D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C18-C18 (unsaturated) acyl] derivs.

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-01-08 to 2014-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000

Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(6E)-N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadec-6-enamide; N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadecanamide

EC Number:

940-373-5

Cas Number:

1591782-99-8

Molecular formula:

C25H49NO6 (C18 unsatd.) C25H51NO6 (C18 satd.)

IUPAC Name:

(6E)-N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadec-6-enamide; N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadecanamide

Test material form:

other: Waxy solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 10-11 weeks old, females: 10-11 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 281 - 333 g (mean: 308.00 g, ± 20% = 246.40 – 369.60 g)
females: 186 - 236 g (mean: 203.80 g, ± 20% = 163.04 – 244.56 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on
Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3°C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0801)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls
at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H,
polysulphone cages on Altromin saw fibre bedding (lot no. 260913)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. All animals were healthy.
Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most
homogenous variation in body weight throughout the groups of males and females.

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

Preparation of the Test Item Formulation
The test item was weighed into a tarred plastic container on a suitable precision balance and carefully transferred to a porcelain mortar.
Required amount of vehicle (corn oil) was added to the mortar and mixed thoroughly with pestle. This procedure was repeated each day for
the formulation preparation. The vehicle was selected as suggested by the sponsor based on the test item’s characteristics.
The test item formulation was prepared freshly on each administration day before the administration procedure.
The vehicle was also used as control item.

Experimental Groups and Doses
The doses for this study were chosen by the study monitor based on repeated dose toxicity study with the analogue test substance.
The doses used for the study with analogue test substance were 100, 250 and 625 mg/kg body weight/ day. Similar dose levels were chosen
for this study after discussion with study monitor. The following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose,
HD = high dose) and 1 control group (C). The animals were treated with the test item formulation or vehicle on 7 days per week for a period of
54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up
to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed.

C 0 mg/kg bw/ day
LD 125 mg/kg bw/ day
MD 300 mg/kg bw/ day
HD 650 mg/kg bw/ day

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending
sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle at the dose volume of
5 mL/ kg body weight.

Administration of Doses
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was
5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of
the mating period to confirm the pregnancy. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle
were analysed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating),
5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples).
Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and
5 (12 samples). Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours
after the preparation (at room temperature), from high and low dose formulations (4 samples).
All formulation samples were collected and were stored at -20° C. These samples were analysed after the completion of the toxicity study at
BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 136166.

Duration of treatment / exposure:

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days,
i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3
in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed.

Frequency of treatment:

7 days/week

Details on study schedule:

Arrival of the Test Item: 18 October 2013
Study Initiation Date: 08 January 2014
1st Amendment to Study Plan: 28 May 2014
Experimental Starting Date: 17 January 2014
Experimental Completion Date: 11 March 2014
Completion Date of Delegated Phase (Histopathology): 11 November 2014
Completion Date of Delegated Phase (Formulation Analysis): 01 August 2014

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 125, 300, 650 mg/kg Body weight/day
Basis:
nominal conc.

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day after dosing. The health condition of the
animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when
observations were made once daily.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation,
diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait,
posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre
behaviour were recorded.

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the
treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within
24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed
prior to the sacrifice. Food consumption was measured weekly on the corresponding days of the body weight measurements after the
beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the
post-mating period in males.

Litter observations:

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after
delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups
were identified by tattoo mark on paw. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.

Postmortem examinations (parental animals):

Pathology
All surviving male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while
female animals were sacrificed on post-natal day using an anaesthesia (ketamine/xylazin, 2:1, Pharmanovo, lot no: 24664, expiry date: 06/2015
and Serumwerk, lot no: 00512, expiry date: 07/2014 and lot no: 00513, expiry date: 05/2015) was used.
Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities and killed by decapitation.
Non-pregnant females were sacrificed on study day 26 from the day of evidence of mating.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices
and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex
organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were
preserved in 10 % neutral buffered formalin, except for testes and epididymides which were preserved in modified Davidson’s Solution and
then transferred in 70% ethanol. All organs (all gross lesions, lung, brain, urinary bladder, stomach, lymph nodes (mesenteric and axillary),
small and large intestines (including Peyer´s patches), trachea, liver, kidneys, thymus, adrenal glands, spleen, heart, ovaries, testes,
accessory sex organs (prostate, seminal vesicle with coagulating gland), vagina, uterus with cervix, epididymides) were collected from the
animal number 64 which was sacrificed prematurely for animal welfare reasons due to the presence of tumor mass in the left flank.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights
The testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland as a whole) of all male adult animals
as well as the ovaries, uterus with cervix of all female adult animals were weighed. Paired organs were weighed together.
Organ weight of animal killed prematurely was not taken.

Histopathology
A full histopathology was carried out on the preserved organs and tissues of animal which was killed prematurely due to the presence of
tumor mass on the left flank. Testes, epididymides, prostate, seminal vesicle with coagulating gland, ovaries, uterus with cervix, vagina were
trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and examined in
Control and HD animals and in non pregnant female animal (no. 58) and its mating parter male (no. 18) of the LD group.
All gross lesion macroscopically identified was examined microscopically in all animals. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
 
Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services,
Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified
contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test
site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1]
(Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the
corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of
compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a
pathology phase report to the study director upon the completion of the study.

Postmortem examinations (offspring):

Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

Reproductive indices:

There were no effects of test item treatment observed for copulation, fertility, delivery and viability indices.
Successful mating resulted in 100% pregnancies in the control, MD and HD groups and 90% pregnancy in the LD group.
A reduced fertility index in the LD group (90%) was considered incidental due to the absence of dose response relation.

Offspring viability indices:

The viablity index was very slightly reduced (approx. by 1%) in the LD and MD groups when compared to control group.
This was within the normal range of biological variation and was considered to be incidental in origin.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

no adverse effects

Mortality:

no mortality observed

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

no adverse effects

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Mortality
There was no mortality considered to be due to test item treatment in any of the treated groups. However, one female (no. 64) of MD group was
sacrificed prematurely for animal welfare reasons due to the presence of tumor mass found in the region of the left flank. The tumor mass was
of a spontaneous nature and considered not to be related to treatment with the test item (see 12.14 Histopathology).
The remainder of animals survived the scheduled study period.

Clinical Observations
In males and females, there were no clinical signs considered adverse due to test item treatment. There were cases of salivation and moving the
bedding in most males and females of LD, MD and HD groups immediately post dose administration and were assumed to be due to discomfort
and not considered to be an adverse effect.
There were also transient incidences of reddish nasal discharge, slight to moderate abnormal breathing, moderate to severe piloerection,
aggressive behavior and broken incisor tooth in a single or very few isolated males and/or females of MD and/or HD groups. These clinical
signs being transient and limited to a single or few animals were not considered to be an adverse effect.
There were also isolated incidences of tail injury and nasal discharge in male controls and slight piloerection and alopecia (shoulder and forepaws)
in female control and/or LD groups. These signs were incidental in nature. One female (no. 64) of MD group had a tumor mass found in the region
of the left flank, which was of a spontaneous nature.

Body Weight Development
In males there was statistically significantly lower body weight gain in MD and/ or HD groups after the 1st and the last week of treatment.
The cumulative weight gain of the entire treatment duration (premating day 1 to postmating day 14) was also affected due to differences in
body weight gain noted after the 1st and last week of treatment. Although a statistically significant lower body weight gain was observed for the
MD and HD animals, no toxicological relevance was attributed to these findings because no differences were observed in a 28-day study
(BSL study 136162) with the same strain of rats and because no comparable effect was seen in females.
In females, there were no effects of test item treatment on body weight and body weight gain observed during the treatment period.

Food Consumption
In males, there was statistically significantly lower food consumption in HD group after the 1st week of treatment. This lower food consumption
correlated to the lower weight gain. Taking into account the food consumption data of 28 days repeated dose oral toxicity study with
C18/ C18 Unsatd. Glucamide (BSL study 136162) wherein the differences in food intake had no statistical significance and biological relevance,
the difference in food intake in the present study was not considered to be an effect of test item treatment.
In females, there was no effect of test item treatment on food consumption observed during the treatment period.

Pathology- Macroscopic Findings
In the survivors, there were no gross lesions that could be attributed to treatment with the test item. There were single incidences of yellow spot
on testes and bloody epididymides in male LD group and enlarged spleen in a female control. All gross lesions recorded were considered to be
incidental changes which occurred spontaneously or were within the range of normal background alterations which may be recorded in animals
of this strain and age. One female (female no. 64), a tumor mass which correlated microscopically with adenocarcinoma of the mammary gland
was found in the region of the left flank.

Organ Weight
In males, there was a statistically significantly higher (26.5%) relative prostate weight (including the weight of seminal vesicle and coagulating glands)
in MD group. There was also a marginally higher (13 to 21%) absolute prostate (including seminal vesicle and coagulating glands) weight in LD, MD
and HD groups and relative prostate weight in LD and HD groups, but there was no statistical significance and no dose response relation observed.
In females, there was marginally lower (-11%) absolute and relative uterus (with cervix) weight in HD group. There was no statistically significant
difference observed. The above mentioned changes in absolute and relative reproductive organ weight of males and females in the absence of
histopathological changes were considered to have no adverse effect of test item treatment.

Histopathology
Mammary gland adenocarcinoma with central necrosis was observed in female no. 64 (MD group) which was sacrificed prematurely for animal
welfare reasons. As discussed later again this malignant tumor was of a spontaneous nature and considered not to be related to treatment with
the test item. Minimal extramedullary hemopoiesis was recorded in the spleen, but this was within the range of normal background finding
which may be recorded in animals of this strain and age. The stages were checked on completeness of cell populations, completeness of stages
and degenerative changes. There were not treatment-related effects on the completeness of stages or cell populations of the testes.
Spermatid retention and/or Sertoli cell vacuolation were recorded at a minimal severity in some animals from both of the control and high-dose
groups and in animal nos. 11 and 18 from the medium-dose group. There were not differences in incidence and severity between groups,
and therefore, all of findings were of spontaneous nature and considered not to be related to treatment with the test item.
Nothing special was observed in ovaries, uterus with cervix and vagina of control female No. 58 which was not pregnant. Nothing special of
lesions that could be attributed to treatment with the test item was recorded in testes, epididymides, prostate, seminal vesicles and coagulating
glands of the pairing partner male No. 18 as well. All microscopic findings recorded in the survivors were within the range of normal
background lesions which may be recorded in animals of this strain and age.

Dose Formulation Analysis
Concentration analysis of formulation samples was determined in study weeks 1, 3, 5 and 7 for all dose groups. The mean recoveries
observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 110%, 109% and 124% of the nominal concentration,
respectively. Nominal concentrations were confirmed for all dose groups, as measured concentration did not differ from nominal concentration
by more than 20%. Stability of formulation samples was investigated in study week 1 based on concentration in the LD and HD dose groups.
After 6 hours storage at -20oC recovery compared to starting value was 103.6% and 97.3%. All samples were stable, as concentration after
storage did not differ from start value by more than 20%. Homogeneity of formulation samples was determined in study weeks 1 and 5 for the
LD and HD dose groups. The mean recovery observed for the LD dose group was 116% and 111% of the nominal value and 116% and 126% of
the nominal value for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 5.0% and 1.3% in
LD dose group and 5.3% and 2.9% in the HD dose group. All samples were homogenous, as COV was below or equal 20%.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 650 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Mortality:

no mortality observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Litter Data
There were no effects of test item treatment observed for litter data including total number of pups born, number of still births, number of runts
on PND 0 and number of male and female pups, sex ratio, number of live pups on PND 0 and PND 4. There were no statistically significant
differences in litter data observed between the test item treated and the concurrent control groups. However, there was very slightly lower
number of male and female pups in LD, MD and HD groups, without statistical significance and dose responsive relation. Hence, the finding
was not related to test item treatment.

Litter Weight Data
There were no effects of test item treatment observed for litter weight data including group mean pup weight, total litter weight and male
and female litter weight on PND 0 and PND 4. There was statistically significantly higher group mean pup weight on PND 0 and PND 4 in LD group.
In the absence of dose response relation, the statistical significance in LD group was not considered to be of biological relevance.
There was marginally lower (-9 to -16%) female litter weight in test item treated groups (LD, MD, and HD groups) when compared to the control
without achieving the statistical significance. There were no dose response relation and in addition the mean and most individual values were
within the range of historical control data. Therefore, this finding was not considered to be an effect of test item treatment.
 
Precoital Interval and Duration of Gestation
There were no treatment-related effect observed for the duration of precoital and the duration of gestation when compared with the control group.
The values were comparable between the groups. All pregnancies resulted in normal births.

Pre- and Post-Natal Data
There was higher percent pre- implantation loss in LD, MD and HD groups when compared to control and achieving statistical significance
only in LD and MD groups. There was no obvious dose response and the mean and individual values were within the range of historical control data. Therefore, the finding was not considered to be an adverse effect of test item treatment. There were no effects of test item treatment observed for
the pre and post natal data including number of corpora lutea, number of implantation loss, number of live pups and percent post implantation loss.

Reproductive Indices
There were no effects of test item treatment observed for copulation, fertility, delivery and viability indices.
Successful mating resulted in 100% pregnancies in the control, MD and HD groups and 90% pregnancy in the LD group. A reduced fertility index
in the LD group (90%) was considered incidental due to the absence of dose response relation. The viablity index was very slightly reduced
(approx. by 1%) in the LD and MD groups when compared to control group. This was within the normal range of biological variation and was
considered to be incidental in origin.

Pup Survival Data
There was no test item related effect on the survival of the pups from PND 0 to PND 4 in any of the treated group when compared to controls.
However, one pup each from animals 80 (HD group, pup no. 8) and 55 (LD group, pup no. 11) were missing on PND 2 and PND 3, respectively.
These missing pups were attributed to the cannibalism by the dam. Given a single isolated fetus of single isolated female of LD and HD groups,
the finding was considered incidental in origin.

Pup External Findings
There were no treatment-related gross external findings observed in any of the treated groups.
However, there were a few incidences of external findings observed in the control (dark spot on head), LD (dark snout),
MD (dark snout and dark tail) and the HD (dark spot on snout and small wound) goups. These findings were in few isolated pups of the isolated
females and hence were considered to be spontaneous and not related to the test item.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 650 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

On the basis of this reproduction/ developmental toxicity screening test with C18/C18 unsatd.-Glucamide in male and female Wistar rats
treated at dose levels of 125, 300, and 650 mg/kg body weight/ day the following conclusions can be made:
There were no adverse effect of test item treatment on adult male and female animals and reproductive parameters.
The NOAEL for the adult animals and reproductive toxicity was 650 mg/ kg body weight/ day.
There was higher percent pre implantation loss in LD, MD and HD groups, achieving statistical significance only in LD and MD groups.
In the absence of obvious dose response and absence of statistical significance in the HD group, as well as the finding that all mean and
individual values being within the range of historical control data, the finding was not considered to be an adverse effect of test item treatment.
Therefore, the NOAEL for the developmental toxicity was considered to be 650 mg/ kg body weight/ day.

Executive summary:

The aim of this study was to assess the possible effects of C18/C18 unsatd.-Glucamide on male and female fertility and embryofetal

development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period

of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed.

Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study.

The 4 groups comprised 10 male and 10 femaleWistarrats.

The following doses were evaluated:

Control:                         0     mg/kg body weight/ day

Low Dose:                 125     mg/kg body weight/ day

Medium Dose:          300     mg/kg body weight/ day

High Dose:                650     mg/kg body weight/ day

The test item formulation was prepared freshly on each day of administration. The test item was suspended in corn oil and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 5 mL/kg body weight.

During the period of administration, the animals were observed each day for the signs of toxicity. Animal killed intercurrently was examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the

mating period in female animals and the mating and post-mating period in male animals.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards

the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated

and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed

on post natal day 4. Non-pregnant females were sacrificed on day 26 from the day of evidence of mating.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on postnatalday 4, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the reproductive organs was performed on high dose and control animals and non pregnant female animal (no. 58) and its mating partner (no. 18) of the LD group. All gross lesions from all groups,were examined by light microscopy.In addition, the following organs and tissues were also processed and examined in female no. 64 of MD group which was sacrificed prematurely for animal welfare reasons due to the presence of tumor mass in the left flank: brain, stomach, small and large intestines (including Peyer’s patches), liver, thymus, spleen, lung, urinary bladder, lymph nodes (mesenteric and axillary), trachea, kidneys, adrenal glands and heart.

Summary Results

One female (no. 64) of MD group was sacrificed prematurely for animal welfare reasons due to the presence of tumor mass in the region of the left flank, which was of a spontaneous nature. There was no other mortality considered to be due to test item treatment.

There were no clinical signs considered adverse due to test item treatment.There were casesof salivation and moving the bedding immediately post dose administration in most males and femalesin LD, MD and HD groups, which were assumed to be due to discomfort and not considered to be an adverse effect.

There were also transient incidences of reddish nasal discharge, slight to moderate abnormal breathing, moderate to severe piloerection, aggressive behavior and broken incisor tooth in a single or very few isolated males and/or females of MD and/or HD groups and these were not considered to be an adverse effect.

There were no effects of test item treatment on body weight, body weight gain and food consumption in both male and female treated groups when compared to the corresponding control groups.

There were no effects of test item treatment on litter data including total number of pups born, number of still births, number of runts on PND 0 and number of male and female pups, sex ratio, number of live pups on PND 0 and PND 4.

There were no effects of test item treatment on litter weight data including group mean pup weight, total litter weight and male and female litter weight on PND 0 and PND 4.

There were no treatment-related effects observed for the duration of precoital and the duration of gestation when compared to control group. The values were comparable between the groups. All pregnancies resulted in normal births.

There were no effects of test item treatment on pre and post natal data including number of corpora lutea, number of implantation loss, number of live pups and percent post implantation loss. However, there was higher percent pre- implantation loss in LD, MD and HD groups when compared to control and achieving statistical significance only in LD and MD groups. There was no obvious dose response and the mean and individual values were within the range of historical control data. Therefore, the finding was not considered to be an adverse effect of test item treatment.

There were no effects of test item treatment on copulation, fertility, delivery and viability indices.

There was no test item related effect on the survival of the pups from PND 0 to PND 4 in any of the treated group when compared to controls.

There were no treatment-related gross external findings observed in any of the treated groups.

In the survivors, there were no gross lesions that could be attributed to treatment with the test item. All gross lesions recorded were considered to be incidental changes which occurred spontaneously or were within the range of normal background alterations which may be recorded in animals of this strain and age. One female (female no. 64), a tumor mass which correlated microscopically with adenocarcinoma of the mammary gland was found in the region of the left flank.

There were no adverse effects of test item treatment on reproductive organ weights (absolute and relative) in male and female animals of treated groups when compared to control. However, there were higher prostate weight in LD, MD and HD groups and a marginally lower in uterus weight in HD group, but there were no associated histopathological changes observed.There were no histological evidences of toxicological properties in the organs and tissues of the reproductive system; i.e. testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus and cervix, and vagina. Asa result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure. Nothing special was observed in the reproductive organs of a female (no. 58, low-dose group) which was not pregnant, and nothing special was recorded in the reproductive organs of the pairing partner male (no. 18) as well.One female (no. 64) with tumor mass on the left flank was histopathologically considered as mammary gland adenocarcinomaknown as one of the most commonly occurring spontaneous neoplasms in females of many strains of rats which are commonly used in safety assessment studies.All other findings recorded in this study were within the range of normal background lesions which may be recorded in animals of this strain and age.

Conclusion

On the basis of this reproduction/ developmental toxicity screening test with C18/C18 unsatd.-Glucamide in male and female Wistar rats treated at dose levels of 125, 300, and 650 mg/kg body weight/ day the following conclusions can be made:

There were no adverse effect of test item treatment on adult male and female animals and reproductive parameters. The NOAEL for the adult animals and reproductive toxicity was 650 mg/ kg body weight/ day.

There was higher percent pre implantation loss in LD, MD and HD groups, achieving statistical significance only in LD and MD groups. In the absence of obvious dose response and absence of statistical significance in the HD group, as well as the finding that all mean and individual values being within the range of historical control data, the finding was not considered to be an adverse effect of test item treatment. Therefore, the NOAEL for the developmental toxicity was considered to be 650 mg/ kg body weight/ day.