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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Relevant information regarding the endpoint reproductive toxicity is available from a guideline conform OECD 421 study on the registration substance as well as from a guideline conform OECD TG 415 study with the read-across compound Glucamide 24. Based on the results from these studies, the overall NOAEL with regard to reproductive effects is considered to be equal or greater 350 mg/kg body weight per day. The NOAEL for parental toxicity is considered to be 150 mg/kg body weight per day, based on effects on body weight and food consumption. Clinical effects observed at this and the highest dose were considered indicative pf gavage (bolus) administration of a substance with irritative properties.

Link to relevant study records

Reference

Reference 1

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

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

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.

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

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

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.

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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%.

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

Key result

Critical effects observed:

no

Mortality:

no mortality observed

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.

Reference 2

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented guideline study according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)IGS BR VAF/Plus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crl:CD(SD)IGS BR VAF/Plus was obtained from Charles River Laboratories, Inc., Kingston, New York, USA
- Age at study initiation: Approximately 37 days
- Average weight range at study initiation: (P) Males: 132 - 160 g; (P) Females: 98 - 116 g
- Fasting period before study: Not reported
- Housing: P1 generation rats were individually housed in stainless steel, wire-bottomed cages except during the cohabitation and postpartum periods. During cohabitation, each pair of rats was housed in the male rat's cage.
- Diet: Certified Rodent Diet#5002; ad libitum.
- Water: Reverse Osmosis Water; ad libitum. Chlorine was added to the processed water as a bacteriostat.
- Acclimation period: The animals were acclimated. However the period of acclimation is not provided in the study report. No contaminants at levels exceeding the maximum concentration limits for certified feed or deviations from expected nutritional requirements were detected during feed analysis. The processed water was analyzed twice annually for possible chemical contamination.

ENVIRONMENTAL CONDITIONS
- Mean temperature range: 67.3 to 69.2°F (target: 64 to 79 °F)
- Mean relative humidity range: 52.5 to 63% (target: 30 to 70%)
- Air changes: A minimum of 10 changes/hour of 100% fresh air
- Photoperiod: 12-hours light:12-hours dark

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared weekly in Reverse Osmosis Membrane Processed Deionized Water on the basis of most recently recorded body weights. Prepared formulations were stirred continuously. Prepared formulations were stored refrigerated, protected from light in amber bottles. Dose calculations were adjusted for the 97.62% purity of the test material.

VEHICLE
- Concentration in vehicle: 1.5, 15 and 35 mg/mL for 15, 150 and 350 mg/kg be respectively
- Amount of vehicle: 10 mL/kg bw
- Purity: Neither the Sponsor nor the Study Director was aware of any potential contaminants likely to be present in the vehicle that would interfere with the results of this study.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Maximum of 21 days
- Proof of pregnancy: Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be Day 0 of gestation and assigned to individual housing.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: Not reported
- After successful mating each pregnant female was caged: Each pregnant female was housed individually in nesting boxes. Bed-o'cobs bedding was used as the nesting material. Each dam and delivered litter was housed in a common nesting box during the postpartum period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Testing of stability of test solution and analytical verification of test concentration: Triplicate samples were taken from the top, middle and bottom of each concentration prior to the start of study and monthly thereafter. Two samples of each triplicate set were shipped for analysis; the remaining samples were retained at the Testing Facility as backup samples.

Further details on results of analytical verification are provided in the study report.

Duration of treatment / exposure:

Approximately 5 months

Frequency of treatment:

Once daily (beginning from 70 days prior to mating until the day before sacrifice)

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 107 days

Remarks:

Doses / Concentrations:
0, 15, 150 and 350 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

25 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were selected by the Sponsor based on the available data prior to the initiation of dosage.
- Rationale for route of administration: (i) in comparison with the dietary route, the exact dosage can be accurately administered, (ii) it is one possible route of human exposure and (iii) the test substance is unpalatable in the diet.
- Rationale for animal assignment: The rats were assigned to four dosage groups, 25 rats per sex per group, based on computer-generated (weight-ordered) randomization procedures.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for viability; Animals were observed for test substance related clinical observations daily before and approximately one hour after administration.
- Cage side observations included were: Viability, mortality and clinical observations

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Daily during the dosage period and at sacrifice

FEED CONSUMTION: Yes
- Time schedule for examinations:
Males: Weekly during the cohabitation and post cohabitation period
Females: Weekly until cohabitation, on Day 0, 6, 10, 15, 20 and 25 (if necessary) of gestation and on Day 1, 4, 7, 10 and 14 of lactation.

Oestrous cyclicity (parental animals):

Estrous cycling was evaluated by examination of vaginal cytology for 21 days prior to cohabitation.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
- Right testis, left testis, left epididymis (whole and cauda), right epididymis, prostate and seminal vesicles with coagulating gland (with or without fluid) were weighed in male parental generations.
- A portion of the left cauda epididymis was used for evaluation of cauda epididymal sperm concentration and motility using computer-assisted sperm analysis. Motility was evaluated by the Hamilton Thorne IVOS with collection of a sample from the left cauda epididymis through a swim-out method. Sperm concentration (sperm per gram of tissue weight) was evaluated by the Hamilton Thorne IVOS.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, number of implantation sites, stillborn pups, and general condition of the litter during the postpartum period. Each litter was evaluated for viability at least twice each day. The pups present in each litter were counted once each day. Pup body weights were recorded on Days 1, 4, 7, 14 and 21. Clinical observations were recorded once daily during the postpartum period (Day 1 to Day 21 of lactation).

GROSS EXAMINATION OF DEAD PUPS: Yes, pups found dead were examined for gross lesions and for the cause of death. All pups found dead on Day 2 to 4 of lactation were preserved in Bouin's solution for possible future evaluation. All pups found dead on Day 5 to 21 of lactation were preserved in neutral buffered 10% formalin.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed by CO2 asphyxiation, after completion of the cohabitation period.
- Maternal animals: All surviving female rats were sacrificed after completion of the Day 21 postpartum period. Rats that did not deliver a litter were sacrificed on Day 25 of gestation and were examined for pregnancy status. Rats that died were examined for the cause of death on the day that the observation was made. Female rats without a confirmed mating date that did not deliver a litter were also sacrificed on an estimated Day 25 of gestation and necropsied.

GROSS NECROPSY
Gross necropsy included an initial physical examination of external surfaces and all orifices, as well as an internal examination of tissues and organs in situ. The following were examined: external and internal portions of all hollow organs; the external surfaces of the brain and spinal column, the nasal cavity and neck with associated organs and tissues; the thoracic, abdominal and pelvic cavities with associated organs and tissues; and the musculo/skeletal carcass.
Gross lesions were retained in neutral buffered 10% formalin and examined histologically.

ORGAN WEIGHTS: The following organs were individually weighed:
Brain, pituitary gland, ovaries, uterus with cervix, right testis, left testis, left epididymis (whole and cauda), right epididymis, prostate and seminal vesicles with coagulating gland (with or without fluid).

HISTOPATHOLOGY:
Histopathological examinations were performed on following tissues of control and high dose group rats:
Brain, pituitary gland, ovaries, vagina, uterus with cervix, testes, epididymides, seminal vesicles with coagulating gland and prostate
The testes were fixed in Bouin’s solution for 48 to 96 hours and then retained in neutral buffered 10% formalin for histopathological evaluation. Histopathology was also performed on the testes of all male rats that failed to induce pregnancy and on ovaries of all female rats that were not pregnant.

Postmortem examinations (offspring):

SACRIFICE AND GROSS NECROPSY
- All pups removed from study due to standardization on Day 4 of lactation were sacrificed and examined for gross lesions. Necropsy included a single cross section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly.
- All pups were preserved in Bouin's solution.
- All other pups were sacrificed and discarded at weaning on Day 21 of lactation.

Statistics:

- All adult and pup incidence data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.
- Body weights, body weight changes, feed consumption data, durations of gestation and delivery, litter averages for pup body weights and percent male or pups and mortality, cumulative survival and parameters involving continuous data were analyzed using Bartlett's Test of Homogeneity of Variances and the Analysis of Variance (when appropriate).
- If the Analysis of Variance was significant (p≤0.05), Dunnett's Test was used. If the Analysis of Variance was not appropriate, the Kruskal-Wallis Test was used. In cases where the Kruskal-Wallis Test was statistically significant, Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the individual groups.
- All other natural delivery data involving discrete data were evaluated using the Kruskal-Wallis Test.
- Sperm motility data were expressed as percentages and analyzed, as indicated above, by parametric methods.

Reproductive indices:

The females were evaluated for following indices:
1) Duration of gestation: Day 0 of gestation to the day the first pup was observed.
2) Precoital index: Length of time for mating to occur
3) Mating index: Percentage of mating
4) Fertility index: Percentage of matings that result in pregnancies
5) Gestation index: Percentage of pregnancies that result in birth of live litters
Maternal behavior was evaluated on Day 1, 4, 7, 14 and 21 of lactation.

Offspring viability indices:

1) Viability indices: Percentages of pups born that survive to Day 1 and 4 of lactation.
2) Lactation indices: Percentage of pups alive after culling on Day 4 of lactation that survives to Day 21 of lactation.
3) Percent survival and sex ratio (tabulated at Day 1, 4, 7, 14 and 21 of lactation)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

at 350 mg/kg body weight (slight clinical effects at 150 mg/kg body weight are not considered substance but treatment related)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

at 350 mg/kg reduced body weight and reduced feed consumption

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

at 350 mg/kg reduced body weight and reduced feed consumption

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY: No treatment related mortality was observed in the male and female animals.
- Males:
Test substance-related clinical observations observed in male rats at 150 and 350 mg/kg/day dosage groups included excess salivation, rales, chromorhinorrhea, a red or dried red perioral substance and chromodacryorrhea (350 mg/kg/day dosage group only).
- Females:
Clinical observations observed during the precohabitation, gestation and lactation periods included excess salivation and rales in 150 and 350 mg/kg/day dosage groups. These observations were considered test substance-related because they were increased in a dosage dependent manner.
All other clinical observations during the precohabitation, gestation and lactation periods were considered unrelated to the test substance.

BODY WEIGHT:
- Males:
Decreased body weight gains in the 350 mg/kg/day dosage group were considered related to the test substance as they were dosage-dependent. Average body weight gains were reduced to 92% of the control group value in the 350 mg/kg/day dosage group on Day 1 to 70 of study. Body weight gains were also reduced to 75% and 89% of the control group values on Day 91 to 119 of study and Day 1 to termination, respectively, in the 350 mg/kg/day dosage group. Terminal body weights and absolute body weights on Day 112 to 119 of study were significantly reduced in the 350 mg/kg/day dosage group.
- Females:
Average body weights and body weight gains during the precohabitation period were comparable among the four dosage groups. Maternal body weight gains were significantly reduced in the 350 mg/kg/day dosage group for the entire gestation period [Days 0 to 20 of gestation] and on Day 0 to 6 of gestation. Absolute maternal body weights were significantly reduced on Day 12 and 20 of gestation, slightly reduced on all other days of the gestation period and significantly reduced on each day of the lactation period in the 350 mg/kg/day dosage group when compared to the vehicle control group values.
Average maternal body weights and body weight gains during the lactation and gestation period were unaffected by dosages of test substance as high as 150 mg/kg/day.

FOOD CONSUMPTION:
- Males: Absolute and relative feed consumption values were unaffected by dosages of test substance as high as 350 mg/kg/day.
- Females: Absolute feed consumption values were significantly reduced in the 350 mg/kg/day dosage group for the entire gestation period. Absolute and relative feed consumption values during the precohabitation period and lactation period were comparable among the four dosage groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
Dosages of the test substance as high as 350 mg/kg/day did not affect estrous cycling (number of estrus stages per 14 days).

REPRODUCTIVE FUNCTION: SPERM MEASURES
No statistically significant or biologically important differences occurred in the number or the percentage of motile sperm, the number of non-motile sperm, and the sum of the motile and nonmotile sperm. Cauda epididymal sperm counts were 86%, 90% and 80% and density values were 93%, 88% and 85% of the vehicle control group values in the four respective dosage groups. These non-statistically significant reductions were not considered biologically important because mating and fertility parameters were comparable among the dosage groups.

REPRODUCTIVE PERFORMANCE
- The precoital index, the fertility and mating indices and the number of rats with confirmed mating dates during the first and second week of cohabitation were comparable in the four dosage groups.
- All female rats were mated. Pregnancy occurred in 23 (92.0%), 23 (92.0%), 21 (100%) and 22 (88.0%) female rats in the four respective dosage groups (0, 15, 150 and 350 mg/kg bw).
- Natural delivery observations were unaffected by dosages of the test substance as high as 350 mg/kg/day. The gestation index, the number of dams delivering litters, the duration of gestation, averages for implantations and dams with stillborn pups were comparable among the four dosage groups and did not differ significantly.

ORGAN WEIGHTS:
The absolute organ weights and the ratios of these organ weights to the terminal body weight or absolute brain weight of the P1 generation male and female rats were comparable in all four dosage groups.

GROSS PATHOLOGY
- Males: All necropsy observations were considered unrelated to the test substance due to following reasons:
(i) The incidences were not dosage-dependent.
(ii) The observation commonly occurs in this strain of rat and/or;
(iii) The observation occurred in the rats that were found dead.
- Females: Necropsy observations in rats that survived until scheduled sacrifice were limited to two clear fluid-filled cysts on the left kidney of one rat in the vehicle control group. This observation was not considered test substance-related because the observation occurred in a vehicle control group rat.

HISTOPATHOLOGY: There were no test substance-related microscopic changes observed in the brain, pituitary or reproductive tissues of male and female rats in the 350 mg/kg/day dosage group. Microscopic examination of the testes of male rats and ovaries of female rats that failed to reproduce that revealed no findings that could be correlated with infertility.

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Reduced body weight gains and slight reductions in feed consumption values at 350 mg/kg bw, slight clinical findings at 150 mg/kg body weight not considered substance but treatment related

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

CLINICAL SIGNS: All clinical observations in the F1 generation pups were considered unrelated to the test substance.

BODY WEIGHT AND ALL OTHER PARAMETERS: All litter parameters were unaffected by dosages of the test substance as high as 350 mg/kg/day. These included the number of pups found dead or presumed cannibalized from Day 1 to 21 of lactation, viability index, lactation index, pup body weights, surviving pups, percent male pups and live litter sizes at weighings. The number of pups found dead or presumed cannibalized was significantly increased (p≤0.01) in the 350 mg/kg/day dosage group on Day 1 of lactation. This increase was not considered test substance-related because the viability and lactation indices were comparable among the dosage groups.

NECROPSY: All necropsy observations in the F1 generation pups were considered unrelated to the test substance. Necropsy of pups that were found dead revealed no milk in stomach in 3, 2, 1 and 1 pup from the four respective dosage groups (0, 15, 150 and 350 mg/kg bw). All pups appeared normal at necropsy on Day 4 and 21 of lactation.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 350 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Overall effects on litter parameters

Reproductive effects observed:

not specified

Conclusions:

The reproductive toxicity NOAEL for Glucamide 24 when administered orally at 0, 15, 150 and 350 mg/kg/day in rats was ≥ 350 mg/kg bw. Based on the results, natural delivery and litter observations were unaffected after administration of the test substance as high as 350 mg/kg/day.All clinical and necropsy observations in the F1 generation pups were considered unrelated to the test substance. All litter parameters were unaffected by dosages of the test substance as high as 350 mg/kg/day. The reproductive toxicity NOAEL for Glucamide 24 in a one generation study was thus ≥ 350 mg/kg bw.

The NOAEL for parental toxicity was established at 150 mg/kg bw (based on reduced body weight gains and slight reductions in feed consumption).

Executive summary:

The one generation reproduction toxicity study of Glucamide 24 was determined following methods comparable to the OECD Guideline 415 (One-Generation Reproduction Toxicity Study).

One hundred male and one hundred female Crl:CD®(SD)IGS BR VAF/Plus rats received from Charles River Laboratories, Inc., Kingston, New York, USA were used in this study. The animals were housed individually in stainless steel, wire-bottomed cages except during the cohabitation and postpartum periods. During the mating period, each pair of rats was housed in the male rat's cage. During the treatment period, the animals were fed a certified rodent diet, ad libitum. The animals were randomly assigned to the following four dosage groups with 25 rats per sex per dosage group:

Group 1 (Vehicle control): 0 mg/kg bw

Group 2 (Low dose group): 15 mg/kg bw

Group 3 (Mid dose group): 150 mg/kg bw

Group 4 (High dose group): 350 mg/kg bw

Reverse osmosis membrane processed deionized water (R.O. deionized water) served as a vehicle control. The test substance was administered orally (via gavage) to male and female rats once daily (beginning 70 days prior to mating until the day before sacrifice) at a constant volume of 10 mL/kg bw.

The P1 generation animals were mated in a 1:1 ratio for a maximum period of 21 days or until the presence of a copulatory plug was confirmed. Following breeding, each pregnant female was housed individually in a nesting box.

All P1 generation rats were observed for viability at least twice daily. Animals were observed for test substance related clinical observations daily before and approximately one hour after administration. Additionally, female rats were observed daily for abortions and premature deliveries. Body weights were recorded daily during the dosage period and at sacrifice. Feed consumption values for male rats were recorded weekly during the study. Feed consumption values for female rats were recorded weekly until cohabitation, on Day 0, 6, 10, 15, 18, 20 and 25 (if necessary) of gestation and on Day 1, 4, 7, 10 and 14 of lactation.

The female rats were evaluated for duration of gestation, precoital index, mating index, viability index, fertility index, gestation index, number and sex of offspring per litter, number of implantation sites, general condition of the dam and litter during the postpartum period. Variations from expected maternal behavior were recorded during the postpartum period.

Each litter/pup was evaluated for viability, viability indices, lactation index and percent survival and sex ratio. Pup body weights were recorded on Days 1, 4, 7, 14 and 21 of lactation. The pups present in each litter were counted once each day. Clinical observations were recorded once daily during the postpartum period.

After completion of the mating period, P1 generation males were subjected to gross necropsy. Sperm evaluations were also conducted in parental male animals. P1 generation female rats were sacrificed after completion of the 21-day postpartum period. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed in both male and female rats and the gross lesions were retained and examined histologically. The following organs were weighed: brain, pituitary gland, ovaries, vagina, uterus with cervix, testes, epididymides, seminal vesicles with coagulating gland and prostate. Histopathological examinations were performed on the above referenced tissues of all control and high dosage group rats.

Further, histopathology was also performed on the testes of all male rats that failed to induce pregnancy and on ovaries of all female rats that were not pregnant.

All F1 generation pups removed from study due to standardization on Day 4 of lactation were sacrificed and examined for gross lesions. Pups found dead were examined for gross lesions and for the cause of death. Further, all surviving F1 generation pups were sacrificed after the postpartum period and discarded.

No P1 generation male or female rats died as a result of treatment with test substance during this study.

Test substance-related clinical observations observed in male rats in the 150 and 350 mg/kg/day dosage groups included excess salivation, rales, chromorhinorrhea, a red or dried red perioral substance and chromodacryorrhea (350 mg/kg/day dosage group only). Clinical observations observed during the precohabitation, gestation and lactation periods of female rats included excess salivation and rales in the 150 and 350 mg/kg/day dosage groups. These observations were considered test substance-related because they increased in a dosage dependent manner.

Reduced body weight gains in males and females at 350 mg/kg/day were considered related to the test substance. Absolute and relative feed consumption values in males were unaffected by the highest test substance dose (350 mg/kg/day). However in females, the absolute feed consumption values were significantly reduced at 350 mg/kg/day for the entire gestation period. Absolute and relative feed consumption values during the precohabitation period and lactation period of females were comparable among the four dosage groups.

The absolute organ weights and the ratios of these organ weights to the terminal body weight or absolute brain weight of the P1 generation male and female rats were comparable in all four dosage groups. No test substance related necropsy observations were noted in P1 generation male and female rats. There were no test substance-related microscopic changes observed in the brain, pituitary or reproductive tissues of male and female rats in the 350 mg/kg/day dosage group. Microscopic examination of the testes of male rats and ovaries of female rats that failed to reproduce revealed no findings that could be correlated with infertility.

Dosages of the test substance as high as 350 mg/kg/day did not affect estrous cycling (number of estrus stages per 14 days). No statistically significant or biologically important differences occurred in the number or the percentage of motile sperm, the number of non-motile sperm, and
the sum of the motile and non-motile sperm. The precoital index, the fertility and mating indices and the number of rats with confirmed mating dates during the first and second week of cohabitation were comparable in the four dose groups. All female rats were mated and pregnancy occurred in 23 (92.0%), 23 (92.0%), 21 (100%) and 22 (88.0%) female rats in the four respective dosage groups (0 (vehicle), 15, 150 and 350 mg/kg bw).

Natural delivery and litter observations were unaffected after administration of the test substance as high as 350 mg/kg/day.All clinical and necropsy observations in the F1 generation pups were considered unrelated to the test substance. All litter parameters were unaffected by dosages of the test substance as high as 350 mg/kg/day.

Based on above, the reproductive toxicity NOAEL for Glucamide 24 in a one generation study was ≥ 350 mg/kg bw.

The parental toxicity NOAEL for Glucamide 24 was established at 150 mg/kg bw (based on adverse clinical observations, reduced body weight gains and slight reductions in feed consumption).

This one-generation reproduction toxicity study is classified as acceptable, and satisfies the guideline requirements of the OECD 415 method.

Reference 3

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Glucamide OL is chemically and biologically equivalent to Glucamide 24, as they deviate only by the number of CH2-units in the chain length of the respective alkyl moiety and thus read-across of available toxicological data from the source molecule to address data gaps of the target molecule is scientifically possible and toxicologically justified

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Reduced body weight gains and slight reductions in feed consumption values at 350 mg/kg bw, slight clinical findings at 150 mg/kg body weight not considered substance but treatment related

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 350 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall litter data

Key result

Reproductive effects observed:

no

Conclusions:

Glucamide OL was investigated for potential effects on male and female fertility and embryo-fetal development after repeated dose administration to rats per OECD TG 421 (reproduction and developmental toxicity screening test). There were no adverse effects of Glucamide OL found up to the highest dose level tested of 650 mg/kg body weight/ day. No fertility studies according to OECD TG 443, 416 or 415 are available with Glucamide OL. However, based on the very consistent toxicity profile of various Glucamides and taking the chemical and biological comparability into account, available fertility data from Glucamide 24 as source molecule can be used for evaluation purposes using read-across princliples (see also comprehensive read-across justification). Taking a conservative approach, the NOAEL of 150 mg/kg body weight per day for parental toxicity and 350 mg/kg body weight per day for reproductive toxicity revealed in the OECD guideline conform one-generation study with Glucamide 24 is used also for Glucamide OL for assessment considerations.

Executive summary:

Glucamide OL is chemically and biologically equivalent to Glucamide 24, as they deviate only by the number of CH₂-units in the chain length of the respective alkyl moiety and thus read-across of available toxicological data from the source molecule to address data gaps of the target molecule is scientifically possible and toxicologically justified. (see also comprehensive read-across justification).

Glucamide OL was investigated for potential effects on male and female fertility and embryo-fetal development after repeated dose administration to rats per OECD TG 421 (reproduction and developmental toxicity screening test protocol) using 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. the NOAEL for the developmental toxicity was considered to be 650 mg/ kg body weight/ day.

No fertility studies according to OECD TG 443, 416 or 415 are available for Glucamide OL. However, based on the very consistent toxicity profile of various Glucamides and taking the chemical and biological comparability into account, available fertility data from Glucamide 24 as source molecule can be used for evaluation purposes of Glucamide OL using read-across princliples (see also comprehensive read-across justification).Based on the results of a one-generation toxicity study according to OECD 415, natural delivery and litter observations were unaffected after administration of the test substance as high as 350 mg/kg/day.All clinical and necropsy observations in the F1 generation pups were considered unrelated to the test substance. All litter parameters were unaffected by dosages of the test substance as high as 350 mg/kg/day. The reproductive toxicity NOAEL for Glucamide 24 in a one generation study was thus ≥ 350 mg/kg bw. The NOAEL for parental toxicity was established at 150 mg/kg bw (based on reduced body weight gains and slight reductions in feed consumption). Taking a conservative approach, the NOAEL of 150 mg/kg body weight per day for parental toxicity and 350 mg/kg body weight per day for reproductive toxicity revealed in the OECD guideline conform one-generation study with Glucamide 24 is used also for Glucamide OL for assessment considerations.

The performed read-across is scientifically and toxicologically valid not only based on shared structural and comparable functional similarities, but also due to common metabolic principles valid not just for the discussed source and target molecules, but for the entire class of glucamides.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available data base is considered sufficient for hazard / risk characterization independent of the exposure route. There are no indications of adverse effects on reproductive organs or tissues from the available data.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

There is no study concerning developmental toxicity available for the registration substance. However, relevant data is available for the structural and biological analogous compound Glucamide 24 which can be used as source molecule for read-across. Administration of Glucamide 24 to female rats by oral gavage during Days 6-15 of gestation at dose levels of 0, 15, 150 and 350 mg/kg bw/day resulted in a NOAEL of 350 mg/kg bw for developmental toxicity. The NOAEL for maternal toxicity was established at 150 mg/kg bw (based on clinical observations and body weight).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline with minor deviation, GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

(Animals were treated during organogenesis phase Day 6-15 of gestation instead of guideline recommendation of dosing through the day of implantation to one day prior to scheduled kill)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD VAF/Plus®

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source: Sprague-Dawley derived Crl:CD® VAF/Plus rats were obtained from Charles River Laboratories, Portage, Michigan
-Age at study initiation: 12.5 weeks (sexually mature virgin females)
-Weight at study initiation (on Day 0 of gestation): 221 – 227 g
-Housing: The females were housed individually in suspended, stainless steel wire-mesh cages from receipt until euthanasia except during mating.
-Diet: Certified Rodent Chow # 5002; ad libitum
-Water: Tap water, ad libitum
-Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
-Temperature: 72 – 73°F (mean temperature ± SD: 73 ± 0.4°F)
-Relative humidity: 39 – 50% (mean relative humidity ± SD: 42 ± 5.4%)
-Air changes: Not reported
-Photoperiod: 12 hours of fluorescent light per day

IN-LIFE DATE: From Jan. 21, 1991 To Feb. 14. 1991

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test substance for each group was dissolved into vehicle by heating on water bath at 50°C. Additional vehicle was added to obtain final volume of test substance preparation and mixed using a magnetic stir bar and stir plate. The test substance preparation was dispensed into amber glass containers and stored refrigerated. All the dosing solution concentrations were adjusted for active ingredient (45% aqueous solid gel).
-Rate of preparation of dose formulation: Weekly

VEHICLE
-Concentration in vehicle: 1.5, 15 and 36.3 mg/mL for dose of 15, 150 and 363 mg/kg bw respectively
-Amount of vehicle: 10 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and periodic analysis of test substance preparations (during Day 6 to 13) were carried out using HPLC method. The details of method are provided in the study report. The test preparation for use during study week 1 and 2 contained 95 to 102% of the desired test substance concentrations.

Details on mating procedure:

-Impregnation procedure: Cohoused
-M/F ratio per cage: 1:1
-Length of cohabitation: Not reported
-Verification of same strain and source of both sexes: Yes
-Proof of pregnancy: Presence of copulatory plug was taken as Day 0 of gestation.

Duration of treatment / exposure:

Gestation days 6-15 (10 days)

Frequency of treatment:

Once daily

Duration of test:

Approximately 25 days

No. of animals per sex per dose:

25 mated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

-Dose selection rationale: The dosages were selected by the study sponsor on the basis of data from a previously conducted study (IRDC 191-1501).
-Rationale for animal assignment: Mated females were consecutively assigned in block design to one control and 3 treatment groups. The order in which mated females were assigned corresponded to the day the copulatory plug was observed and the order in which the animal appeared on the breeding record.
-Rationale for test system: The rat is acceptable model for developmental toxicity studies. The laboratory has historical control data on the incidence of parameters in this strain from this source. This strain is susceptible to known developmental toxicants.
-Dose volume: 10 mL/kg bw

Maternal examinations:

MORTALITY AND SIGNS OF OVERT TOXICITY: Yes
-Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
-Time schedule: At least once daily from Day 6 to 20 of gestation

BODY WEIGHT: Yes
-Time schedule: The individual maternal body weights were recorded on gestation Days 0, 6, 9, 12, 16 and 20. Body weights of non gravid animals were recorded but were not included in the mean calculations.

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
-Sacrifice on gestation Day # 20; all animals were euthanized by CO2 inhalation.
-Organs examined: The abdominal and thoracic cavities and organs of females were examined for gross morphological changes.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes, the uterus was excised and gravid uterine weights were recorded.
Examinations included:
-Number of corpora lutea: Yes
-Number of implantations: Yes
-Location of viable and non viable fetus: Yes
-Number of live fetuses: Yes
-Early resorptions: Yes
-Late resorptions: Yes
-Other: Uteri from females that appeared non gravid were opened and placed in 10% ammonium sulphide solution for detection of implantation.

Fetal examinations:

-External examinations: Yes, individual fetuses were weighed, sexed, tagged and examined for external malformations and variations.
-Soft tissue examinations: Yes, about 1/2 of the fetuses were placed in Bouin’s solution for subsequent soft tissue examination using Wilson’s razor blade sectioning techniques.
-Skeletal examinations: Yes, about 1/2 of the fetuses were fixed in ethanol, macerated with KOH, stained with Alizarin Red S and cleared with glycerin for subsequent skeletal examinations. Fetal findings were classified as malformations or developmental variations.
-Head examinations: No

Statistics:

All statistical analysis compared the treatment groups with the control with the level of significance at p≤0.05 and p≤0.01. All means were accompanied by standard deviations.
Mean maternal body weight, body weight changes, mean number of corpora lutea, total implantations, live fetuses, gravid uterine weight and mean fetal body weights were compared by one-way analysis of variance, Bartlett’s test for homogeneity of variance, and the appropriate t-test. Significance of difference was determined by Dunnet’s multiple comparison tables or pair wise comparison with a Bonferroni’s correction.
Male to female sex ratios and proportions of litters with malformations and developmental variations were compare using Chi-square test or Fisher’s exact probability test to determine the significance of difference.
The proportions of resorbed and dead fetuses and post-implantation losses were compared by the Mann-Whitney U-test to determine the significance of difference.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: (at 363 mg/kg bw)

Details on maternal toxic effects:
MORTALITY: No mortality was observed during the study.

CLINICAL OBSERVATIONS: Presence of test material around the nose and/or mouth was observed at the high dose level in four animals. Test material around the mouth was observed in a single animal at mid dose level but was not considered to be treatment related. Post dose pharmacological signs of increased salivation in all animals and decreased activity in two animals were observed in the high dose group. Increased salivation was also observed in low incidence in themid dose group.

MATERNAL BODY WEIGHTS AND BODY WEIGHT CHANGES:
A statistically and biologically significant inhibition of body weight gain was observed in the high dose group relative to the control group during the first treatment interval of gestation Days 6–9 and during the overall treatment period of gestation Days 0–20.
A slight inhibition of maternal body weight gain was observed for the treatment interval of gestation Days 6–9 and Days 12–16 in the high dose group when compared with the control group.
A slight increase in maternal body weight gain was observed for treatment interval of gestation Days 16-20 at the high dose level when compared with the control group. This increase was considered to be compensatory for the earlier inhibition and therefore considered to be related to treatment.

NECROPSY FINDINGS: No treatment related differences were noted at necropsy; all findings were observed in low incidence.

CESAREAN SECTION EXAMINATION:
No treatment related differences were observed with regards to cesarean section parameters; values in the treated group were generally comparable with those of control group.

Key result

Dose descriptor:

NOAEL

Effect level:

150 other: mg/kg/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
MALFORMATIONS
No biologically meaningful difference was observed between the incidence of fetal malformations in the treated groups and that of control groups. The malformation occurred were primarily in single incidences were sporadically dispersed among the control and treated groups. No dose related trend was observed with regard to malformations.

DEVELOPMENTAL VARIATIONS:
The incidence and type of developmental variations observed among fetuses from treated group were generally comparable with those of the control group, or observed in sporadic instances with no apparent dose related trend. There was a slight increase in incidence of fetuses observed with unossified sternabrae in the mid and high-dose groups, however, no dose related trend was observed. The variation observed were considered to be due to normal biological variability and not related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 363 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall effects

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Developmental effects observed:

no

Conclusions:

Administration of Glucamide 24 to female rats by oral gavage during Days 6-15 of gestation at dose levels of 0, 15, 150 and 363 mg/kg bw/day resulted in a NOAEL of ≥ 363 mg/kg bw for developmental toxicity.

The NOAEL for maternal toxicity was established at 150 mg/kg bw (based on clinical observations and body weight).

Executive summary:

The developmental toxicity study of Glucamide 24 was conducted following methods comparable to the OECD Guideline 414 (Prenatal Developmental Toxicity Study).

Sexually mature female Crl:CD VAF/Plus® rats (Source: Charles River Laboratories, Portage, Michigan), weighing 221–227 g were used in this study. After receipt, animals were acclimated for 10 days and maintained under standard laboratory conditions (temperature: 7 –73°F, relative humidity: 39 – 50%, photoperiod: 12 hrs dark / 12 hrs fluorescent light). The animals were housed individually in suspended, stainless steel wire-mesh cages from receipt until euthanasia except during mating. The animals were fed on Certified Rodent Chow # 5002 and tap water, ad libitum.

One male and one female rat of same source and same strain were placed together for mating. The presence of a copulatory plug was taken as Day 0 of gestation. Mated females were consecutively assigned to the following treatment groups of 25 females each:

0 (vehicle control), 15, 150 and 363 mg/kg bw

Deionized water served as the vehicle control. The test substance was administered to mated female rats by oral gavage once daily beginning from Day 6 through to Day 15 of gestation. All animals were observed twice daily for mortality and at least once daily for detailed clinical observations. Body weights were recorded on Days 0, 6, 9, 12, 16, and 20 of gestation.

On Day 20 of gestation, animals were euthanized and cesarean section examinations were performed. The ovaries and uterine content (number of corpora lutea, number of implantations, and numbers of early and late resorptions, number and distribution of implantation sites) were examined after sacrifice.

Individual fetuses were weighed, sexed, tagged and examined for external malformations and variations. About half of all fetuses were examined for soft tissue changes using Wilson’s razor blade sectioning techniques. The other half of fetuses were fixed in ethanol, macerated with KOH, stained with Alizarin Red S and cleared with glycerin for subsequent skeletal examinations. Fetal findings were classified as malformations or developmental variations.

No mortality was observed during the study. No treatment related differences were noted at necropsy; all findings were observed in low incidence.

No treatment related differences were observed with regards to cesarean section parameters; values in the treated group were generally comparable with those of control group.

Maternal toxicity manifested as test material around the nose and/or mouth, post dose increased salivation and decreased activity, and significant inhibition of maternal body weight gain at the 363 mg/kg bw dose level.

The incidence of malformations and variations observed among fetuses in the treated group were generally comparable with those of the control group. The observations were sporadic with no apparent dose related trend. Therefore, no apparent developmental toxicity was observed at any dose level evaluated in the study.

Based on above, administration of Glucamide 24 to female Crl:CD VAF/Plus rats by oral gavage during Days 6-15 of gestation at dose levels of 0, 15, 150 and 363 mg/kg bw/day resulted in a NOAEL of ≥ 363 mg/kg bw for developmental toxicity

The NOAEL for maternal toxicity was established at 150 mg/kg bw (based on clinical observations and body weight).

This developmental toxicity study is acceptable and satisfies the OECD Guideline 414 requirement.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Glucamide OL is chemically and biologically equivalent to Glucamide 24, as they deviate only by the number of CH2-units in the chain length of the respective alkyl moiety and thus read-across of available toxicological data from the source molecule to address data gaps of the target molecule is scientifically possible and toxicologically justified.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Key result

Dose descriptor:

NOAEL

Effect level:

>= 363 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall effects

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Developmental effects observed:

no

Conclusions:

Glucamide OL was investigated for potential effects on male and female fertility and embryo-fetal development after repeated dose administration to rats per OECD TG 421 (reproduction and developmental toxicity screening test). There were no adverse effects of Glucamide OL found up to the highest dose level tested of 650 mg/kg body weight/ day. No developmental toxicity study according to OECD TG 414 is available with Glucamide OL. However, based on the very consistent toxicity profile of various Glucamides and taking the chemical and biological comparability into account, available developmental toxicity data from an OECD TG 414 study with Glucamide 24 as source molecule can be used for evaluation purposes of Glucamide OL using read-across princliples (see also comprehensive read-across justification). In this study no developmental toxicity was observed. The NOAEL was thus greater than 363 mg/kg body weight per day (highest dose tested). The NOAEL for maternal toxicity was established at 150 mg/kg body weight per day. Taking a conservative approach, the results from the developmental toxicity revealed in this OECD guideline conform 414 study with Glucamide 24 is used also for Glucamide OL for assessment considerations.

Executive summary:

Glucamide OL is chemically and biologically equivalent to Glucamide 24, as they deviate only by the number of CH₂-units in the chain length of the respective alkyl moiety and thus read-across of available toxicological data from the source molecule to address data gaps of the target molecule is scientifically possible and toxicologically justified. (see also comprehensive read-across justification).

Glucamide OL was investigated for potential effects on male and female fertility and embryo-fetal development after repeated dose administration to rats per OECD TG 421 (reproduction and developmental toxicity screening test protocol) usingdose 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. the NOAEL for the developmental toxicity was considered to be 650 mg/ kg body weight/ day.

No developmental toxicity study according to OECD TG 414 is available for Glucamide OL. However, based on the very consistent toxicity profile of various Glucamides and taking the chemical and biological comparability into account, data from an available OECD TG 414 study for Glucamide 24 as source molecule can be used for evaluation purposes of Glucamide OL using read-across princliples (see also comprehensive read-across justification). Based on the results of this study, administration of Glucamide 24 to female rats by oral gavage during days 6-15 of gestation at dose levels of 0, 15, 150 and 363 mg/kg bw/day resulted in a NOAEL of ≥ 363 mg/kg bw for developmental toxicity. The NOAEL for maternal toxicity was established at 150 mg/kg bw (based on clinical observations and body weight). Taking a conservative approach, these NOAELs revealed in the OECD guideline conform developmental toxicity study with Glucamide 24 is used also for Glucamide OL for assessment considerations.

The performed read-across is scientifically and toxicologically valid not only based on shared structural and comparable functional similarities, but also due to common metabolic principles valid not just for the discussed source and target molecules, but for the entire class of glucamides.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available data base is considered sufficient for hazard / risk characterization for this endpoint. There are no indications of adverse effects on reproductive organs or tissues from repeated dose toxicity studies as well as reproductive and/or developmental toxicity studies.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Glucamide OL was investigated for potential effects on male and female fertility and embryo-fetal development after repeated dose administration to rats per OECD TG 421 (reproduction and developmental toxicity screening test protocol). The test item was administered daily via gavage at doses of 0, 125, 300 or 650 mg/kg bw/day (doses selected on the basis of available acute and repeated dose toxicity data) to 10 male and 10 female animals per group. The treatment period was 54 days and comprised 14 days of pre-mating with a maximum of 14 days mating in both males and females, as well as the gestation period and up to post-natal day 3 in females.

Higher tierfertility studies according to OECD 415 or TG 416 were not performed with Glucamide OL. However, based on close structural similarities and comparable physicochemical properties a read-across to data with Glucamide 24 is not only scientifically justified but advisable also for animal welfare reasons. 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.

The potential effects of Glucamide 24 on reproductive function were investigated following OECD TG 415 in a one-generation reproductive study in rats. The test substance was administered once daily via gavage from 70 days prior to mating, during cohabitation (up to 21 days) until the day before sacrifice to 3 groups of rats (25 Crl:CD®(SD)IGS BR VAF/Plus®/sex/dose) at dose levels of 14.8, 147.8, or 344.9 mg/kg bw/day. Males were sacrificed following cohabitation, females were sacrificed on postpartum day 21 and pups were culled on post-natal day (PND) 4 and those maintained were sacrificed on PND 21. Control animals received the vehicle (water) only.

No parental male or female rats died during the course of the study. Clinical observations considered test item related in males treated with 150 and 350 mg/kg bw/day included excess salivation, rales, chromorhinorrhea, red or died red perioral substance and chromodacryorrhea (350 mg/kg bw/day group only). Clinical observation in female rats considered test item related included excessive salivation and rales at 150 and 350 mg/kg bw/day. Though considered treatment and dose related at 150 mg/kg bw/day and above, findings of excess salivation, rales and red or dried red perioral substance were statistically significant in males treated with 350 mg/kg bw/day group as compared to control animals. 

Justification for classification or non-classification

Administration of Glucamide OL and/or the read-across compound Glucamide 24 up to maternal toxic doses did not result in any significant changes of reproductive and/or developmental effects. Based hereupon classification and labelling of the registered substance concerning reproductive toxicity is not warranted.