Fatty acids, C16-18 and C18-unsatd., branched and linear, reaction products with diethanolamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31-Jul-2012 to 08-Mar-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to GLP in compliance with internationally accepted test guidelines

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isostearamide DEA

IUPAC Name:

Isostearamide DEA

Test material form:

other: Liquid

Details on test material:

- Identification: Isostearamide DEA
- Description: Clear amber liquid
- Batch Number: OE11124 (6/202593/00)
- Purity as Supplied: 100%
- Correction for Purity: No
- Salt/Base Correction Factor: None
- Expiry Date: Nov-2013 (handled by Harlan Laboratories Ltd. as 30-Nov-2013)
- Storage Conditions: At ambient temperature (20 ± 5 °C) in air in the dark, but may be used in the light
- Safety Precautions: Routine hygienic procedures (gloves, goggles, face mask)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group). Additionally, 2 male and 2 female reserve animals.
- Total Number of Animals: 46 males and 46 females
- Age (at delivery): 10 weeks
- Body Weight Range (at Start of Treatment): 315 to 357 g (males) and 194 to 227 g (females)
- Identification: Cage card and individual animal number (ear tattoo). On day 1 post partum, pups were individually tattooed with Indian ink.
- Randomization: Randomly allocated to groups by body weight
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier&Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK), batch CS-100099. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Analyses for contaminants were performed.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of representative samples were performed.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

DOSE FORMULATIONS

The dose formulations were not corrected for purity and were prepared as supplied by the Sponsor. The dose formulations were prepared weekly.

Isostearamide DEA was weighed into a glass beaker on a tared Mettler balance. The vehicle was added and the mixtures were stirred using a magnetic stirrer and used at room temperature (15 - 25 °C).

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS

- Stability of Dose Formulations: For at least one week, based upon the results of stability analyses performed during the non-GLP Harlan Laboratories study D55761.
- Storage of Dose Formulations: At room temperature (20 ± 5 °C) in brown glass bottles.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
- Frequency of Administration: Once daily
- Daily Target Dose Level: 0 mg/kg/day (Group 1), 20 mg/kg/day (Group 2), 100 mg/kg/day (Group 3) and 500 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Wistar rats, Harlan Laboratories study D55761.
- Dose Volume: 5 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (Group 1), 4 mg/mL/day (Group 2), 20 mg/mL/day (Group 3) and 100 mg/mL/day (Group 4)
- Duration of Treatment Period: At least 4 weeks (males) and approximately 7 weeks (females)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD

The dose formulations were analyzed using a method provided by the Sponsor. The samples (generally 2 g each) were delivered to the analytical laboratory.

- Transport of Dose Formulations to Analytical Laboratory: At ambient temperature (20 ± 5 °C)
- Storage of Dose Formulations in Analytical Laboratory: Refrigerated (ca. 5 ± 3 °C)

Samples of dose formulations were not discarded without the written authorization of the study director. The results were summarized by the responsible person for dose formulation analysis.

Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start as follows:

- Concentration and Homogeneity After Experimental Start: 07-Aug-2012
Group 1: b (middle)
Groups 2 to 4: a (top), b (middle) and c (bottom)

- Stability (after 4 Hours and 8 Days)
Groups 2 to 4: Stability 1 - d (4 hr at 20°± 5 °C) and Stability 2 - e (8 d at 20°± 5 °C)

- Concentration and Homogeneity During Week 6: 11-Sep-2012
Groups 2 to 4: a (top), b (middle) and c (bottom)

RESULTS

The Isostearamide DEA peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of Isostearamide DEA and, therefore, the absence of the test item in the vehicle control samples (corn oil) was confirmed.

The application formulations investigated during the study were found to comprise Isostearamide DEA in the range of 87.1% to 114.5% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of Isostearamide DEA in the preparations was approved because single results found did not deviate more than 12.5% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept up to eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item Isostearamide DEA and corn oil as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

The test item was used as analytical reference item.

Duration of treatment / exposure:

- Males: At least 4 weeks
- Females: Approximately 7 weeks

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 100 and 500 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

GENERAL

The purpose of this study was to generate preliminary information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

This study provided information to assess the need to conduct further investigations and guidance in the design of possible subsequent studies.

Rationale for Choice of Species, Route of Administration and Dose Levels

The rat is a suitable species for repeated dose and reproduction/developmental toxicity studies required by regulatory authorities. The oral route is one possible route for human exposure.

Dose levels were selected in agreement with the Sponsor, based on the results of a non-GLP dose range-finding study (Harlan Laboratories study D55761).

SCHEDULE FOR MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Blood Sampling: End of pre-pairing
- Pairing: 14 days maximum
-Treatment End: On day before sacrifice
- Necropsy: After treatment of at least 28 days, when no longer needed for assessment of reproductive effects

SCHEDULE FOR FEMALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Blood Sampling: End of pre-pairing
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment End: On day 3 post partum
- Necropsy: Dams and pups on day 4 post partum.

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

VIABILITY/MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

- Males: Weekly during pre-pairing period
- Females: Pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum.

No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS

- Males: performed once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage.
- Females: performed once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

FUNCTIONAL OBSERVATIONAL BATTERY

At one time during the study (males shortly before the scheduled sacrifice and females on day 3 post partum), relevant parameters from a modified Irwin screen test were performed with five P generation males and five P generation females from each group (five surviving animals with the lowest identification number) in place of the usual weekly behavioral observation. This FOB assessment was conducted following the daily dose administration.

Additionally, hind limb / fore limb grip strength, rectal temperature and locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 10-minute intervals over a period of 60 minutes. These data and the total activity over 60 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS

Blood samples were obtained at the end of the pre-pairing period from 5 males and 5 females from each group (five surviving animals with the lowest identification number). Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The assay was performed at Harlan Laboratories Ltd. (Füllinsdorf) under internal laboratory quality control conditions to assure reliable test results.

Day 14 (End of Pre-Pairing Period): 20-Aug-2012

The following hematology parameters were determined:

Complete Blood Cell Count:

- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count
- Platelet count

Coagulation

- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

Sacrifice and pathology:

TERMINATION AND NECROPSY

Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

All animals sacrificed or found dead were weighed and subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS

At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.

In addition, from 5 males and 5 females killed at the end of the study which were selected from each group (five surviving males with the lowest identification number and the first five females which gave birth) following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.

- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION

The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:

- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)
- Epididymides (in Bouin’s fixative)

The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:

In addition, from the 5 males and 5 females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:

- Gross lesions
- Brain
- Spinal cord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

From animal no. 37 only the reproductive organs were preserved and analyzed by error.

HISTOTECHNIQUE

All organ and tissue samples to be examined by the principal investigator for histopathology were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.

HISTOPATHOLOGY

Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the prinicipal investigator for histopathology. The same applied to all occurring gross lesions and to all animals which died spontaneously.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Because of possible test item-related findings noted in the high dose group, the liver in both sexes and the kidneys in males were examined also in five animals of intermediated groups. Histological examination of ovaries was carried out on any females that did not give birth.

A histopathology peer review was performed

Other examinations:

MATING, GESTATION AND LACTATION

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually, if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was initiated.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

REPRODUCTIVE AND OFFSPRING VIABILITY INDICES

From the on-line recorded reproduction data, the following parameters were calculated: mean precoital time, percentage mating, fertility index, conception rate, post-implantation loss, gestation index, birth index and viability index, dead/live pups at first litter check, pup sex ratio and viability index.

LITTER OBSERVATIONS

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

TERMINATION AND NECROPSY OF OFFSPRING

Pups were sacrificed on day 4 post partum by an injection of sodium pentobarbital. Dead pups, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Statistics:

The following statistical methods were used to analyze food consumption, body weights, locomotor activity, grip strength, body temperature, clinical laboratory data, organ weights, macroscopical findings, and reproduction data:

- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied, if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied, if the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day, salivation recorded in both genders during the study as well as ruffled fur in one male and one female for one day each.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 500 mg/kg/day, salivation recorded in both genders during the study as well as ruffled fur in one male and one female for one day each.

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased albumin and decreased globulin concentration in males at 500 mg/kg/day considered test item related and as a consequence also increased albumin/globulin ratio.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day, increased absolute and relative (to body weight) liver and kidney weights in males and increased liver weights relative to body weight in females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Increased incidence of enlarged livers in males at 500 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day minimal to slight centrilobular hepatocellular hypertrophy in males and females considered to be of metabolic nature and adaptive character and increased incidence of hyaline droplets of the kidneys in males.

Histopathological findings: neoplastic:

not examined

Details on results:

1. IN-LIFE DATA

VIABILITY / MORTALITY

One female in the control group died spontaneously on day 11 of the pre-pairing period. At necropsy, dark red discoloration of the lungs was recorded. Histologically, the respiratory disorder consisted of congestion and interstitial edema of the lung was recorded. The exact cause of death could not be established from the tissues and organs examined.

All other animals survived their scheduled study period.

DAILY CLINICAL SIGNS OR OBSERVATIONS

At 500 mg/kg/day, salivation was recorded in males at the end of the pairing period and in females during the gestation and lactation periods as well as in one female during the pre-pairing period. Ruffled fur was observed on one day in one male during the pre-pairing period and in one female during the gestation period.

At 100 mg/kg/day, one female had salivation for one day in the lactation period and a further female had chromodacryorrhea for the last 2 days of the lactation period. Since these findings were isolated, they were considered to be incidental.

No clinical signs of toxicological relevance were recorded in animals treated at 20 mg/kg/day.

DETAILED WEEKLY CLINICAL OBSERVATIONS

No toxicologically relevant clinical signs were noted during the weekly observations.

FUNCTIONAL OBSERVATIONAL BATTERY

No test item-related effects were recorded during functional observational battery in males and females at any dose level.

- Grip Strength: No effects of the treatment with the test item on grip strength were observed.
- Body Temperature: No effects of the treatment with the test item on body temperature were observed.
- Locomotor Activity: No effects of the treatment with the test item on locomotor activity were observed.

FOOD CONSUMPTION OF MALES

There were no differences of toxicological relevance between the mean food consumption of test item-treated or control males.

FOOD CONSUMPTION OF FEMALES

There were no differences of toxicological relevance between the mean food consumption of test item-treated or control females.

BODY WEIGHTS OF MALES

There were no differences of toxicological relevance between the mean body weights of test item-treated or control males.

BODY WEIGHTS OF FEMALES

There were no differences of toxicological relevance between the mean body weights of test item-treated or control females.

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

No changes in hematology parameters of toxicological relevance were observed at any dose level. Changes in a few parameters reaching statistical significance were not considered to be test item-related, because the values remained within the range of the historical control data and changes were not dose-related.

BIOCHEMISTRY

An increased albumin (+11%) and decreased globulin (-13%) concentration observed in males treated at 500 mg/kg/day was considered to be test item-related. As a consequence of these changes the albumin / globulin ratio was also increased (2.45 versus 2.04 in the controls).

No changes of toxicological relevance in biochemistry parameters were observed in males treated at 20 and 100 mg/kg/day or in females at any dose level.

Changes in a few parameters reaching statistical significance were not considered to be test item-related, because the values remained within the range of the historical control data and changes were not dose-related.

3. TERMINAL FINDINGS

ORGAN WEIGHTS

Increased absolute and relative (to body weight) liver (+19%/+15%) and kidney (+20%/+15%) weights were recorded in males treated at 500 mg/kg/day. Increased liver weights relative to body weight (+11%) were also recorded in females treated at 500 mg/kg/day.

No effects of the treatment with the test item on organ weights were recorded in animals treated at 20 and 100 mg/kg/day.

MACROSCOPICAL FINDINGS

Macroscopically, the incidence of enlarged livers was increased in males treated at 500 mg/kg/day (for details see table in the section “Any other information on results incl. tables” below).

HISTOPATHOLOGY FINDINGS

- Liver: Centrilobular hepatocellular hypertrophy was recorded at minimal to slight severity in both males and females treated at 500 mg/kg/day (for details see table in the section “Any other information on results incl. tables” below).
- Kidney: Incidence of hyaline droplets of the kidneys was increased in males treated at 500 mg/kg/day (for details see table in the section “Any other information on results incl. tables” below).
- Other Findings: No test item-related histological findings were recorded in the ovaries of females which did not give birth (animal nos.: 54, 59, 67 and 79). The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Sperm Staging:

No differences in the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

for general toxicity

Effect level:

100 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

for reproduction

Effect level:

500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No test item-related effects affected reproduction or development parameters.

3

Dose descriptor:

NOAEL

Remarks:

for development

Effect level:

500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No test item-related effects affected reproduction or development parameters.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

1. REPRODUCTION, BREEDING AND PUP DATA

 

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litter

 

Group (mg/kg/day)	1 (0)	2 (20)	3 (100)	4 (500)
Female numbers	45 - 55	56 - 66	67 - 77	78 - 88
Number of females paired	10	11	11	11
Number of females mated	10	11	11	11
Number of females which died before the scheduled necropsy (A)	1	0	0	0
Number of females not pregnant (B)	0	1	1	1
Number of females with implantation sites only (C)	1	0	0	0
Number of females which reared their pups until day 4 post partum	9	10	10	10

(A)      Female No. 49 died spontaneously on day 11 of the pre-pairing period.

(B)      Female Nos. 59, 67 and 79 were not pregnant.

(C)     Female No. 54 had implantation sites only.

 

MATING PERFORMANCE AND FERTILITY

 

9, 10, 10 and 10 females at 0, 20, 100, and 500 mg/kg/day respectively, were mated within the first or second pairing period.

 

The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 3.3, 3.7, 2.9 and 2.1 days at 0, 20, 100, and 500 mg/kg/day, respectively. The median precoital time was 3, 3, 4 and 1 days in order of ascending dose level. In the two females that mated during the second pairing period, precoital times were 3 and 12 days in at 20, and 100 mg/kg/day, respectively.

 

The fertility index was 90%, 91%, 91%, and 91% and the conception rate showed exactly the same values in the groups treated with 0 mg/kg/day, 20 mg/kg/day, 100 mg/kg/day or 500 mg/kg/day, respectively.

 

CORPORA LUTEA COUNT

 

Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (14.6, 14.3, 14.8 and 14.2 in order of ascending dose level) and gave no indication of a test item-related effect.

 

DURATION OF GESTATION

 

The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.1, 21.3, 21.7 and 21.3 days, in order of ascending dose level.

 

IMPLANTATION RATE AND POST IMPLANTATION LOSS

 

No effects on implantation rate were recorded at any dose levels.

 

A higher post-implantation loss was recorded in group 4 (15.2, as a percentage of implantation sites) when compared to the control group (6.8, as a percentage of implantation sites). This was mainly due to female No. 83, which had 8 post-implantation losses and No. 88, which had 5 post-implantation losses. Since all other females at this dose level had similar incidences of post-implantation losses as in the control group, the higher losses in females 83 and 85 were considered likely to be incidental.

 

LITTER SIZE AT FIRST LITTER CHECK

 

No effects on litter size and birth index were recorded at any dose level.

 

A lower mean litter size (10.6 vs. 12.1) recorded in group 4 when compared with the controls was not considered to be test item-related, because the reduction was within the range of the historical control data and it was not statistically significant.

 

A lower birth index (84.8% vs. 93.2%) recorded in group 4 was due to litters 83 and 88, which had incidentally higher post-implantation loss and was therefore not considered to be test item-related.

 

POSTNATAL LOSS DAYS 0 – 4 POST PARTUM

 

At 500 mg/kg/day, post natal loss was increased without statistical significance (mean 0.5 compared to 0.2 in the control group). However, the mean litter size was similar to the control group after controlling for 2 animals that had high post-implantation losses, which were not considered to be related to the test item.

 

At 100 mg/kg/day, post natal loss was also increased without statistical significance (mean 0.5). However, the mean litter size on day 4 post partum was similar to the control group and therefore this was not considered to be a test item-related effect.

 

No effects on postnatal loss were noted at 20 mg/kg/day.

 

EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related abnormal findings were noted at the first litter check or during the first 4 days post partum.

 

PUP SEX RATIOS

 

Sex ratios at the first litter check and on day 4 post partum were unaffected by exposure to the test item.

 

BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM

 

Mean pup weights were unaffected by treatment of the females with the test item.

 

MACROSCOPICAL FINDINGS OF PUPS

 

No macroscopic findings were observed in pups at necropsy.

 

2. MACROSCOPICAL AND MICROSCOPIC SUMMARY TABLES

 

MACROSCOPICAL FINDINGS

 

- Incidence of Enlargement in Liver

 

Finding	Group 1		Group 2		Group 3		Group 4
Incidence	11M	11F	11M	11F	11M	11F	11M	11F
Enlarged	1	-	2	-	1	-	4	-

 

MICROSOPIC FINDINGS

 

- Incidence and Mean Severity Grade of Main Findings in Liver

 

Finding	Group 1		Group 2		Group 3		Group 4
Incidence / Mean Severity	5M	5F	5M	5F	5M	5F	4M	5F
Hepatocellular hypertrophy	-	-	-	-	-	-	3/1.0	5/1.2

 

- Incidence and Mean Severity Grade of Main Findings in Kidneys

Finding	Group 1		Group 2		Group 3		Group 4
Incidence / Mean Severity	5M	5F	5M		5M		4M	5F
Hyaline droplets	1/1.0	-	1/1.0		-		3/1.3	-

Applicant's summary and conclusion

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Isostearamide DEA to rats. Isostearamide DEA was administered in corn oil as a vehicle at dosages of 20, 100, and 500 mg/kg body weight/day, and controls received the vehicle only. Isostearamide DEA was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 3 post partum.

One female in the control group died spontaneously on day 11 of the pre-pairing period. The exact cause of death could not be established from the tissues and organs examined.

At 500 mg/kg/day, salivation was recorded in males and females during the study as well as ruffled fur in one male and one female for one day each. Mean food consumption, body weight and body weight gain were not affected by treatment with the test item. Clinical laboratory investigations revealed an increased albumin and decreased globulin concentration in males. As a consequence of these changes, the albumin / globulin ratio was also increased.

At 500 mg/kg/day, an increased post natal loss was recorded, but it was not statistically significant. No test item-related effects were observed in the pups at this dose level.

Increased absolute and relative (to body weight) liver and kidney weights were recorded in males treated at 500 mg/kg/day. Increased liver weights relative to body weight were also recorded in females treated at this dose level. At macroscopical examination, the incidence of enlarged livers was increased in males. Microscopical examination revealed centrilobular hepatocellular hypertrophy at minimal to slight severity in both males and females. This lesion was considered to be of metabolic nature and adaptive character.

In addition, there was an increased incidence of hyaline droplets of the kidneys in males at this dose level. These hyaline droplets were considered to be induced by overload of synthetic protein, that’s specific in male rat like as alpha-2 microglobrin, derived from hyperfunction of the liver.

At 20 and 100 mg/kg/day, no test item-related effects were observed during the study.

Based on these results, a NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females was considered to be 100 mg/kg bw/day. The NOAEL for reproduction/developmental toxicity was considered to be 500 mg/kg bw/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of Isostearamide DEA on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Isostearamide DEA was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

The following dose levels were applied:

Group 1:     0 mg/kg body weight/day (control group)

Group 2:   20 mg/kg body weight/day

Group 3: 100 mg/kg body weight/day

Group 4: 500 mg/kg body weight/day

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 

One female in the control group died spontaneously on day 11 of the pre-pairing period. All other animals survived their scheduled study period.

 

At 500 mg/kg/day, salivation was recorded in males and females during the study as well as ruffled fur in one male and one female for one day each.

 

No further clinical signs of toxicological relevance were recorded.

 

FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS

 

No test item-related effects were observed from the functional observational battery or locomotor activity in males and females at any dose level.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 

There were no differences of toxicological relevance between the mean food consumption of test item-treated or control males or females.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 

There were no differences of toxicological relevance between the mean body weights of test item-treated or control males or females.

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

 

No changes in hematology parameters of toxicological relevance were observed at any dose level.

 

An increased albumin and decreased globulin concentration observed in males treated at 500 mg/kg/day was considered to be test item-related. As a consequence of these changes the albumin / globulin ratio was also increased.

 

No changes of toxicological relevance in biochemistry parameters were observed in males treated at 20 and 100 mg/kg/day or in females at any dose level.

 

REPRODUCTION AND BREEDING DATA OF PARENTAL ANIMALS

 

At 500 mg/kg/day, a slightly increased post natal loss was seen, but this difference was not statistically different from the control. No effects were seen in the individual pups.

 

At 20 and 100 mg/kg/day, no test item-related effects were observed in the reproduction and breeding data.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

 

Increased absolute and relative (to body weight) liver and kidney weights were recorded in males treated at 500 mg/kg/day. Increased liver weights relative to body weight were also recorded in females treated at this dose level.

 

No effects of the treatment with the test item on organ weights were recorded in animals treated at 20 and 100 mg/kg/day.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGY EXAMINATION OF PARENTAL ANIMALS

 

At macroscopical examination, the incidence of enlarged livers was increased in males treated at 500 mg/kg/day.

 

Microscopical examination revealed centrilobular hepatocellular hypertrophy at minimal to slight severity in both males and females treated at 500 mg/kg/day. This lesion was considered to be of metabolic nature and adaptive character.

 

In addition, there was an increased incidence of hyaline droplets in the kidneys in males at this dose level. These hyaline droplets were considered to be induced by overload of synthetic protein, that’s specific in male rat like as alpha-2 microglobrin, derived from hyperfunction of the liver.

 

No other test item-related findings were observed in males or females at any dose level.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related abnormal findings were noted at the first litter check or during the first 4 days post partum. Sex ratios at the first litter check and on day 4 post partum were unaffected by exposure to the test item.

 

BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM

 

Mean pup weights were unaffected by treatment of the females with the test item.

 

MACROSCPICAL FINDINGS OF PUPS

 

No macroscopic findings were observed in pups at necropsy.

 

CONCLUSION

 

Based on these results, a NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females was considered to be 100 mg/kg bw/day. The NOAEL for reproduction/developmental toxicity was also considered to be 500 mg/kg bw/day.