Fatty acids, C16-18 and C18-unsatd., Me esters

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:

From 2010, April 05 to 2010, June 11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP with certificate.The study is clear and complete in any parts. The substance is well identified, the method is suitable for the substance.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2010, April 11 to 2010, May 30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP with certificate. The study is clear and complete in any parts. The substance is well identified, the method is suitable for the substance. Used for read across

Reason / purpose for cross-reference:

reference to same study

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:

Test Item Name: C16-C18 and C18 Unsaturated Alkyl Methyl EsterBatch/Lot No: 110-1ECAS Number: 67762-38-3
Description: Yellow Liquid
Expiry / Retest: A sample of the test item was sent to ASG Analytik Service forester content (stability testing) following the end of the dosingperiod.
Purity: 99.35%Storage Conditions: 2-8°C protected from light

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
One batch of 42 male and 42 female Sprague-Dawley rats (Crl: CD®(SD) strain) were received on 30 March 2010 from Charles River UK Limited, Margate, Kent, UK. The animals were ca 6 weeks of age and weighed 201-239 g for males and 150-179 g for females, on despatch.

ANIMAL IDENTIFICATION
Each animal received a subcutaneously implanted electronic chip, which identified it individually within the study, and which corresponded to that animal’s number. Each animal was given a cage card which was colour coded for treatment group, and was marked with the study number, cage and animal numbers, sex and relevant treatment group. In addition, a second cage card was given to each animal identifying that animal by study
number, neurotox identification, animal number and sex. This cage card was not colour coded.

ACCLIMATISATION
The animals were acclimatised in the Charles River animal room for 12 days prior to commencement of treatment. All the animals were examined on arrival for signs of abnormality or disease. No such signs were found and the animals were accepted for use on the study.

ENVIRONMENTAL CONDITIONS
There was automatic control of light cycle, temperature and humidity in the animal room. Light hours were 0700-1900 h. The target ranges for temperature and humidity were 21°C ± 2°C and 55% ± 15% respectively, with a minimum of 15 air changes per hour.
Daily monitoring indicated that temperature remained within the target range of 21°C ± 2°C (actual range 20°C-23°C) and humidity was slightly above the target range on 6 occasions (actual range 35-68%). This deviation from target range was considered to be minor and had no significant impact on the outcome and integrity of the study.

ROOM SANITATION
Each day, floors were swept and then mopped with a 0.5% solution of Tego 2000 (Th.Goldschmidt Limited, Ruislip, Middlesex, UK), an amphoteric biocide/cleanser. The room was washed with this solution at approximately weekly intervals.

CAGING
The animals were initially housed 2 per cage, in polycarbonate cages, with solid bottoms and stainless steel mesh tops and measured ca 48 x 37.5 x 25 cm. A stainless steel food hopper and a polycarbonate water bottle were provided for each cage and sterilised wood shavings were provided as bedding. Male and female cages were racked separately. A few days prior to pairing for mating, males were transferred to individual cages of a
stainless steel grid insert measuring ca 48 x 37.5 x 25 cm. Excreta were collected on a tray lined with absorbent paper suspended beneath each cage. The mated females were transferred to individual solid bottomed cages measuring ca 48 x 37.5 x 25 cm. White paper tissue was supplied as nesting material from Day 20 of gestation. Females with litters retained this cage type until termination. After mating the males remained singly housed until termination.

CAGE SANITATION
Cages, absorbent papers and water bottles were changed at regular intervals, as appropriate. White tissue paper nesting material was changed when it was unacceptably soiled. Clean wood shavings were provided at each change of solid-bottomed cage.

ENVIRONMENTAL ENRICHMENT
To provide environmental enrichment, wooden chewsticks were made available to the animals as appropriate.

DIET AND WATER
Rat and Mouse Breeder Diet No. 3 (Expanded) SQC supplied by Special Diets Services Limited, Stepfield, Witham, Essex, UK was available to the animals ad libitum. The diet was supplied with a batch analysis for nutritive constituents and a range of significant contaminants. The analytical certificate for a batch of diet used in this study is retained in the study archive.
Due to technical error, Animal 74 (Group 4 female) was fed expired diet from 02-07 June 2010 prior to terminal kill. The diet given expired on the 01 June 2010; therefore this deviation was considered to be minor given that the food had expired on the previous day to use and the short length of time the animal had consumed the expired diet. This protocol deviation had no impact on the outcome or integrity of the study.
The food was not considered to contain any additional substances in sufficient concentration to influence the outcome of the study.
The animals had access to domestic, mains quality water ad libitum. The supply is analysed regularly for dissolved and suspended materials, including a range of significant contaminants. The analytical certificate for a typical recent analysis is retained in the study archive.

TREATMENT GROUP
Cages were allocated to treatment group by the use of randomly sequenced numbers, in such a way that each complete rack contained representatives from all treatment groups.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

DOSE LEVELS
Dose levels were agreed upon with the Sponsor after evaluation of existing relevant toxicological data, including Charles River Study 495283; a one week dose range finding study in rats. Results from this study indicated no adverse effect of treatment at 2000 mg/kg/day. However, for the purposes of this study a high dose level of 1000 mg/kg/day was considered appropriate.

ROUTE AND MEANS OF ADMINISTRATION
The animals were dosed once daily by oral gavage at a dose volume of 5 mL per kg body weight, using a plastic gavage. The volume to be administered to each animal was determined on each day by the weight of the animal as measured at the time of administration, except during late gestation; from Day 16 of gestation until parturition was complete, the dose volume of the females was determined by the weight of the animal on Day 16 of gestation.
The males were dosed once daily for 4 weeks overall, commencing 2 weeks prior to mating. The females were dosed once daily from 2 weeks prior to mating then continued until at least Day 4 of lactation. Dosing for males and females continued until the day prior to termination.

Details on mating procedure:

MATING PROCEDURE
A few days prior to the initiation of mating, the males were separated into individual grid bottomed cages.
Pairing was on a 1 male to 1 female basis, within the same treatment group. Each female was transferred to the cage of an appropriate co-group male near the end of the working day, and remained there until mating was detected or 14 nights had elapsed.
Vaginal lavages were taken daily early each morning from the day of pairing until mating had occurred and the stage of oestrus observed in each lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated Day 0 of gestation.
The time taken for each female to show a positive mating sign was evaluated.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

4 weeks for males,
2 weeks before pairing, during pairing, during gestation and until 4 days post partum for female

Frequency of treatment:

Once daily

Details on study schedule:

OBSERVATIONS OF FEMALES WITH LITTERS DURING LACTATION
The females were allowed to litter normally. The day of birth of the litter was designated Day 0 of lactation. The duration of gestation in days was calculated and evaluated. The numbers of live and dead pups born in each litter were recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily up
to Day 4 of lactation. Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation. Pups killed on Day 7 of lactation were also weighed en masse by sex; this additional data has not been reported and will be retained in the raw data. This protocol deviation had no significant impact on the outcome or integrity of the study.
When possible, any pups that were found dead or killed during lactation were sexed and appropriately examined as above. Any externally normal decedent pups were discarded. Any deficiencies in maternal care were recorded. Points looked for were inadequate construction and cleaning of the nest, pups left scattered and cold, physical harm of pups, or apparently inadequate lactation or feeding. Detailed information is retained in the study data.

Remarks:

Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested

Remarks:

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

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

All adults were subject to a detailed necropsy under the guidance of a veterinary pathologist. The necropsy consisted of an external and internal examination to include body orifices (ears, nostrils, mouth, anus, vulva) and cranial, thoracic and abdominal organs and tissues. All gross lesions were recorded in terms of location, size, shape, colour, consistency and number.

Adults were examinated for the followings (for more details, see the related ) Repeated doese toxicity study):
Mortality check
Clinical observation
Body weight
Food consumption
Water consumption
Ophthalmic examinations
Functional observation
Function test
Hematology
Coagulation
Clinical chemistry
Histological
Bone marrow smears



All observation didn't reveal any effect related to the treatment

Litter observations:

Mating performance or fertility (as measured by the fertility indices) was not affected by
treatment.
There were no obvious effects on the duration of gestation at any dose level applied.
There were no effects on the number of live young born at any of the dose levels, or on the
mean number of implant sites per pregnancy.

The following abnormalities were observed among pups:
Litter 45 (Group 1): Day 1-2: One female pup with no tail. Pup was found dead on Day 2.
The following observations were observed among pups:
The following is a summary of the observations recorded among pups. Full details of the
observations noted within the litters are retained within the raw data.
Litter 41 (Group 1): Day 1: One female pup with bruising to the head.
Litter 47 (Group 1): Day 1-2: One female pup scabbing on head
Litter 48 (Group 1): Day 3: One male pup scabbing present on muzzle
Litter 58 (Group 2): Day 1-2: One male pup scabbing on anus.
Litter 58 (Group 2): Day 1: One male and one female pup bruising on dorsal surface.
Litter 61 (Group 3): Day 1: One male pup had bruising on muzzle
Litter 74 (Group 4): Day 1-2: One male pup with bruising to the back.
Litter 74 (Group 4): Day 3-7: One male pup with scabbing to the muzzle
Litter 79 (Group 4): Day 2-3: One male pup with bruising to the dorsal surface.
These observations were considered to be typical for pre-weanlings.

Litter Survival, Litter and Pup Weights:
At 1000 mg/kg/day the survival of pups (as indicated by the viability index) was slightly
lower (88%) compared to Control. This was due to Animal 72 which had a total litter loss;
this incidence and percentage are well within the background data for this laboratory.
Mean litter and pup weights in treated groups were similar to Control.

Postmortem examinations (parental animals):

See repeated toxicity study results

Postmortem examinations (offspring):

Offspring killed or found dead were sexed, then checked for the presence of milk in the stomach and the presence of externally visible abnormalities. Any abnormal pups were preserved in 10% formalin or methylated ethyl alcohol as appropriate for possible future examination. Externally normal decedents were discarded.

Reproductive indices:

The following indices of fertility were evaluated:

For each group:
Fertility Index (male) = Number siring a litter/Number paired
Fertility Index (female) = Number pregnant/Number paired
Gestation Index = Number bearing live pups/Number pregnant

For each litter and group:
Birth Index = Total number of pups born (live and dead)/Number of implantation scars
Live Birth Index =Number of pups live on Day 0 of lactation/Total number born (live and dead)
Viability Index = Number of pups live on Day 4 of lactation/Number live on Day 0

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: none adverse effect

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

1000 mg/kg/day

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: none adverse effect

Reproductive effects observed:

not specified

Conclusions:

The tested substance revealed no effect in combined Repeated Dose Toxicity Study with the reproduction/Developmental Toxicity screening Test in Ratsfor a dose of until 1000 mg/kg/bw

Executive summary:

A total of 80 rats (40 males and 40 females) Sprague-Dawley rats,Crl CD® (SD)were allocated to four groups, three of which received the test item, Heptadecanoic Fatty Acid Methyl Ester (C₁₆-C₁₈) while the other group received the vehicle (corn oil) by oral route (gavage) once daily under a dosage volume of 5 mL/kg. Animals will be dosed for 2 weeks prior to pairing, during pairing, during gestation and until at least day 4 post-partum for females or until sacrifice for non pregnant females and until necropsy for males following 4 weeks of treatment.

The following dose-levels were used:

. Group 1 (10 males and 10 females): 0 mg/kg/day,

. Group 2 (10 males and 10 females):100mg/kg/day,

. Group 3 (10 males and 10 females):300mg/kg/day,

. Group 4 (10 males and 10 females):1000mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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:

Fatty acids, C16-18 and C18-unsatd., Me esters

EC Number:

267-015-4

EC Name:

Fatty acids, C16-18 and C18-unsatd., Me esters

Cas Number:

67762-38-3

Molecular formula:

UVCB substance, not univocal molecular formula available

IUPAC Name:

UVCB substance, no IUPAC name avalilable Chemical name: C16-C18 and C18 unsaturated alkyl carboxylic acids methyl esters

Details on test material:

Test Item Name: C16-C18 and C18 Unsaturated Alkyl Methyl Ester
Batch/Lot No: 110-1E
CAS Number: 67762-38-3
Description: Yellow Liquid
Expiry / Retest: A sample of the test item was sent to ASG Analytik Service for
ester content (stability testing) following the end of the dosing
period.
Purity: 99.35%
Storage Conditions: 2-8°C protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
One batch of 42 male and 42 female Sprague-Dawley rats (Crl: CD®(SD) strain) were received on 30 March 2010 from Charles River UK Limited, Margate, Kent, UK. The animals were ca 6 weeks of age and weighed 201-239 g for males and 150-179 g for females, on despatch.

ANIMAL IDENTIFICATION
Each animal received a subcutaneously implanted electronic chip, which identified it individually within the study, and which corresponded to that animal’s number. Each animal was given a cage card which was colour coded for treatment group, and was marked with the study number, cage and animal numbers, sex and relevant treatment group. In addition, a second cage card was given to each animal identifying that animal by study
number, neurotox identification, animal number and sex. This cage card was not colour coded.

ACCLIMATISATION
The animals were acclimatised in the Charles River animal room for 12 days prior to commencement of treatment. All the animals were examined on arrival for signs of abnormality or disease. No such signs were found and the animals were accepted for use on the study.

ENVIRONMENTAL CONDITIONS
There was automatic control of light cycle, temperature and humidity in the animal room. Light hours were 0700-1900 h. The target ranges for temperature and humidity were 21°C ± 2°C and 55% ± 15% respectively, with a minimum of 15 air changes per hour.
Daily monitoring indicated that temperature remained within the target range of 21°C ± 2°C (actual range 20°C-23°C) and humidity was slightly above the target range on 6 occasions (actual range 35-68%). This deviation from target range was considered to be minor and had no significant impact on the outcome and integrity of the study.

ROOM SANITATION
Each day, floors were swept and then mopped with a 0.5% solution of Tego 2000 (Th.Goldschmidt Limited, Ruislip, Middlesex, UK), an amphoteric biocide/cleanser. The room was washed with this solution at approximately weekly intervals.

CAGING
The animals were initially housed 2 per cage, in polycarbonate cages, with solid bottoms and stainless steel mesh tops and measured ca 48 x 37.5 x 25 cm. A stainless steel food hopper and a polycarbonate water bottle were provided for each cage and sterilised wood shavings were provided as bedding. Male and female cages were racked separately. A few days prior to pairing for mating, males were transferred to individual cages of a
stainless steel grid insert measuring ca 48 x 37.5 x 25 cm. Excreta were collected on a tray lined with absorbent paper suspended beneath each cage. The mated females were transferred to individual solid bottomed cages measuring ca 48 x 37.5 x 25 cm. White paper tissue was supplied as nesting material from Day 20 of gestation. Females with litters retained this cage type until termination. After mating the males remained singly housed until termination.

CAGE SANITATION
Cages, absorbent papers and water bottles were changed at regular intervals, as appropriate. White tissue paper nesting material was changed when it was unacceptably soiled. Clean wood shavings were provided at each change of solid-bottomed cage.

ENVIRONMENTAL ENRICHMENT
To provide environmental enrichment, wooden chewsticks were made available to the animals as appropriate.

DIET AND WATER
Rat and Mouse Breeder Diet No. 3 (Expanded) SQC supplied by Special Diets Services Limited, Stepfield, Witham, Essex, UK was available to the animals ad libitum. The diet was supplied with a batch analysis for nutritive constituents and a range of significant contaminants. The analytical certificate for a batch of diet used in this study is retained in the study archive.
Due to technical error, Animal 74 (Group 4 female) was fed expired diet from 02-07 June 2010 prior to terminal kill. The diet given expired on the 01 June 2010; therefore this deviation was considered to be minor given that the food had expired on the previous day to use and the short length of time the animal had consumed the expired diet. This protocol deviation had no impact on the outcome or integrity of the study.
The food was not considered to contain any additional substances in sufficient concentration to influence the outcome of the study.
The animals had access to domestic, mains quality water ad libitum. The supply is analysed regularly for dissolved and suspended materials, including a range of significant contaminants. The analytical certificate for a typical recent analysis is retained in the study archive.

TREATMENT GROUP
Cages were allocated to treatment group by the use of randomly sequenced numbers, in such a way that each complete rack contained representatives from all treatment groups.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

DOSE LEVELS
Dose levels were agreed upon with the Sponsor after evaluation of existing relevant toxicological data, including Charles River Study 495283; a one week dose range finding study in rats. Results from this study indicated no adverse effect of treatment at 2000 mg/kg/day. However, for the purposes of this study a high dose level of 1000 mg/kg/day was considered appropriate.

ROUTE AND MEANS OF ADMINISTRATION
The animals were dosed once daily by oral gavage at a dose volume of 5 mL per kg body weight, using a plastic gavage. The volume to be administered to each animal was determined on each day by the weight of the animal as measured at the time of administration, except during late gestation; from Day 16 of gestation until parturition was complete, the dose volume of the females was determined by the weight of the animal on Day 16 of gestation.
The males were dosed once daily for 4 weeks overall, commencing 2 weeks prior to mating. The females were dosed once daily from 2 weeks prior to mating then continued until at least Day 4 of lactation. Dosing for males and females continued until the day prior to termination.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

4 weeks for males,
2 weeks before pairing, during pairing, during gestation and until 4 days post partum for female

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested

Doses / concentrations 4

Remarks:

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

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

MORTALITY CHECKS
All the animals were checked for viability early in the morning and again as late as possible on each day.

CLINICAL OBSERVATIONS
All the animals were examined for reaction to treatment on each day from commencement of dosing. The onset, intensity and duration of any signs were recorded. In addition, once each week (starting during Pretrial), all adult animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.
BODY WEIGHTS
For both sexes, body weights were recorded one week prior to the start of treatment then daily throughout the dosing period until termination.
For brevity of reporting, only weekly weights for males and for females during the pre-mating period have been presented. Additionally, weights for females on Days 0, 7, 14, 16 and 20 of gestation and Days 1 and 4 of lactation have been presented.

FOOD CONSUMPTION
For all animals, the weight of food consumed by each cage was recorded once weekly commencing during the week prior to the start of dosing, until pairing for mating. For mated females, the amount of food consumed was recorded over Days 0-7, 7-14 and 14-20 of gestation, and Days 0-4 of lactation.

WATER
Water consumption was monitored by visual inspection of the water bottles on a weekly basis throughout the study. No intergroup differences were noted, therefore measuring water consumption gravimetrically twice weekly was not considered necessary.

OPHTHALMIC EXAMINATION
The eyes were examined using indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl®). The following areas were evaluated: anterior, lenticular and fundic areas. An ophthalmic examination was undertaken from all animal during Pretrial, all males during Week 4 and all females shortly prior to sacrifice.

FUNCTIONAL OBSERVATION
Once during the pre-treatment week (Week -1) and weekly thereafter, a more detailed examination was made on all animals. These examinations were conducted by a technician not involved in the dosing procedures or in the collection of body weight and food consumption data, and were performed at an approximately standardised time of day. Before the independent technician entered the animal room on each occasion to perform the examinations, the cage card showing treatment group location was removed from each cage, leaving the second pre-prepared card as the neurotox animal identifier. In each cage one or two animals will have their tail marked to allow the independent technician to identify each animal.

CAGESIDE OBSERVATION
Posture/condition on first approach (animal undisturbed), checking for:
Prostration
Lethargy
Writhing
Circling
Breathing abnormalities
Gait abnormalities
Tremor
Fasciculation
Convulsions
Biting (of cage components or self mutilating)
Vocalisations
Piloerection
Ease of removal from cage.
Body temperature:
This was taken and recorded from the implanted electronic identification chip. If the electronic chip was not functioning, a rectal temperature was recorded.
Condition of the eyes, checking for:
Pupillary function (reaction to visual stimulus)
Miosis
Mydriasis
Page 20
Test Facility Study No. 495325
Exophthalmos
Encrustation
Lacrimation
Condition of the coat.
Presence of salivation.
Overall ease of handling.
Observations in a standard arena (2 min observation period):
Latency (time to first locomotory movement).
Level of mobility.
Rearing.
Grooming.
Urination/defecation.
Arousal (level of alertness).
Posture, tremor/convulsions, vocalisation, piloerection – recorded as for
cageside observatons.
Palpebral closure.
Gait abnormalities.
Stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

FUNCTION TEST
The following additional functional assessments were performed on 5 males and 5 females per group during Week 4 for males (prior to blood sampling) and during lactation for females. For males, the animals selected were the first 5 males in the group. For females, the animals
selected for assessment were the first 5 females to reach at least Day 3 of lactation. Again, these assessments were performed at an approximately standardised time of day. Reaction to sudden sound (click above the head) Reaction to touch on the rump with a blunt probe

GRIP STRENGHT
This was measured using a method derived from that of Meyer et al (1979). A strain gauge was used, to which was attached a wire pull-bar. Once the animal gripped the bar, the body was pulled until its grasp was broken; the strain gauge recorded the force required. The procedure was repeated 3 times for the forelimbs and 3 times for the hindlimbs, and the mean fore and hind grip strengths calculated.

PAIN PERCEPTION
This was assessed by measurement of the tail flick response, using a technique based on the method devised by D’Amour and Smith (1941). The apparatus used shined a calibrated infrared heat source onto the tail and automatically measured the reaction time of the animals (accurate to 0.1 s). It was ensured that no visible injury was caused to the tail by this test.

LAMDING FOOT SPLAY
Maize oil was applied to the hind paws of each animal. The animal was then held in a horizontal, prone position with the nose ca 30 cm above a bench surface covered with absorbent paper. When the animal was calm, it was dropped. The distance between the prints of the central footpads was measured. The procedure was repeated 3 times. If the rat did not land properly on its feet, it was recorded.

MOTOR ACTIVITY
Each animal was placed in an individual monitoring cage, scanned by a motion sensor utilising infra-red pyroelectric detectors. Movement was detected in 3 dimensions anywhere in the cage, and was differentiated into large and small movements. Each animal was monitored for one session for 1 h for males and for females it was 0.5 h. Activity counts were recorded over successive periods of 5 min each.

OTHER PHYSICAL/FUNCTIONAL ABNORMALITIES
Any other abnormality not already recorded in the above screening battery.

Sacrifice and pathology:

TERMINAL PROCEDURES
The males were sacrificed following 4 weeks of treatment. The females were sacrificed with their litters between Days 5 and Day 7 of lactation.
Adult animals were sacrificed by exposure to carbon dioxide followed by exsanguination. The pups were sacrificed by intra-peritoneal or intra-thoracic injection of sodium pentobarbitone.

CLINICAL PATHOLOGY

TIMING OF INVESTIGATION
Samples were obtained during Week 4 for males, and on or after Day 3 of lactation for females.
The animals were not deprived of food overnight prior to sampling.

ANIMAL USED
Blood samples were obtained from 5 males and 5 females from each dose group. For males, the first 5 animals in each group were tested. For females, the first 5 animals to have reared their litter to at least Day 3 of lactation were tested.

BLOOD SAMPLES AND PARAMTERS INVESTIGATED
Samples for haematology, coagulation and clinical chemistry were collected via the tail vein after careful cleaning of the sampling site to avoid any possible contamination. Sampling was by sterile, disposable plastic syringes and vygon infusion sets. Blood was then transferred into plastic tubes containing anticoagulant as follows:

HEMATOLOGY
Ca 0.5 mL was taken into tubes containing EDTA and assayed for:
Haemoglobin
Red Blood Cell Count
Haematocrit
White Blood Cell Count
Mean Cell Volume
Mean Cell Haemoglobin
Mean Cell Haemoglobin Concentration
Platelets
Red Cell Distribution Width
Reticulocytes
Differential White Blood Cell Count:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Large Unclassified Cells
A blood film smear was produced from each haematology specimen. Blood smears were labelled, stained for possible examination and stored and archived. No abnormal haematological findings were obtained or treatment related effects observed. Blood cell morphology, therefore, was not performed.

COAGULATION
Ca 0.9 mL blood into tubes containing 0.1mL 3.8% (w/v) trisodium citrate. The final sample volume will be as close as possible to 1.0 mL to give a final concentration of 0.38% (blood to citrate ratio of 9:1). The citrated blood samples will be centrifuged and the plasma separated into plain plastic tubes and analysed for:
Prothrombin Time
Activated Partial Thromboplastin Time
Fibrinogen

CLINICAL CHEMISTRY

Ca 1.0 mL into tubes containing lithium heparin which were then centrifuged and assayed for:
Urea
Glucose
Aspartate Aminotransferase
Alanine Aminotransferase
Alkaline Phosphatase
Gamma Glutamyl Transferase
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Test Facility Study No. 495325
Glutamate Dehydrogenase
Lactate Dehydrogenase
Sodium
Potassium
Chloride
Total Protein
Albumin
Globulin – derived
AG Ratio – derived
Cholesterol
Triglycerides
Creatine Kinase
Creatinine
Total Bilirubin
Calcium
Phosphate


MATING PROCEDURE
A few days prior to the initiation of mating, the males were separated into individual grid bottomed cages.
Pairing was on a 1 male to 1 female basis, within the same treatment group. Each female was transferred to the cage of an appropriate co-group male near the end of the working day, and remained there until mating was detected or 14 nights had elapsed.
Vaginal lavages were taken daily early each morning from the day of pairing until mating had occurred and the stage of oestrus observed in each lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated Day 0 of gestation.
The time taken for each female to show a positive mating sign was evaluated.

OBSERVATIONS OF FEMALES WITH LITTERS DURING LACTATION
The females were allowed to litter normally. The day of birth of the litter was designated Day 0 of lactation. The duration of gestation in days was calculated and evaluated. The numbers of live and dead pups born in each litter were recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily up
to Day 4 of lactation. Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation. Pups killed on Day 7 of lactation were also weighed en masse by sex; this additional data has not been reported and will be retained in the raw data. This protocol deviation had no significant impact on the outcome or integrity of the study.
When possible, any pups that were found dead or killed during lactation were sexed and appropriately examined as above. Any externally normal decedent pups were discarded. Any deficiencies in maternal care were recorded. Points looked for were inadequate construction and cleaning of the nest, pups left scattered and cold, physical harm of pups, or apparently inadequate lactation or feeding. Detailed information is retained in the study data.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

2 PREMATURE DECEDENTS THROUGHOUT THE STUDY, NOT POSITIVELY RELATED TO THE TREATMENT

Mortality:

no mortality observed

Description (incidence):

2 PREMATURE DECEDENTS THROUGHOUT THE STUDY, NOT POSITIVELY RELATED TO THE TREATMENT

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Slight intergroup differences in the body weight gain of the males were too small to be attributed to treatment with C16-C18 and C18 Unsaturated Alkyl Methyl Ester.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Group mean food consumption in all treated males was similar to Control. Group mean food consumption for females prior to mating and throughout gestation and lactation were similar to Control.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmic findings which were considered to be related to treatment with C16-C18 and C18 Unsaturated Alkyl Methyl Ester

Haematological findings:

no effects observed

Description (incidence and severity):

In males treated at 100 mg/kg/day, there was a statistically significant increase in % red cell distribution width. In the absence of any other changes in haematological parameter this increase was considered to be incidental and not related to treatment

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In males treated at 100 mg/kg/day, there was a statistically significant increase in potassium levels. In the absence of any other changes in clinical chemistry parameter indicators and a dose level response; this increase was considered to be incidental

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no significant neurotoxicity clinical observations noted in any of the treated groups in either sex compared to Control

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Slight intergroup differences in male group mean organ weights were too small to be attributed to treatment (P>0.05). At 1000 mg/kg/day there was an increase in female absolute heart weight which did not attain statistical significance (P>0.05).

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no histology findings that could be attributed to treatment with C16-C18 and C18 Unsaturated Alkyl Methyl Ester

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The tested substance revealed no effect in Repeated dose oral toxicity for a dose of until 1000 mg/kg/bw

Executive summary:

A total of 80 rats (40 males and 40 females) Sprague-Dawley rats,Crl CD® (SD)were allocated to four groups, three of which received the test item, Heptadecanoic Fatty Acid Methyl Ester (C₁₆-C₁₈) while the other group received the vehicle (corn oil) by oral route (gavage) once daily under a dosage volume of 5 mL/kg. Animals will be dosed for 2 weeks prior to pairing, during pairing, during gestation and until at least day 4 post-partum for females or until sacrifice for non pregnant females and until necropsy for males following 4 weeks of treatment.

The following dose-levels were used:

. Group 1 (10 males and 10 females): 0 mg/kg/day,

. Group 2 (10 males and 10 females):100mg/kg/day,

. Group 3 (10 males and 10 females):300mg/kg/day,

. Group 4 (10 males and 10 females):1000mg/kg/day.