Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

On the basis of a reproduction / developmental toxicity screening test with the registration substance according to OECD TG 421 in male and female Wistar rats with dose levels of 100, 300 and 1000 mg/kg body weight/day the following conclusions can be made: In male animals slight effects were found at a dose level of 1000 mg/kg body weight/day in haematology parameters (neutrophil and lymphocyte ratios). Although these effects were only marginally and no comparable findings were observed in female animals, the parental NOAEL of the registration substance in male animals of this study is conservatively considered to be 300 mg/kg body weight/day. The maternal NOAEL for female animals is considered to be 1000 mg/kg body weight/day. Due to a slightly higher mortality of pups and a lower total number of live pups on post-natal day 4 at a dose level of 1000 mg/kg body weight/day compared to controls, the NOAEL for reproduction toxicity in this study is conservatively considered to be 300 mg/kg body weight/day.

Link to relevant study records
Reference
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:
2013-10-31 to 2015-04-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to
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. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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: 278 - 313 g (mean: 294.70 g, ± 20% = 235.76 – 353.64 g)
females: 182 - 211 g (mean: 195.45 g, ± 20% = 156.36 – 234.54 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. 060613)
- 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. No animal arriving at BSL
showed pathological signs. 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 vial on a precision balance. The test item was dissolved in corn oil.
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
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
According to the results of the dose range finding study (BSL project no. 133207), a 28 days repeated dose oral toxicity study
(BSL project no. 133208) and in consultation with the sponsor 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 was completed.

C 0 mg/kg bw/ day (Male No.:1-10/ Female No.:41-50)
LD 100 mg/kg bw/ day (Male No.:11-20/ Female No.:51-60)
MD 300 mg/kg bw/ day (Male No.:21-30/ Female No.:61-70)
HD 1000 mg/kg bw/ day (Male No.:31-40/ Female No.:71-80)

The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. T
hereafter, 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 using
the same volume as used for the dose groups (4 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 4 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. If the vaginal smear of a particular female was not found to be sperm-positive,
the actual stage of the estrus cycle on that day was documented. 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 p
reparation (at room temperature), from high and low dose formulations (4 samples).
Each sample was retained twice (sample A, sample B, each of at least 5 mL).
The dose formulation analysis was performed by BSL BIOSERVICE Scientific Laboratories GmbH, in accordance with GLP. All formulation samples
were stored at -20° C. The A samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH
under the BSL study no. 133212.
The B samples will be retained at BSL until the analysis has been performed, and discarded after completion of the final study report.

Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum 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 was completed.
Frequency of treatment:
7 days/week
Details on study schedule:
Arrival of the Test Item: 07 June 2013
Study Initiation Date: 31 October 2013
1st Amendment to Study Plan: 28 November 2013
2nd Amendment to Study Plan: 21 January 2014
3rd Amendment to Study Plan: 17 March 2014
Experimental Starting Date: 05 November 2013
Experimental Completion Date: 30 December 2013
Proposed Completion Date of Delegated Phase (Histopathology): 26 May 2014
Completion Date of Delegated Phase (Formulation Analysis): 03 March 2015

Remarks:
Doses / Concentrations:
0, 100, 300, 1000 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, approximately at the same time each day. 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. One female showing signs of abortion prior to the scheduled scarification of the animals was sacrificed and subjected
to a thorough macroscopic examination. 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.

Haematology
Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC),
mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC),
reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos),
basophils (Baso) and large unstained cells (Luc).

Blood Collection for a possible Determination of Metabolites
Before sacrifice 2 ml blood were sampled from the abdominal aorta of 5 male and 5 female animals per group and collected into serum
separator tubes. After blood collection, the samples were allowed to clot at room temperature and then centrifuged for 10 min at room
temperature at 4000 rpm. After centrifugation, serum was transferred into a tube labeled with the study no., animal ID number, gender
and “serum”. All samples are stored at ≤ -70°C for a possible determination of metabolites.
In addition before sacrifice 2 ml blood from the abdominal aorta of each of the remaining 5 male and 5 female animals per group was
collected in tubes containing Lithium-Heparin, followed by centrifugation for 10 min at room temperature at 4000 rpm. Plasma was
transferred into a tube labeled with the study no., animal ID number, gender and “Li-Hep-Plasma”. All samples are stored at ≤ -70°C
for a possible determination of metabolites.




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 tattooing their paws. In addition to the observations of parent animals, any abnormal behaviour of the offspring
was recorded.
Postmortem examinations (parental animals):
Pathology
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while all surviving
female animals were sacrificed on post-natal day 4 using anaesthesia (ketamine/xylazin, 2:1, Pharmanovo GmbH, lot no: 24431,
expiry date: 01/2015, Pharmanovo GmbH, lot no. 24664, expiry date: 06/2015 and Serumwerk, lot no: 00512, expiry date: 07/2014).
All surviving pups were sacrificed by decapitation on post-natal day 4.
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
A non-pregnant female was sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating.
All animals were subjected to a detailed gross necropsy which includes 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, except from animal no. 22, no. 39 and no. 68, 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.
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) of an animal which was euthanized during the course of the study for animal welfare reasons were preserved and examined histopathologically in order to determine the cause of death.
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.


Histopathology
A full histopathology was carried out on the preserved organs and tissues of an animal which was euthanized due to morbidity.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed,
embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and were examined in Control
and HD animals. Any 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):
All surviving pups were sacrificed by decapitation on post-natal day 4.
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 as well as
of the prenatal and litter weight data 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:
No effects of toxicological relevance were noted for copulation, fertility and delivery indices in the dose groups, when compared to the
control group. No abnormality was observed histopathologically in the reproductive organs of female no. 44 of the control group which
was not pregnant, and no abnormality was observed in its male pairing partner no. 4.
Offspring viability indices:
A slightly reduced viability index (no. of live offspring at day 4 / no. of live offspring at birth x 100) was observed in the HD group (89.7%) as
compared to 100% in the control group. As values did not achieve statistical significance and were within range of historical control data,
this was not considered as an adverse effect of the test item.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Mortality
Female no. 56 of the LD group was euthanized on gestation day 23 for animal welfare reasons during littering of dead pups.
This animal’s morbidity (anaemia, severe piloerection, slightly reduced spontaneous activity, kyphosis, abnormal breathing and wasp waist)
was considered to be related to an endometrial hyperplasia with focal necrotizing inflammation (see 12.2). Abnormal hyperplastic changes
in the reproductive organs were not observed in any HD females, and therefore, the above finding recorded in the uterus of a LD female animal
is not considered to be associated with the treatment of the test item. With this exception, all animals survived the treatment period.


Clinical Observations
There were no clinical signs of toxicological relevance in the dose groups, when compared to the control group.
However, there were occasional incidences of slight to severe salivation in all males and females of the LD, the MD and the HD group.
Moving the bedding was also noted transitorily in all male and female animals of the LD, the MD and the HD group. As the symptoms of
salivation and moving the bedding were noted mainly immediately after administration, they are considered to be signs of discomfort due to
a local reaction to the treatment with the test item rather than a test item-related systemic effect.
Piloerection was noted in one male of the MD group as well as in one female of the control group, two females of the LD, one female of the MD
and three females of the HD group. As the clinical sign of piloerection was transitory in nature and did not follow a dose-related pattern,
it is not considered to be test item-related. Low incidences of slight clinical signs like red nasal discharge, abnormal breathing and slight
diarrhea were noted in isolated males and/or females of the dose groups and/or the control group. No toxicological relevance is considered
for these findings as these were only observed in isolated animals on particular days.
Alopecia in various parts of the body was seen in each one female of the LD, the MD and the HD group. This finding was considered to be incidental.
Prior to euthanasia for animal welfare reasons on gestation day 23, female no. 56 of the LD group showed anaemia, severe piloerection,
slightly reduced spontaneous activity, kyphosis, abnormal breathing and a wasp waist. This was caused by an incidental endometrial
hyperplasia not related to the test item.

Body Weight Development
In both males and females, the mean body weight increased with the progress of the study during premating, mating/postmating and
gestation period in the control, the LD, the MD and the HD group. Values were within the normal range of variation of historical control data.
There were no test item-related effects of toxicological relevance noted on body weight and body weight gain in both males and females.
However, after the first week of treatment until the end of the study, the body weight of the female HD group was slightly higher compared
to the control group. At the end of premating and beginning of gestation period, this difference reached statistical significance with a 6% and
7% higher body weight, when compared to the controls. Corresponding to its higher mean body weight, the body weight gain in the female
HD group was slightly higher during premating and lactation period with a significant difference in the first week of premating (13.7 g in the
HD group compared to 5.3 g in the control group). As values were within the normal range of variation for animals of this strain, this effect is
not assumed to be toxicologically relevant. There were no statistically significant differences in body weight and body weight gain between
the male dose groups and the respective control group.

Food Consumption
There were no statistically or biologically significant effects on food consumption during the treatment period in both males and
females of the dose groups, when compared to the respective controls.

Haematology
At the end of the treatment period, haematological parameters RBC, HB, HCT, MCV, MCH, MCHC, PLT, WBC, Mono, Eos, Baso, Luc and Re
were in the normal range of variation for male and female animals of this strain. Slight differences between the groups are not assumed to
be toxicologically relevant. In the male animals of the HD group, there was a slightly higher mean value for neutrophils (21.13%) compared
to the control group (12.94%) without a left shift. A slightly, but statistically significant, lower value of lymphocytes was observed in the male
HD group (75.68%) compared to 84.54% in the corresponding control group. However, individual values were mainly within a normal range
of variation. As no such effects on both neutrophils and lymphocytes were noted in female animals, the slightly diverging values in male animals
could be induced by stress due to the treatment, however, a relation to the test item cannot be excluded.
Female no. 80 from the HD group showed a markedly lower relative value of neutrophils and a markedly higher value of monocytes.
As this finding was observed only in one single animal, it is not considered to have toxicological relevance.

Pathology- Macroscopic Findings
There were no macroscopic findings of toxicological relevance in any of the groups.
All gross lesions recorded, such as yellow spots on the epididymides, are considered to be incidental changes, which are within the
range of normal background alterations that may be recorded in animals of this strain and age. Gased intestines noted in female no. 68
from MD group are also considered to be incidental, as these changes were observed only in one single animal without following a dose
related pattern.

Organ Weight
In males, there were no statistically or biologically significant effects on the absolute and relative organ weights in the dose groups,
when compared to the control group. In females, there was a marginal tendency towards a lower mean weight of uterus with cervix in the
MD group (absolute 89%, relative to body weight 87%). A slightly, statistically significantly lower mean weight of uterus with cervix in the
HD group (absolute 85%, relative to body weight 80%) was noted, when compared to the corresponding control group. This could be attributed
to an effect of the test item, but without a histopathological correlate and as values were within the range of historical control data,
it was not considered adverse. There were no effects of toxicological relevance on the mean weight of ovaries.

Histopathology
There was no histological evidence 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.

No abnormality was observed in ovaries, uterus with cervix and vagina of control female no. 44 which was not pregnant. No abnormality was
recorded in testes, epididymides, prostate, seminal vesicles and coagulating glands of its pairing partner male no. 4.
There were no 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 control and high-dose males.
These changes as well as focal hypoplastic tubules are considered to be within the range of normal background lesions, which are occasionally
observed in males 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 LD, MD and HD groups were 262.9%, 72.1% and 49.3% of the nominal concentration, respectively. Nominal concentrations could not be
confirmed. Whereas significant outliers were observed each in the LD and HD group, a more consistent and uniform range of values was
recorded for the MD group. 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 room temperature recovery compared to starting value was 72.9% and 177.0%. No conclusive statement on stability
can be given based on these values. 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 70.2% and 60.7% of the nominal value and 66.1% and 73.3% of the nominal value for
HD dose group. Although diverging results concerning the stability of N-(2-Ethylhexyl)isononan-1-amide in the formulation samples were
revealed, stability is proven in additional tests using various vehicles including sesame oil (Reference: Stability in Sesame Oil, Corn Oil and
Dimethylsulfoxide Clariant Rep. No. 13-148451). With regard to the homogeneity results, the physical chemical properties of
N-(2-Ethylhexyl)isononan-1-amide may have contributed to the conflicting recovery values obtained for the dose groups.
Because of the high surface tension N-(2-Ethylhexyl)isononan-1-amide tends to form droplets and may easily adhere to the surface
(e.g. walls of glass vessels). In the quantitative analytic measurements this may have led to underestimation of the actual concentration
of the test item. No triplicate reanalysis was performed with samples not meeting the acceptance criteria. In order to confirm the results
reanalysis was performed using a different technique at another laboratory (Reference: “Concentration in Corn Oil” Clariant Rep. No. 14-176684).

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: The NOAEL for female animals is considered to be 1000 mg/kg body weight/day.
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects
Histopathological findings:
not examined
Litter Data
There were no effects of toxicological relevance for the total number of pups born, still births and runts on PND 0 as well as for the sex ratio
of pups on PND 0 and PND 4. No statistically significant changes were noted for the litter data. However, the total number of live pups on PND 4
was slightly lower in the dose groups compared to the control group in a dose-dependent manner (9.70 in the LD, 9.60 in the MD and 8.70 in
the HD group compared to 11.11 in the control group). Although this effect was not statistically significant, due to dose dependency
it could be attributed to the treatment with the test item. The total number of pups born (PND 0) was also very slightly lower in the dose groups
compared to the control group with a dose dependent pattern (10.70 in the LD, 10.10 in the MD and 9.90 in the HD group compared to 11.11 in the
control group). As this tendency was not supported by statistical significance and the numbers were within the normal range of variation for
animals of this strain, this effect is not assumed to be toxicologically relevant. Sex ratio (male/female) of pups born was slightly but
dose-dependently higher in the dose groups compared to the control group (0.95 in the LD, 1.14 in the MD and 1.23 in the HD group
compared to 0.71 in the control group). Values were within the normal range of variation of historical control data and thus, this finding
is assumed to be incidental.

Litter Weight Data
There were no effects of toxicological relevance noted for the litter weight data including pup mean weight, total litter weight, male litter
and female litter weight on PND 0 and PND 4. However, on PND 0 and on PND 4 a slightly – but not statistically significantly – lower total litter weight
was noted dose-dependently for the dose groups, when compared to the control group. This finding corresponded to the lower number of
pups in the dose groups. The female litter weight on PND 4 was found to be statistically significantly lower in the HD group (43.68 g) and the
MD group (43.86 g), when compared to the control group (66.90 g). This effect was also attributed to the lower number of female pups of the
dose groups on PND 4 and, therefore, was not considered to be an adverse effect of the test item.
There were no further statistically significant differences in these litter weight data.

Precoital Interval and Duration of Gestation
There were no statistically or biologically significant effects on the duration of precoital interval and the duration of gestation in the dose groups,
when compared to the control group. Values were within the normal range of variation of historical control data.

Pre- and Post-Natal Data
There were no statistically or biologically significant effects on the number of corpora lutea, number of implantation sites, number of
live pups on PND 0 and percentage of pre- and post-implantation loss. Although there was a slight dose-dependent increase in pre-implantation
loss in the dose groups (13.02 in the LD, 19.33 in the MD and 22.95 in the HD group) compared to the control group (11.05),
due to lack of statistical significance and based on historical control data, this finding is assumed to be incidental.

Reproductive Indices
No effects of toxicological relevance were noted for copulation, fertility and delivery indices in the dose groups, when compared to the control group.
No abnormality was observed histopathologically in the reproductive organs of female no. 44 of the control group which was not pregnant,
and no abnormality was observed in its male pairing partner no. 4. A slightly reduced viability index (no. of live offspring at day 4 / no. of live
offspring at birth x 100) was observed in the HD group (89.7%) as compared to 100% in the control group. As values did not achieve statistical
significance and were within range of historical control data, this was not considered as an adverse effect of the test item.

Pup Survival Data
No statistically significant effect on the survival of the pups from PND 0 to PND 4 was observed in any treatment group, when compared to the
control group. However, a moderately higher – but not statistically significant – mortality of pups between PND 1 and PND 4 was noted in the
HD group (10.5%) compared to 0.0% in the control group. This finding was attributed to female no. 74 with 3 pups missing and 2 pups found
dead as well as to female no. 76 with also 3 pups missing and 2 pups found dead. The missing of the pups was attributed to cannibalism by the dam.
Though not statistically significant, this effect on survival can be considered as test item-related with a slightly higher mortality on post-natal
day 2 (2.5%) and 3 (0.8%) compared to 0.0% in historical control data.

Pup External Findings
No gross external abnormalities of toxicological relevance were observed in the dose groups.
Few external findings were noted in the control group (dark spots at snout and back), the LD (small body size), the MD (red spots at body
side and snout) and the HD group (red spots at head and nose, bite injury at jaw, cold, small body size), which are considered incidental and
not related to the test item.

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Reproductive effects observed:
not specified
Conclusions:
On the basis of this reproduction / developmental toxicity screening test with N-(2-Ethylhexyl)isononan-1-amide in male and female Wistar rats with dose levels of 100, 300 and 1000 mg/kg body weight/day the following conclusions can be made: In male animals slight effects of N-(2-Ethylhexyl)isononan-1-amide were found at a dose level of 1000 mg/kg body weight/day in haematology parameters (neutrophil and lymphocyte ratios). Although these effects were only marginally and no comparable findings were observed in female animals, the NOAEL of N-(2-Ethylhexyl)isononan-1-amide in male animals of this study is conservatively considered to be
300 mg/kg body weight/day. The NOAEL for female animals is considered to be 1000 mg/kg body weight/day.Due to a slightly higher mortality of pups and a lower total number of live pups on post-natal day 4 at a dose level of 1000 mg/kg body weight/daycompared to controls, the NOAEL for reproduction toxicity in this study is conservatively considered to be 300 mg/kg body weight/day.

Executive summary:

The aim of this study was to assess the possible effects of N-(2-Ethylhexyl)isononan-1-amideon 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 female Wistar rats.

The following doses were evaluated:

Control:                        0        mg/kg body weight

Low Dose:                    100     mg/kg body weight

Medium Dose:              300     mg/kg body weight

High Dose:                   1000   mg/kg body weight

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in corn oil and administered daily during14 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 dosedfor 28 days. Dose volumes were adjusted individually based on weekly body weight measurements.

The administration volume was 4 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. One animal that was euthanized for animal welfare reasons 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 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 29and the females along with their pups were sacrificed on post-natal day 4. Non-pregnant females were sacrificed on day 26. Haematological examinations were made on blood samples obtained from all adult animals at terminal sacrifice. After sacrifice, all adult animals were subjected to necropsy. The wet weight of a subset of organs was taken and a set of organs/tissues was preserved. The number of implantation sites and corpora lutea were recorded for each parental female at necropsy. Pups sacrificed on post-natalday 4 and those found dead, were carefully examined for gross external abnormalities.

A histopathological evaluation of testes, epididymides, ovaries, uterus with cervix, vagina and accessory sex organs (prostate, seminal vesicle with coagulating gland) was performed on high dose and control animals.Any gross lesion macroscopically identified was examined microscopically in all animals.

Summary Results

Femaleno. 56 of the LD groupwas euthanized in a moribund condition on gestation day 23 for animal welfare reasons.This animal’s morbidity was considered to be related to an incidental endometrial hyperplasia with focal necrotizing inflammation, which is not considered to be associated with the treatment of the test item.With this exception, all animals survived the treatment period.

There were no clinical signs of toxicological relevance in the dose groups. However, there were occasional incidences of slight to severe salivation in all males and females of the LD, the MD and the HD group. Moving the bedding was also noted transitorily in all male and female animals of the LD, the MD and the HD group.As the symptoms of salivation and moving the bedding were noted mainly immediately after administration, they are considered to be signs of discomfort due to a local reaction to the treatment with the test item rather than a test item-related systemic effect.

There were no test item-related effects of toxicological relevance on body weight and body weight gain in both males and females.

There were no test item-related effects of toxicological relevance on food consumption during the treatment period in both males and females.

There were no test item-related effects of toxicological relevance on the total number of pups born, still births and runts on PND 0 or on the sex ratio of pups on PND 0 and PND 4. However, the total number of live pups on PND 4 was slightly lower in the dose groups compared to the control group in a dose-dependent manner (9.70 in the LD, 9.60 in the MD and 8.70 in the HD group compared to 11.11 in the control group).

lthough this effect was not statistically significant, due to dose dependency it could be attributed to the treatment with the test item.

There were no test item-related effects of toxicological relevance on litter weight data including pup mean weight, total litter weight, male litter and female litter weight on PND 0 and PND 4. However, the female litter weight on PND 4 was found to be statistically significantly lower in the MD group and the HD group, when compared to the control group. This effect was attributed to the lower number of female pups in the dose

groups on PND 4.

There were no test item-related effects of toxicological relevance on the duration of precoital interval and the duration of gestation.

There were no test item-related effects of toxicological relevance on the number of corpora lutea, number of implantation sites, number of live pups on PND 0 and percentage of pre- and post-implantation loss.

There were notest item-relatedeffects of toxicological relevance on copulation, fertility and delivery indices.

A slightly reduced viability index (no. of live offspring at day 4 / no. of live offspring at birth x 100) was observed in the HD group (89.7%) as compared to 100% in the control group. As values did not achieve statistical significance and were within range of historical control data, this was not considered as an adverse effect of the test item.

A moderately higher mortality of pups between PND 1 and PND 4 was noted in the HD group (10.5%) compared to 0.0% in the control group. Though not statistically significant, the effect on survival on single days can be considered as test item-related with a slightly higher mortality on post-natal day 2 (2.5%) and 3 (0.8%) compared to 0.0% in historical control data.

There were no gross external abnormalities of toxicological relevance observed in pups of the dose groups, when compared to the control group.

At the end of the treatment period, haematological parameters RBC, HB, HCT, MCV, MCH, MCHC, PLT, WBC, Mono, Eos, Baso, Luc and Re were in the normal range of variation for male and female animals of this strain. Slight differences between the groups were not assumed to be toxicologically relevant.

In the male animals of the HD group, there was a slightly, statistically significantly higher neutrophil rate (without left shift) and a slightly, statistically significantly lower value of lymphocytes compared to the control group. However, individual values were mainly within a normal range of variation. As no such effects on both neutrophils and lymphocytes were noted in female animals, the slightly diverging values in male animals could be induced by stress due to the treatment, however, a relation to the test item cannot be excluded.

A slightly but statistically significantly lower value of lymphocytes was observed in the male HD group compared to the corresponding control group. This finding could be considered as an effect of the test item.

In females, there were no effects of toxicological relevance on both neutrophils and lymphocytes.

At necropsy, there were no macroscopic findings of toxicological relevance in any of the groups.

In males, there were no differences of toxicological relevance in the absolute and relative organ weights between the dose groups and the control group.

A slightly, statistically significantly lower mean weight of uterus with cervix in the HD group (absolute 85%, relative to body weight 80%) was noted when compared to the corresponding control group. This could be attributed to an effect of the test item, but without a histopathological correlate and as values were within the range of historical control data, it was not considered adverse.

There were no differences of toxicological relevance in the absolute and relative ovary weights between the dose groups and the control group.

There was no histological evidence oftoxicologicalproperties in the organs and tissues of the reproductive system; i.e. testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus and cervix, and vagina.

There were no treatment-related effects on the completeness of stages or cell populations of the testes.

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 262.9%, 72.1% and 49.3% of the nominal concentration, respectively. Nominal concentrations could not be confirmed. Whereas significant outliers were observed each in the LD and HD group, a more consistent and uniform range of values was recorded for the MD group.

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 room temperature recovery compared to starting value was 72.9% and 177.0%.Despite the diverging results concerning the stability of

N-(2-Ethylhexyl)isononan-1-amide in the formulation samples, stability is proven in additional tests using various vehicles in another laboratory.

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 70.2% and 60.7% of the nominal value and 66.1% and 73.3% of the nominal value for HD dose group.

The physical chemical properties of N-(2-Ethylhexyl)isononan-1-amide may have contributed to the conflicting recovery values obtained for the dose groups. Because of the high surface tension N-(2-Ethylhexyl)isononan-1-amide tends to form droplets and may easily adhere to the surface (e.g. walls of glass vessels).In the quantitative analytic measurements this may have led to underestimation of the actual concentration of the test item.

No triplicate reanalysis was performed with samples not meeting the acceptance criteria. In order to examine the results, reanalysis was performed using a different technique at another laboratory.

Conclusion

On the basis ofthis reproduction / developmental toxicity screening test withN-(2-Ethylhexyl)isononan-1-amidein male and female Wistar rats with dose levels of 100, 300 and 1000 mg/kg body weight/day the following conclusions can be made:

In male animals slight effects of N-(2-Ethylhexyl)isononan-1-amidewere found at a dose level of 1000 mg/kg body weight/day in haematology parameters (neutrophil and lymphocyte ratios). Although these effects were only marginally and no comparable findings were observed in female animals, the NOAEL of N-(2-Ethylhexyl)isononan-1-amidein male animals of this study is conservatively considered to be 300 mg/kg body weight/day. The NOAEL for female animals is considered to be 1000 mg/kg body weight/day.

Due to a slightly higher mortality of pups and a lower total number of live pups on post-natal day 4 at a dose level of 1000 mg/kg body weight/day compared to controls, the NOAEL for reproduction toxicity in this study is conservatively considered to be 300 mg/kg body weight/day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data reliable and plausible.
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, from all available data no indications of a significant developmental toxic potential exist. This view is supported by computational structural alert analysis.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Administration of the registration substance up to maternal toxic doses did not result in any significant changes of reproductive and/or developmental effects in a guideline conform OECD TG 421 screening study. Based hereupon, no indication for classification and labelling of the registered substance concerning reproductive toxicity exist.