value were within historical control ranges. Although there was a microscopic liver change evident in males treated with 1000 mg/kg
bw/day, no such effects were detected in 300 or 30 mg/kg bw/day males and the effect detected was considered to be adaptive, therefore the intergroup differences were considered not to be of toxicological importance.

Histopathology:
The following microscopic change was evident: Liver: A reduction in the amount of glycogen-type rarefaction was evident in four males treated with 1000 mg/kg bw/day compared to the control animals and the remaining 1000 mg/kg bw/day male. No such effects were detected in females treated with 1000 mg/kg bw/day or in animals of either sex treated with 300 or 30 mg/kg bw/day.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

The oral administration of PU Thickener to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg bw/day did not result in any effects that were considered to be toxicologically significant. The No Observed Adverse Effect (NOAEL) for either sex was therefore considered to be 1000 mg/kg bw/day.

Executive summary:

In a GLP compliant, guideline sub-acute oral toxicity test the oral administration of the registered substance to rats for a period of twenty-eight consectuive days at dose levels of 30, 300 and 1000 mg/kg bw/day did not result in any effects that were considered to be toxicologically significant.

No clinical signs of toxicity were evident in any animal. Overall body weight gains were slightly reduced in animals of either sex treated with 1000 mg/kg bw/day however this was considered to be the result of a reduced body weight gain in females during the first week of treatment and in males during the final week of treatment. Females showed recovery in body weight gain thereafter and no effect on body weight gain was evident in males prior to the final week of treatment. The intergroup differences may therefore represent normal biological variation rather than a true adverse effect of treatment.

There were no treatment-related effects detected in the blood chemical parameters measured and although there were a couple of statistically significant differences in treated animals from controls for the hematological parameters measured, the majority of these differences were within historical control ranges and were considered not to be of toxicological significance.

Histopathological examination of liver sections revealed a reduction in the amount of glycogen-type rarefaction in four males treated with 1000 mg/kg bw/day compared to the control animals and the remaining 1000 mg/kg bw/day male. The amount of glycogen type rarefaction is variable across studies of this type however it was considered to be particularly high in control animals in this study. Therefore, it is not clear if the change in the 1000 mg/kg bw/day males was related to treatment and as such a change of this nature would be considered to be an adaptive response indicating a minor metabolic change.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant guideline sub-acute study with a klimisch score of 1

Additional information

In a GLP compliant sub-acute toxicity study to OECD test guideline 407 the oral administration of the regsistered substance to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg bw/day did not result in any effects that were considered to be toxicologically significant.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study available