Reaction products of amines, dicoco alkyl and glycollic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-10-25 to 2006-03-16; Amended 2007-08-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rats

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Chemical Substance Control Law Guidelines of 21 November 2003 (28-day repeat oral dose study).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Chemical name: Reaction product of poly (n=1-3) cocamine (C8, C10, C12, C14, C16, C17, and C18, linear and branched) with glycolic acid including N,N_Didodecyl-2-hydroxyacetamide as a main component.
- Physical state: dark amber slightly viscous liquid
- Analytical purity: 100%
- Storage condition of test material: room temperature in the dark
- Stability during study: Stability of test concentrations performed during study. Formulations stable for at least 14 days. Prepared weekly and stored at +4°C in the dark.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent UK
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: males: 145 to 185 g and females: 128 to 158 g
- Fasting period before study: No
- Housing: groups of five by sex in polyproylene grid-floor cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour dark and light cycle

IN-LIFE DATES: From: 2005-10-05 To: 2006-01-03

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): stability and solubility
- Concentration in vehicle: 6.25, 62.5, 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

GC using an external standard.

Samples and standards extracted in acetone to give a concentration of 0.05 mg/mL

GC system: Agilent Technologies 5890
Column: DB-1 (30 x 0.32 mm id x 0.25µm film)
Oven temperature: Initial 200°C for 0 mins, rate 10°C/min, final 325°C for 15 mins
Injection temp: 250°C
Flame ionisation detector temp: 300°C
Injection volume: 1 µL
Specificity: No interference
Accuracy: 86 - 118%

Stability determination: analysed initally and after 14 days in the dark at +4°C.
Stability: 92 - 105% of initial concentration after 14 days

Formulation concentrations:
Formulation: 86 - 104% of nominal

Duration of treatment / exposure:

28-days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
25 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

five male/five female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: range-finder
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: Not stated
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: Prior to start of treatment and on Days 7, 12, 16 ansd 23
- Battery of functions tested: sensory activity / grip strength / motor activity
- Cage side observations: behavioural assessments, funtional performance tests, sensory reactivity .

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, immediately post dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends).

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 (prior to the start of treatment) and on Days 8, 15,22 and 29 prior to terminal kill, and, in the case of recovery group animals, on Days 36 and 43 prior to terminal kill.

FOOD CONSUMPTION:
- Food consumption: recorded for each cage group at weekly intervals throughout the study.

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 :
- Time schedule for examinations: daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of the treatment period (Day 28) and on all surviving recovery group animals at the end of the treatment-free period (Day 42). Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All animals
- Parameters examined:
EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)

Total leucocyte count (WBC)
Differential leucocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)

Platelet count (PLT)
Reticulocyte count (Retic)
- Cresyl blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Thrombomax HS with calcium’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.1 1 moV1)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of the treatment period (Day 28) and on all surviving recovery group animals at the end of the treatment-free period (Day 42). Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43
- Animals fasted: No
- How many animals: All
- Parameters examined:
Urea
Glucose
Total protein (Tot.Prot.)
Albumin
AlbumidGlobulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Gamma glutamyltranspeptidase (yGT)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Triglycerides (Tri)
Total cholesterol (Chol)
Total bilirubin (Bili)

URINALYSIS: Yes
- Time schedule for collection of urine: non-recovery test and control group animals during Week 4 and on all surviving recovery group animals during Week 6.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:
Volume
Specific Gravity
Protein
Glucose
PH
Ketones
Bilirubin
Urobilinogen
Reducing Substances
Blood


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Organ weights:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus

HISTOPATHOLOGY: Yes
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Colon
Duodenum
Epididymides
Eyes
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)#
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles
Skin (hind limb)
Spinal cord (cervical)
Spleen
Stomach
Testes
Thymus
Thyroiflparathyroid
Trachea
Lymph nodes (cervical and mesenteric)
Muscle (skeletal) Uterus
Urinary bladder

Other examinations:

None

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
All data were summarised in tabular form. Where appropriate, quantitative data were analysed by the ProvantisTM Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed 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 showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Mortality. One control male was killed on humane grounds due to the severity of its clinical observations on Day 26. There were no other unscheduled deaths during the study.

Mortality:

no mortality observed

Description (incidence):

Mortality. One control male was killed on humane grounds due to the severity of its clinical observations on Day 26. There were no other unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis 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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Remarks:

No Observed Effect Level (NOEL)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of 1000 mg/kg/day did not result in any toxicologically significant changes. The ‘No Observed Effect Level’ (NOEL) was considered to be 1000 mg/kg/day.

Executive summary:

Test Guidance

i) Commission Directive 96/54/EC (Method B7).

ii) The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004.

iii) The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 27 July 1995).

iv) USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

Method and materials.

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD@ (SD) IGS BR strain rats, for up to twenty-eight consecutive days, at dose levels of 25, 250 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for up to twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all surviving recovery group animals at the end of the treatment-free period.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from non-recovery high dose and control animals was performed.

Results.

Mortality. One control male was killed on humane grounds due to the severity of its clinical observations on Day 26. There were no other unscheduled deaths during the study.

Clinical Observations. No clinically observable signs of toxicity were detected throughout the study period.

Behavioural AssessmentWeekly behavioural assessments revealed no treatment-related changes in the parameters measured.

Functional Performance Tests. There were no treatment-related changes in the functional performance parameters measured.

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

BodyweightNo adverse effect on bodyweight or bodyweight gains were detected.

Food Consumption. No adverse effect on dietary intake or food efficiency was detected.

Water Consumption. Daily visual inspection of water bottles revealed no intergroup differences.

Haematology.There were no toxicologically significant changes in the haematological parameters investigated.

Blood Chemistry.There were no toxicologically significant effects on the blood chemistry parameters investigated.

Urinalysis. No treatment-related urinalytical effects were detected.

Organ Weights. No toxicologically significant treatment-related organ weight changes were detected.

Necropsy. No treatment-related macroscopic abnormalities were detected.

Histopathology. No treatment-related changes were detected.

Conclusion.

Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of 1000 mg/kg/day did not result in any toxicologically significant changes. The ‘No Observed Effect Level’ (NOEL) was considered to be 1000 mg/kg/day.

In accordance with EU CLP Regulation (EC) No. 1272/2008, classification of this substance is not required for subacute repeat oral dose toxicity