Octene, hydroformylation products, low-boiling

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In life: 25/11/2020 until 28/07/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

June 2018

GLP compliance:

yes

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS]:

Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method OECD TG 443) in rats, oral route with the registered substance specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce systemic toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation; and
- Cohorts 2A and 2B (Developmental neurotoxicity).
- Premating exposure duration for parental (P0) animals :
There was no substance specific information supporting shorter premating exposure duration as advised in the ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a, Section R.7.6 (version 6.0, July 2017).
- Basis for dose level selection :
The dose levels have been selected based on available toxicological data and the results of a preliminary subchronic repeated dose toxicity study (0-200-400-750 mg/kg bw/day) in rats: 3 test-item related deaths (males) were observed at the high dose of 750 mg/kg b.w.. Reduced body weights were noted for males at the low, the intermediate and the high dose group (approx. 4% (200 mg/kg b.w.), approx. 8% (400 mg/kg b.w.) or approx. 15% (750 mg/kg b.w.) below the control on test day 57; statistically significant at the high dose level with p < 0.01). Reduced body weights (statistically significant or not) were noted for females for all 3 treatment groups on gestation days 0, 7 and 14 (at maximum approx. 8% (200 mg/kg b.w.), approx. 6 (400 mg/kg b.w.) or approx. 13% (750 mg/kg b.w.) below the control).
Hence, doses of 50, 150, 450 mg/kg b.w./day are proposed for the OECD 443 study.
- Inclusion/exclusion of extension of Cohort 1B:
Due to exposure considerations extension of cohort 1B to mate F2 generation is needed.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B :
Cohorts 2A and 2B need to be conducted because there is a concern on (developmental) neurotoxicity based on the results from the above-identified in vivo study on the registered substance.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 :
No trigger for the inclusion of Cohort 3 were identified.
- Route of administration :
The oral route is the most appropriate route of administration for substances except gases to focus on the detection of hazardous properties on reproduction as indicated in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a, Section R.7.6 (version 6.0, July 2017).

Test material

Test material information

Constituent 1

Reference substance name:

Octene, hydroformylation products, low-boiling

EC Number:

273-110-1

EC Name:

Octene, hydroformylation products, low-boiling

Cas Number:

68938-03-4

IUPAC Name:

Octene, hydroformylation products, low-boiling

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: approximately 20 weeks

- Housing:
- Diet: ad libitum
- Water: ad libitum)
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3 °C
- Humidity (%): 55% ± 10%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12h/12h
IN-LIFE DATES: From: 25/11/2020 To: approx. 28/07/2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

According to international guidelines.

Details on mating procedure:

Sexually mature male and female rats of the same dose group are paired randomly monogamously, i.e. 1 male and 1 female animal are placed in one cage during the dark period. The female is placed with the same male until evidence of mating is observed
or two weeks have elapsed. The females are examined each morning for the presence of sperm. The day of conception (gestation day (GD) 0) is considered to be the day on which sperm is found.

Duration of treatment / exposure:

F0 males: 10 weeks prior to mating, during the mating period, and at least until weaning of the F1 generation; F0 females: 10 weeks prior to mating, during the mating, gestation and lactation period and until termination after weaning of their litters
F1 cohort 1A: Until a dosing period of 10 weeks has been completed (up to and including the day before sacrifice, i.e. around PND 90); F1 cohort 1B: Until sacrifice of the F2 generation (up to and including the day before sacrifice, i.e. F2 PND 21); F1 cohort 2A: Up to and including the day before sacrifice, i.e. around PND 75 (after neurological screening); F1 cohort 2B: Until sacrifice at PND 21/22
F2 animals: Until sacrifice at PND 21

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

control

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day

Remarks:

mid dose

Doses / concentrations 4

Dose / conc.:

450 mg/kg bw/day

Remarks:

high dose

No. of animals per sex per dose:

F0 animals: 24
F1 animals: cohort 1A: 20; cohort aB: 20; cohort 2A: 10; cohort 2B:10

Control animals:

yes, concurrent vehicle

Details on study design:

administration volume: 2 ml/kg bw/day

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cageside observations will include skin/fur, eyes, mucous membranes, respiratory and
circulatory systems, somatomotor activity and behaviour patterns.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: daily (pre-mating and mating period, males+females), GD 0, 7, 14, 21 (gestation, females), PND 1, 4, 7, 14, 21 (lactation, females), daily (post-mating, males)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measured weekly, except for mating

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations: Water consumption was measured weekly, except for mating
:

Oestrous cyclicity (parental animals):

14-day pre-exposure period to select 96 animals with regular oestrus cycles (4-5 days). During 10 weeks of premating until evidence of mating.

Sperm parameters (parental animals):

Parameters examined in [all F0, all F1 cohort 1A] male parental generations:
testis weight, epididymis weight, sperm count, sperm motility, sperm morphology, epididymis not preserved for histopathology is used for enumeration of cauda epididymis sperm reserves

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible)

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, sexual maturation (balano-preputial separation or vaginal patency, abnormatilies of genitals)an

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Screening of sensory reactivity (auditory, visual and proprioceptive stimuli; (Gad, 1982)), assessment of grip strength (Meyer, 1979) and motor activity assessment. Auditory startle response at PND 24±1, Functional observational battery and motor activity at PND 63-75
Righting reflex, Body temperature, Salivation, Startle response, Respiration,
Mouth breathing, Urination, Convulsions, Pilo-erection, Diarrhea, Pupil size, Pupil response, Lacrimation, Impaired gait, Stereotypy, Toe pinch, Tail pinch, Wire maneuver, Hind-leg splay, Positional passivity, Tremors, Positive geotropism, Limb rotation, Auditory function, Posture, Palpebral closure, Vocalisation, Arousal, Bizarre behavior, Ease of removal/handling, Muscle tone, Approach response, Touch response
Grip strength, Locomotor activity
Neuro-histopathology of the F1 Cohort 2A:
Brain (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus,
mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum), eye with optic nerve and retina, Muscle (skeletal), Nerve (sciatic), Spinal cord (3 sections)
Neuro-histopathology of the F1 Cohort 2B:
Brain (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum)

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Cohort 3 was not included in this study.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after weaning of the F1 animals
- Maternal animals: All surviving animals shortly after weaning of the F1 animals

GROSS NECROPSY
- Full necropsy

HISTOPATHOLOGY / ORGAN WEIGHTS
Full histopathology on preserved organs.
Eye with optic nerve, Epididymis, Testicle, Adrenal gland, Oviducts, Bone Pituitary, Bone marrow (os femoris) Prostate, Brain (cerebrum, cerebellum, pons), Seminal vesicles with coagulating glands, Gross lesions observed Spinal cord (3 sections), Heart (3 levels: right and left ventricle, septum), Spleen, Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method), Stomach, Intestine, large (colon, rectum), Thyroid (including parathyroids), Kidney and ureter, Thymus, Liver, Trachea (including larynx), Lungs (with mainstem bronchi and bronchioles), Urinary bladder, Mammary gland, Uterus (including cervix), Muscle (skeletal), Vagina, Nerve (sciatic), Vas deferens, Oesophagus, Identified target organs, Ovary

Postmortem examinations (offspring):

SACRIFICE
- F1 animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
“surplus” pups All On PND 4 Gross necropsy
“surplus” pups All On PND 22 Gross necropsy Including assessment of reproductive organs
Cohort 1A All At the end of the dosing period (PND 91) Full necropsy
Cohort 1B All Shortly after weaning of the F2 animals Full necropsy
Cohort 2A All After behavioural testing, around PND75 Full necropsy
Cohort 2B All On PND 21/22 Full necropsy


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
Eye with optic nerve, Epididymis, Testicle, Adrenal gland, Oviducts, Bone Pituitary, Bone marrow (os femoris) Prostate, Brain (cerebrum, cerebellum, pons), Seminal vesicles with coagulating glands, Gross lesions observed Spinal cord (3 sections), Heart (3 levels: right and left ventricle, septum), Spleen, Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method), Stomach, Intestine, large (colon, rectum), Thyroid (including parathyroids), Kidney and ureter, Thymus, Liver, Trachea (including larynx), Lungs (with mainstem bronchi and bronchioles), Urinary bladder, Mammary gland, Uterus (including cervix), Muscle (skeletal), Vagina, Nerve (sciatic), Vas deferens, Oesophagus, Identified target organs, Ovary

Reproductive indices:

Gestation Index = Number of litters with live pups/Number pregnantx 100
Fertility Index female [%] =Number of pregnant rats/Number of females usedx 100

Offspring viability indices:

Birth Index =Total number of pups born (live +dead)/Number of implantation scarsx 100
Live Birth Index =Number of pups born alive on day 0/1/Total number born (live + dead)x 100
Viability Indexpre-select =Number of pups alive on day 4 (pre-select)/Number of pups live on day 0/1x 100
Viability Indexpost-select =Number of pups alive on day 21 (post-select)/Number of pups live on day 0/1x 100
Post-implantation loss [%] =Implantations - number of pups born alive/Implantationsx 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males
A post-dosing salivation was noted for several low dosed males (6 of 24), nearly all males of the intermediate dose group (22 of 24) and all surviving male animals of the high dose group (21 of 21) (50, 150 or 450 mg Oxooil LS9/kg b.w./day). In nearly all cases salivation was a short lasting post-dosing symptom. The post-dosing salivation was considered as test item-related but not adverse.
Changes in the consistency of the faeces in the form of soft faeces and / or diarrhea were noted for several males of the control group and the low and the intermediate dose group, but only for one male of the high dose group. As no dose-response relationship was noted and also several control males were affected, the observed changes in the consistency of the faeces were considered to be spontaneous.
The other observations that were noted for the male animals at the intermediate (breathing sounds (1 of 24), reduced faeces excretion (1 of 24)) and at the high dose level (haemorrhagic canthus (1 of 21), soft faeces, reduced faeces excretion (1 of 21)) were considered to be spontaneous due to their low incidences.

Females
A post-dosing salivation was noted for a few females of the low dose group (3 of 24 during pre-mating / mating, 1 of 21 during lactation), nearly all females of the intermediate dose group (22 of 24 during pre-mating / mating, 5 of 22 during gestation, 8 of 22 during lactation) and all females of the high dose group (23 of 23 during pre-mating / mating, 11 of 12 during gestation, 12 of 12 during lactation) (50, 150 or 450 mg Oxooil LS9/kg b.w./day).
The post-dosing salivation was considered to be test item-related but not adverse.
A swollen fore paw (low dose group) and breathing sounds or pale eyes (intermediate dose group) were also noted but considered to be spontaneous due to their low incidences.


Detailed clinical observations
No further observations in addition to those made during the daily cage side observations were noted for the male and female animals of the control group and the treatment groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

Males
No test item-related premature death or sacrifice was noted for the male animals at 50 or 150 mg Oxooil LS9/kg b.w./day.
At 450 mg Oxooil LS9/kg b.w./day 1 male was pre-prematurely sacrificed on test day 125 due to a loss of hindlimb function due to an atrophy of the muscle fibre that was caused by degeneration of the nerve fibres. The observed muscle atrophy and the degeneration of the nerve fibres were considered to be test item-related.
Hence, the animal was prematurely sacrificed due to test item-related adverse effects.
The death of 2 further high dosed males was not considered to be test item-related. The death was probably caused by a misgavage.
Females
No test item-related premature death or sacrifice was noted for the female animals at 50, 150 or 450 mg Oxooil LS9/kg b.w./day.
One high dosed female died by a pituitary adenoma, which was considered to be spontaneous as no further animals were noted with a pituitary adenoma.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males
A marginally reduced body weight (statistically not significant) was noted at the intermediate dose level (150 mg Oxooil LS9/kg b.w./day) from test day 50 until test day 146 (2.7 % or 5.4 % below the control).
The reduced body weight at the intermediate dose level was considered to be test item-related but not adverse due to the small differences.
At the high dose level (450 mg Oxooil LS9/kg b.w./day), the body weight of the males was clearly reduced from test day 43 until test day 146 (6.4 % or 18.9 % below the control, p ≤ 0.05 / 0.01).
The reduced body weight at the high dose level was considered to be test item-related and adverse.
A markedly reduced body weight at autopsy was noted at 450 mg Oxooil LS9/kg b.w./day (20.5 % below the control, p ≤ 0.01).
The changes were in accordance with the last live body weight. The marked reduction of the last live body weight at the high dose level was considered to be test item-related and adverse.


Body weight gain - Males
Accordingly, a marginally reduced body weight gain was noted at the intermediate dose level and a severely reduced body weight gain at the high dose level (30.1 % or 10.9 % in comparison to 37.0 % in the control group) between test days 15 to 146.
Statistically significantly reduced absolute values in body weight gain were noted for the low, the intermediate and the high dose group (159.75 g, 152.24 g and 55.71 g in comparison to 188.82 g in the control group, p ≤ 0.05 / 0.01).

Females
Slight to marginal reductions in body weight were noted at the high dose level (450 mg Oxooil LS9/kg b.w./day). The reductions in body weight were noted during the pre-mating period (at maximum 4.0 % below the control, statistically not significant), the gestation period (6.0 % below the control on gestation day 0, p ≤ 0.05) and the lactation period (at maximum 7.5 % below the control on lactation day 21, p ≤ 0.05).
Due to the small difference in body weight and body weight gain (see below) the observed reduction in body weight was considered to be test item-related but not adverse or only of low toxicological relevance.
At 450 mg Oxooil LS9/kg b.w./day, the body weight at autopsy was 11.5 % below the control (p ≤ 0.01). This was in accordance with the last live body weight (7.5 % below the control, p ≤ 0.05) at the end of the lactation period.
The reduction in the body weight at autopsy that was noted at the high dose level was considered to be test item-related. However, as the differences in live body weight between the control group and the high dose group were considered to be of low toxicological relevance.

Body weight gain - Females
At the high dose level, a marginally reduced body weight gain was noted during the pre-mating period (9.3 % in comparison to 12.8 % for the control) and during the lactation period (3.9 % in comparison to 6.9 % in the control).
The absolute values of body weight gain revealed a slight, but statistically significantly reduced value at the high dose level in comparison to the control group (26.76 g in comparison to 36.63 g, p ≤ 0.05) during the pre-mating period.
No statistically significant differences were noted between the absolute values of body weight gain during the lactation period (12.83 g at the high dose level in comparison to 23.98 g in the control group, not statistically significant).
A moderate reduction in body weight gain was noted at the high dose level during the gestation period (28.8 % in comparison to 47.9 % in the control).
The absolute value of body weight gain at the high dose level was statistically significantly reduced in comparison to the control group (87.04 g in comparison to 155.27 g, p ≤ 0.01) during the gestation period.

The slight differences in body weight gain between the control group and the high dose group that were noted during the pre-mating period and the lactation period were considered as non-adverse. The more pronounced reduction in body weight gain that was noted during the gestation period was due to a reduced number of pups.

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males
At the high dose level (450 mg Oxooil LS9/kg b.w./day) reduced numbers of white blood cells and lymphocytes were noted (40.8 % or 38.6 % below the control, p ≤ 0.01).

Females
No test item-related changes were noted.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females
No adverse effects were noted for the male and female animals.
However, for the male animals a statistically significantly increased cholesterol concentration was noted at the intermediate dose level (31.5 % above the control, p ≤ 0.05).
At the high dose level statistically significantly increased cholesterol and bilirubin concentrations were noted for the male animals (45.4 % or 101.8 % above the control, p ≤ 0.01).
For the female animals a statistically significantly increased bilirubin concentration was noted at the high dose level (41.7 %, above the control, p ≤ 0.01).

However, the observed increased cholesterol and bilirubin concentrations were considered to be non-adverse. It is most likely that they were due to 2 non-adverse effects. These 2 non-adverse effects were the metabolism of the oily test substance Oxooil and the observed liver hypertrophy which was considered to be an adaptive effect to liver enzyme induction.

Endocrine findings:

no effects observed

Description (incidence and severity):

Males and females
No test item-related changes were noted.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males
At 450 mg Oxooil LS9/kg b.w./day, a test item-related peripheral neuropathy was noted for several males in the form of sciatic nerve fiber degeneration (9 of 17) and muscle fiber atrophy (9 of 18).
In addition, axonal swelling and nerve fiber degeneration (ventral root) were noted for a few to several of these 9 males for nerves that originated from the cervical, thoracic or lumbar segment of the spinal cord.
These findings, which were summarised as peripheral neuropathy were considered as a test item-related and adverse effect.
Additional examinations in groups 2 and 3:
The additional examination of the sciatic nerves and the skeletal muscles from the male animals dosed with 50 or 150 mg Oxooil LS9/kg b.w./day showed no test item related lesions for the sciatic nerves (nerve fiber degeneration) and the skeletal muscles (muscle fiber atrophy).

Test item-related findings that were not considered to be adverse or non-relevant to humans were noted at the high dose level for the kidneys (enhanced hyaline droplet deposits in the proximal tubular epithelia, only relevant for male rats and not for humans) and the liver (centrilobular hepatocellular hypertrophy, considered to be of metabolic nature and of adaptive character.

Examination of reproductive organs - Males
A reduced secretion was noted in the prostate gland, the coagulating glands and the seminal vesicles. This was considered to be secondary to a decreased body weight.
No test item-related observations were noted for the testes and the epididymis of the male animals of group 4.
Detailed histopathological examination of one testis and one epididymis
With one exception (no. 67 of group 2, see below), all males examined histologically in this study showed completeness of stages and cell population.

Females
At 450 mg Oxooil LS9/kg b.w./day, a centrilobular hepatocellular hypertrophy was noted in the liver.
This was considered to be of metabolic nature and non-adverse.

Examination of reproductive organs - females
No test item-related findings were noted.


Male and female pairs
The reproductive organs from 9 pairs with reduced fertility that were not scheduled for full histopathology, were histopathologically examined.
The male (no. 67) of one pair of the low dose group showed atrophic changes in the testis. As this observation was a singular finding, it was considered to be spontaneous.
No histopathological findings were noted for 5 pairs (2 of the low dose group, 2 of the intermediate dose group and 1 of the high dose group).
Unphysiological changes were observed in the uterus from the females of 3 high dosed pairs. It remains unclear whether these findings were related to the negative reproductive outcome.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the mean number and the mean length of the oestrous cycles.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

A reduced fertility index was noted at the high dose level (450 mg Oxooil LS9/kg b.w./day) (78 % in comparison to 100 % in the control group).
At 450 mg Oxooil LS9/kg b.w./day, a reduced gestation index of 61 % was noted in comparison to 100 % in the control group.
In total, 7 pregnant females did not deliver live pups, due to the resorption of all implants (2 females, for the respective females only 1 or 2 implants per female were noted at all), the intra-uterine death of their only one fetus (3 females), the delivery of their only one fetus as a stillbirth (one female) or unobserved abortion/littering (one female with placental parts in the stomach).
The loss of only a small number of implants by resorption can be considered as a physiological effect due to an insufficient level of gonadotropine. This was due to the low number of placentae, which were not able to produce enough gonadotropine to maintain pregnancy. Hence, this effect was not considered to be test item-related.
However, the finding of a fully developed fetus in the uterus from 3 females at necropsy on gestation day 25 (in all cases it was their only one fetus, developed from their only one implantation site) was considered as a hint of littering problems and hence, considered as an adverse effect on the gestation index.
No effects on pre-coital time, gestation length.

F1 Pups - Pre- and postnatal development - Prenatal development (from conceptus to birth):
At 450 mg Oxooil LS9/kg b.w./day, a decreased number of implantation sites was noted (5.6 ± 5.4 implantation sites per dam in comparison to 14.8 ± 4.0 in the control group, p ≤ 0.01). Altogether, 9 of 18 pregnant females revealed only 1 or 2 implantation sites. The reduced number of implantation sites was considered as a test item-related and adverse effect.
In addition, an increased post-implantation loss and a decreased birth index were noted at the high dose level due to 7 females with only 1 or 2 implantation sites and an implantation loss of 100 %.
The implantation loss of 100 % was due to the failing of the delivery of a live pup from their only 1 or 2 implantation sites due to resorptions, the intra-uterine death of the fetus or the delivery of a stillbirth.
As described under ‘Gestation index’ these effects were physiologically or due to problems with littering and hence, not considered to be an adverse effect on the development of the embryo or fetus after implantation.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

body weight and weight gain

haematology

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

1

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (nominal)

System:

peripheral nervous system

Organ:

other: sciatic nerve in males

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males
A post-dosing salivation was noted for 18 of 20 males of the high dose group (300 mg Oxooil LS9/kg b.w./day).
The post-dosing salivation was considered as test item-related but not adverse.
Cuts or wounds, a reddened canthus, a haemorrhagic eye lid or canthus, hair loss and a decreased water consumption were noted for individual males of all treatment groups and considered to be spontaneous.

Females
A post-dosing salivation was noted for 1 of 20 females of the intermediate dose group and for 13 of 20 females of the high dose group during the pre-mating / mating period (150 or 300 mg Oxooil LS9/kg b.w./day). During the gestation and the lactation period, a post-dosing salivation was only noted for 2 different high dosed females on one test day each, either during the gestation period or the lactation period.
The post-dosing salivation was considered as test item-related but not adverse.

Other observations like cuts or wounds, hair loss and a haemorrhagic or reddened canthus were only noted for individual animals of the low, the intermediate and the high dose group and considered to be spontaneous.

Start and duration
In nearly all cases, salivation was a short-lasting post-dosing symptom.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Cohort 1B
Males
No test item-related premature death was noted for the male animals at 50, 150 or 300 mg Oxooil LS9/kg b.w./day.
One male of the low dose group was prematurely sacrificed due to a palpable mass on the left forelimb. As this finding was not noted for any other animal, the occurrence of a palpable mass was considered to be spontaneous.
Females
No premature death was noted for the female animals at 50, 150 or 300 mg Oxooil LS9/kg b.w./day.
Two premature deaths were noted for the females of the control group. One was prematurely sacrificed due to a spontaneous palpable mass near the mamma complex and one died during littering.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males
A marginally reduced body weight (statistically not significant) was noted at the intermediate dose level (150 mg Oxooil LS9/kg b.w./day) from post-natal day 64 until post-natal day 140 (2.0 % or 3.0 % below the control).
The marginally reduced body weight was considered as test item related but not as adverse.
At the high dose level (300 mg Oxooil LS9/kg b.w./day), the body weight of the males was reduced from post-natal day 57 until post-natal day 140 (7.4 % or 11.4 % below the control, p ≤ 0.05 / 0.01), with a maximum difference on post-natal day 106 (13.0 % below the control, p ≤ 0.01).
This was considered as test item-related and adverse.

Body weight gain - Males
Accordingly, a reduced body weight gain was noted at the intermediate and the high dose level (894.2 % or 829.5 % in comparison to 978.7 % in the control group) between post-natal days 22 and 140.
A statistically significant reduction (p ≤ 0.01) in comparison to the control group was noted at the high dose level for the absolute value of body weight gain. The absolute value of body weight gain was 561.59 g in the control group, 579.31 g at the low, 539.86 g at the intermediate and 489.34 g at the high dose level.
At 300 mg Oxooil LS9/kg b.w./day a reduced body weight at autopsy was noted (12.2 % below the control, p ≤ 0.01). This was in accordance with the reduction for the last live body weight, which was considered to be test item-related and adverse.

Females
No test item-related changes were noted during the pre-mating, the gestation and the lactation period.
No statistically significant differences were noted for the absolute values of body weight gain between the control group and the treatment groups during the pre-mating, the gestation and the lactation period.
A marginally reduced body weight at autopsy was noted at the high dose level (4.7 % below the control, statistically not significant). This small difference was in accordance with the last live body weight and not considered to be test item-related.

Body weight gain - Females
The body weight gains from all dose groups were in the range of the control group during the pre-mating, the gestation and the lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males
Statistically significantly increased relative organ weights were noted at the high dose level. These changes were secondary to a reduced body weight and not an adverse effect on the organs itself.

Females
No test item-related influence was noted.

Gross pathological findings:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test item-related influence was noted.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

A reduced fertility index was noted at 300 mg Oxooil LS9kg b.w./day (79 % in comparison to 95 % in the control group).

Gestation index, gestation length, pre-coital time: No test item-related influence was noted.

F2 pups: Pre- and postnatal development - Pre-natal development (from conceptus to birth):
No test item-related influence was noted on the number of implantation sites, the number of live born pups, the birth index, the live birth index, and the percentage of post-implantation loss.
No increased number of resorptions or stillborn were noted in the treatment groups in comparison to the control group.

Effect levels (P1)

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

body weight and weight gain

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A
Males
A post-dosing salivation was noted for 4 of 20 males of the intermediate dose group and all males (20 of 20) of the high dose group (150 or 450 mg Oxooil LS9/kg b.w./day).
The observation of post-dosing salivation was considered to be test-item-related, but not adverse.
Cuts or scratch wounds on the neck, head or body were noted for one male each of the treatment groups, which were considered to be idiopathic and or self-inflicted and not related to treatment.
With the exception of post-dosing salivation and cut wounds, no further observations were noted for the male animals of the treatment groups.

Females
A post-dosing salivation was noted for 7 of 20 females of the intermediate dose group and for nearly all females (17 of 19 females) of the high dose group (150 or 450 mg Oxooil LS9/kg b.w./day).
The observation of post-dosing salivation was considered to be test-item-related, but not adverse.
No further observations were noted for the female animals of the low and the high dose level.
At the intermediate dose level one female with cut wounds (1 of 20) and another female with a reddened and / or haemorrhagic eye lid (1 of 20) were noted.
These observations were considered to be spontaneous.
Start and duration
In all cases, salivation was a short lasting post-dosing symptom.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Cohort 1A
Males
No test item-related premature death or sacrifice was noted for the male animals at 50, 150 or 450 mg Oxooil LS9/kg b.w./day.
One male of the control group was prematurely sacrificed due to cuts / wounds on the head.
Females
No test item-related premature death or sacrifice was noted for the female animals at 50, 150 or 450 mg Oxooil LS9/kg b.w./day.
One female of the high dose group was prematurely sacrificed due to cuts / wounds on the head and the body.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A
Males
A marginally reduced body weight (statistically not significant) was noted at the intermediate dose level (150 mg Oxooil LS9/kg b.w./day) from post-natal day 36 until post-natal day 87 (3.1 % or 5.0 % below the control).
The reduced body weight at the intermediate dose level was considered to be test item-related but non-adverse due to the small differences.
At the high dose level (450 mg Oxooil LS9/kg b.w./day), the body weight of the males was clearly reduced from post-natal day 43 until post-natal day 87 (6.9 % or 14.2 % below the control, p ≤ 0.05 / 0.01), with a maximum difference on post-natal day 78 (15.7 % below the control, p ≤ 0.01).
The reduced body weight at the high dose level was considered to be test item-related and adverse.

Body weight gain - Males
Accordingly, a reduced body weight gain was noted at the intermediate and the high dose level (702.0 % or 638.4 % in comparison to 745.3 % in the control group) between post-natal days 22 and 87.
The absolute value of body weight gain at the high dose level was statistically significantly reduced (367.09 g in comparison to 436.71 g in the control group, p ≤ 0.01).

Females
No test item-related changes were noted.

Body weight gain - Females
The body weight gain that was noted at the low, the intermediate and the high dose level (395.9 %, 393.3 % or 386.9 % in comparison to 383.0 % in the control) was in the range of the control group.
No statistically significant differences were noted for the absolute values of body weight gain between the treatment groups (low, intermediate and high dose) and the control group (225.54 g, 229.74 g and 207.04 g in comparison to 215.99 g in the control group, statistically not significant).

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A
Males and females
No adverse effects were noted for the male and female animals.
However, for the male animals a statistically significantly increased cholesterol concentration was noted at the intermediate dose level (32.5 % above the control, p ≤ 0.05).
At the high dose level statistically significantly increased cholesterol and bilirubin concentrations were noted for the male animals (41.3 % or 101.8 % above the control, p ≤ 0.01).
For the female animals statistically significantly increased cholesterol and bilirubin concentrations were noted at the high dose level (24.3 % or 30.5 % above the control, p ≤ 0.01).

However, the observed increased cholesterol and bilirubin concentrations were considered to be non-adverse. It is most likely that they were due to 2 non-adverse effects. These 2 non-adverse effects were the metabolism of the oily test substance Oxooil and the observed liver hypertrophy which was considered to be an adaptive effect to liver enzyme induction.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Cohort 1A
Males and females
No test-item related changes were noted for the specific gravity, the pH value and the urine volume.
However, no usable results for the specific gravity and the pH value were obtained for the males of the high dose group, as the urine samples were contaminated with gel food.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

Balano-preputial separation:
Males (Cohort 1A, 1B and 2A combined)
No test item-related influence was noted for the day of preputial separation and the body weight on the day of preputial separation.

Day of vaginal opening:
Females (Cohort 1A, 1B and 2A combined)
A statistically significant delay in vaginal opening was noted at the high dose levels of Cohort 1A and Cohort 1B (450 or 300 mg Oxooil LS9/kg b.w./day). Day of vaginal opening at 450 mg/kg was on PND 34.8 ± 3.1 (p ≤ 0.01) and at 300 mg/kg on PND 34.5 ± 3.2 (p ≤ 0.05) in comparison to PND 32.3 ± 1.9 for the females of the control group.
The body weight on the day of vaginal opening was increased at 450 mg/kg and at 300 mg/kg (10.3 % or 13.2 % above the control, statistically not significant or p ≤ 0.01).
The delays in vaginal opening were considered to be test item-related but not adverse, as with the exception of a reduced fertility index at the high dose level, no adverse effects were noted at the reproductive endpoints of the females (14 day oestrous cycles, weights of the reproductive organs, the histopathological examination of the reproductive organs, the number of corpora lutea).

First appearance of cornified cells:
Females (Cohort 1A, 1B and 2A combined)
A delay (statistically not significant) in the first appearance of cornified cells was noted at 450 mg Oxooil LS9/kg b.w./day (PND 36.6 ± 4.1 in comparison to PND 34.7 ± 3.5 for the females of the control group).
The period between vaginal opening and the appearance of cornified cells was similar for the females that were dosed with 450 mg/kg and the females of the control group (1.8 ± 1.6 days at 450 mg/kg in comparison to 2.3 ± 2.7 days in the control group).
Hence, the delay in the appearance of cornified cells was considered to be secondary to the delay of vaginal opening and not an adverse effect on sexual maturation.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A
Males
Test item-related increased relative and / or absolute organ weights, that were not relevant for humans or not considered to be adverse, were noted for the kidneys and the liver.
For the kidneys, statistically significantly increased absolute and / or relative weights were noted at the intermediate and the high dose level (150 or 450 mg Oxooil LS9/kg b.w./day).
For the left kidney, the relative organ weights at the intermediate and the high dose level were 20.5 % and 43.3 % above the control (p ≤ 0.01). The absolute organ weights were 18.1 % above the control at the intermediate dose level (p ≤ 0.01) and 5.6 % above the control at the high dose level (not statistically significant).
For the right kidney, the relative organ weights at the intermediate and the high dose level were 16.1 % and 42.2 % above the control (p ≤ 0.01). The absolute organ weights were 13.8 % and 4.8 % above the control at the intermediate and the high dose level (not statistically significant).
The increased kidney weights may be due to hyaline droplet deposits that were noted during the histopathological examination of the kidneys from the high dosed males. The finding of hyaline droplet deposits was considered to be test item-related and adverse. However, the finding is unique to male rats and is not relevant for humans.
The markedly increased relative kidney weights at the high dose level of 43.3 % (left) and 42.2 % (right) were additionally caused by the reduced body weight at autopsy.
For the liver, statistically significantly increased relative organ weights were noted at 50, 150 or 450 mg Oxooil LS9/kg b.w./day (14.9 %, 22.3 % or 27.8 % above the control, p ≤ 0.01).
The increased relative liver weight may be due to a centrilobular hypertrophy that was noted at the high dose level (F0 Generation, Co1A) and the intermediate dose level (Co2A). As centrilobular hypertrophy was an adaptive effect of the liver the resulting increase in the liver weight was considered to be test item-related but not adverse.

For the absolute liver weight, statistically significantly increased liver weights were noted at the low and the intermediate dose level (17.7 % or 19.4 % above the control, p ≤ 0.05), whereas a slightly decreased absolute liver weight was noted at the high dose level (6.4 % below the control, statistically not significant). This was due to a decreased body weight at autopsy.
Furthermore, at 450 mg Oxooil LS9/kg b.w./day statistically significantly increased relative organ weights were noted for several organs. This was secondary to the reduced body weight at autopsy and not considered to be adverse for the affected organs with an increased relative organ weight.

Females
No test item-related changes were noted for the females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A
Males and females
No adverse test item-related findings were noted.

Light-red discoloured seminal vesicles were noted for 3 of 20 male animals of the high dose group. This finding was considered to be test item-related but non-adverse, as no histopathological correlation was noted.
One of those 3 high dosed male animals additionally showed light-red discoloured sciatic nerves. Due to the low incidence, this observation was considered to be spontaneous.

Females
No test item-related findings were noted.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A
Males and females
Non adverse or non-relevant observations were noted for the male and female animals that were dosed with 450 mg Oxooil LS9/kg b.w./day.
Findings were noted at the high dose level for all male and female animals in the form of centrilobular hepatocellular hypertrophy and for all male animals in the form of an alpha2micro-globulin nephropathy.
Both observations were considered as test item-related, but non-adverse (liver hypertrophy) or not relevant for human (alpha2micro-globulin nephropathy).

Examination of reproductive organs
No test item-related observations were noted for the examined reproductive organs of the male and female animals of group 4.

Detailed histopathological examination of one testis and one epididymis
All testes examined histologically from the Cohort 1A high dosed males showed completeness of stages and cell population.
Quantitative evaluation of ovarian primordial and small growing follicles and corpora lutea
No statistically significant differences between the females of the control group and the high dose group were noted for the number of corpora lutea, the number of growing follicles and the number of antral follicles.

A statistically significantly (p ≤ 0.05) reduced number of primordial follicles was noted at the high dose level. This observation was considered to be spontaneous, as no statistically significant changes were noted for the number of antral follicles and the number of corpora lutea.

Other effects:

no effects observed

Description (incidence and severity):

Cohort 1A
Males and females
Thyroid hormone levels (postnatal days 87 to 96, at necropsy):
No test item-related changes were noted.
Lymphocyte typing (spleen): No test-item related changes were noted.

Oestrus cyle, Testosterone levels, sperm parameters: No test-item related changes were noted.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Neurohistopathology (Co1A)
Males and females
Qualitative examination
No test item-related findings were noted for the brain, the eye, the skeletal muscle, the sciatic nerve and the spinal cord from the Cohort 1A male and female animals dosed with 450 mg Oxooil LS9/kg b.w./day.
Histomorphometry of the brain
No test item-related differences were noted for the morphometric parameters of the brain between the male and female animals of the control group and the male and female animals that were dosed with 450 mg Oxooil LS9/kg b.w./day.

Neurohistopathology (Co2A) (groups 1 and 3, prepared using perfusion fixation of the whole animal):
Males and females
Qualitative examination
No test item-related findings were noted for the brain, the eye, the skeletal muscle, the sciatic nerve and the spinal cord from the Cohort 2A male and female animals dosed with 150 mg Oxooil LS9/kg b.w./day.
Histomorphometry of the brain
No test item-related differences were noted for the morphometric parameters of the brain between the male and female animals of the control group and the male and female animals that were dosed with 150 mg Oxooil LS9/kg b.w./day.


Auditory startle response (Cohort 2A group 1 to 3 plus Cohort 1A group 4):
Males and females
No test-item related influence was noted on the auditory startle response. A similar process of habituation to the acoustic signal was noted for all groups.

Observational screening, Grip strength, Locomotor activity (PND 63 - 66) (Cohort 2A group 1 to 3 plus Cohort 1A group 4):
Males and females
No test-item related changes were noted.

Cohort 2B
Neurohistopathology (groups 1 and 3)
Males and females
Qualitative examination
No test item-related findings were noted for the brain, from the Cohort 2B male and female animals dosed with 150 mg Oxooil LS9/kg b.w./day.
Histomorphometry of the brain
No test item-related differences were noted for the morphometric parameters of the brain between the male and female animals of the control group and the male and female animals that were dosed with 150 mg Oxooil LS9/kg b.w./day.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

body weight and weight gain

Results: F2 generation

General toxicity (F2)

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index:
Pre-cull period:
A decreased viability index was noted at the low and the high dose level for the pre-cull
period. In detail, the viability index during the pre-cull period was 93.91 % in the control
group, 79.15 % at the low dose level, 98.26 % at the intermediate dose level and 87.76 %
at the high dose level.
The reduced viability index that was noted at the low dose level was mostly caused by 3
dams (nos. 429, 436 and 440) with 14, 10 or 15 prematurely deceased pups during the
pre-cull period.
Considering all these facts (only a slightly reduced body weight on lactation day 1, found
dead without milk in the stomach and / or found cold to touch), it is most probably that the
death of the pups was caused by an insufficient maternal care and not due to a bad health
status of the pups itself. May be the litters were too large for the dams. This assumption
is in accordance with the observation that 8 pups from dam no. 436 and 2 pups from dam
no. 440 survived until lactation day 21.
Finally, the behaviour of these 3 low dosed dams can be considered as spontaneous and
not as a test item-related adverse effect on maternal care, as no dam with such a high
number of dead pups was noted at the intermediate dose level (no dose-response
relationship).
The reduced viability index at the high dose level was mostly due to female no. 502, with
a total loss of all 16 live born pups on lactation day 2. It is unlikely that all pups died during the
night because of a bad health status of the pups itself. It is more likely that the dam has
killed their pups overnight (11 pups partly cannibalized) due to an unknown reason (may
be the dam was too stressed with the care of her large litter). However, this is probably
not an adverse effect on pup viability. Furthermore, as such a behaviour was only noted
for one dam of the high dose group, it was considered to be spontaneous.

Body weight and weight changes:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Gross pathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

External and internal examination:
No test item-related malformations or variations were noted.

T4, TSH determination (PND 22):
No test item-related difference was noted.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

> 300

Based on:

test mat.

Sex:

not specified

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The effects seen in this extended one generation study with test item Oxooil LS9 were considered not sufficient for classification and labeling for reproductive toxicity according to CLP Regulation (EC) No 1272/2008.

Executive summary:

General toxicity (adverse and test item-related effects)

At 450 mg/kg b.w./day, one male animal of the high dose group was prematurely sacrificed due to a loss of hindlimb function. The loss of hindlimb function was correlated with histopathological findings for the male rats at 450 mg/kg b.w./day for the sciatic nerves and skeletal muscle as well as for the spinal cords and its ventral nerve root and correlated with macroscopic post-mortem findings at necropsy at 450 mg/kg b.w./day in the form of a reduced muscle size at the left and right forelimb. This pattern of disease can be designated as a peripheral neuropathy that was noted for the male animals at 450 mg/kg b.w./day. As the peripheral neuropathy was only reported in F0 males and not in F1 animals or females, this effect is likely considered test item-related being more prominent in older male rats but is not associated with reproductive toxicity.

No test item-related findings were noted during the additional histopathological examination of the sciatic nerves and skeletal muscles from male animals that were dosed with 50 or 150 mg/kg b.w./day.

Further adverse findings for the males at 450 mg/kg b.w./day were in the form of a reduced body weight and changes in the haematological parameters (reduced numbers of white blood cells and lymphocytes).

Changes in male kidney weight and correlating histopathology findings at the high dose level were considered adverse but non-relevant for human. Due to a known male rat specific mode of action, this type of renal lesions, which is known as male rat specific alpha2micro-globulin nephropathy, is of adverse nature in the rat model, however, not relevant to human.

No adverse effects on general toxicity were noted for the female animals of the F0 Generation.

Reproductive toxicity

For the reproductive toxicity of the females a reduced fertility index and a subsequently reduced gestation index were noted at 450 mg/kg b.w./day.

For the prenatal development of the pups a clearly reduced number of implantation sites was noted at 450 mg/kg b.w./day. Reduced implantation sites is a maternal derived effect and not a sign of prenatal developmental toxicity.

No test item-related adverse effects were noted for the postnatal development of the pups during the lactation period until weaning.

 

General toxicity during post-weaning development (F1 - Cohorts 1A, 1B, 2A, 2B) (adverse and test item-related effects)

No test item-related premature death or premature sacrifice was noted.

For the male animals a reduced body weight was noted at the high dose levels of Cohort 1A and 1B (450 or 300 mg/kg b.w./day).

No adverse effects were noted during the laboratory examinations of the Cohort 1A animals (haematology, biochemistry, lymphocyte typing in spleen, urinalysis, thyroid hormone levels).

No adverse effects were noted on the parameters of sexual development that were examined for the animals of Cohort 1A, 1B and 2A (balano-preputial separation, vaginal opening, appearance of cornified cells, testosterone level).

Also, the reproductive parameters that were examined for the Cohort 1A females (oestrous cycle data between test days 53 to 66) and the Cohort 1A males (sperm parameters) revealed no test item-related or adverse effects.

No adverse findings were noted during the macroscopic examinations at necropsy for the Cohort 1A animals (necropsy between PNDs 87 and 96), the Cohort 1B animals (necropsy between PNDs 136 and 151), the Cohort 2A animals (necropsy between PNDs 71 and 75) and the Cohort 2B animals (necropsy on PND 22).

Also, the examined organ weights of the Cohort 1A, 1B, 2A (brain, liver, seminal vesicles, coagulating glands) and 2B (brain) animals revealed no adverse findings

No adverse findings were noted during the histopathological examination of the Cohort 1A animals dosed with 450 mg/kg including a quantitative analysis of the number of corpora lutea and ovarian follicles.

The histopathological examination of the liver, the seminal vesicles and the coagulating gland of the Cohort 2A animals dosed with 150 mg/kg also revealed no adverse findings.

Comparison between F1 Cohort and F0 Generation males:

The observation that the effect on the haematological parameters (reduced numbers of white blood cells and lymphocytes) and the finding of a peripheral neuropathy, which were both noted for the F0 Generation males at 450 mg/kg were not noted for the Cohort 1A males at 450 mg/kg may be due to the older age of the F0 Generation males at necropsy (between PNDs 231 to 233) in comparison to the Cohort 1A males (between PNDs 87 to 96). Peripheral neuropathy is not associated with reproductive toxicity in high dosed male rats.

 

Reproductive toxicity (F1 – Cohort 1B)

For the reproductive toxicity of the females, a reduced fertility index was noted at 300 mg/kg b.w./day.

No test item-related effect was noted on the pre- and post-natal development of the pups.

 

Neurological toxicity (F1 – Cohorts 2A and 2B and group 1 and 4 of Cohort 1A)

(In Cohort 2A, the high dose group was replaced by animals from Cohort 1A)

Neurological screening (Cohort 2A groups 1 to 3 and Cohort 1A group 4):

No test item-related influence was noted on the auditory startle response, the observational screening, the grip strength and the locomotor activity for the Cohort 2A animals of the low and the intermediate dose group (50 or 150 mg/kg b.w./day) and the animals of the Cohort 1A animals of the high dose group (450 mg/kg b.w./day).

Neuro-histopathology (Cohort 2A and 2B groups 1 and 3 and Cohort 1A groups 1 and 4):

The qualitative neurohistopathological examination of the brain, the eye, the skeletal muscle, the sciatic nerve and the spinal cord of the Cohort 2A animals dosed with 150 mg/kg b.w./day and the Cohort 1A animals dosed with 450 mg/kg b.w./day revealed no test item-related findings.

Also, no test item-related findings were noted for the brains from the Cohort 2B animals dosed with 150 mg/kg b.w./day.

Also, the histomorphometric analysis of the brain from the Cohort 2A and Cohort 2B animals dosed with 150 mg/kg b.w./day and the Cohort 1A animals dosed with 450 mg/kg b.w./day revealed no test item-related differences in comparison to the control groups of Cohort 2A, 2B and Cohort 1A.

 

The following no-observed-adverse-effect levels (NOAEL) were established for the parental animals of the F0 Generation and the F1 Generation:

 

F0 Generation:

General toxicity (for systemic toxicity)

NOAEL: 150 mg Oxooil LS9/kg b.w./day

Due to changes that were noted for the male animals at 450 mg/kg in form of a reduced body weight, haematological findings and a peripheral neuropathy. The peripheral neuropathy was diagnosed due to a reduced muscle size for the hindlimbs that was noted during the macroscopic examination at necropsy and histopathological findings in the form of sciatic nerve fiber degeneration, muscle fiber atrophy and findings in the spinal cord in the form of axonal swelling and nerve fiber degeneration (ventral roots). Due to the development of a peripheral neuropathy one high dosed male had to be prematurely sacrificed on test day 125. Changes in kidney weights due to hyaline droplet accumulation for the male animals at 450 mg/kg were considered adverse but not relevant for human.

Reproductive toxicity

1. a) adverse effects on the reproductive parameters of the parental females:

NOAEL 150 mg Oxooil LS9/kg b.w./day

Due to a reduced fertility and a subsequently reduced gestation index at 450 mg/kg.

1. b) adverse effects on the pre-natal development of the pups:

NOAEL 150 mg Oxooil LS9/kg b.w./day

Due to a reduced number of implantation sites triggered by a reduced fertility index seen in F0 females at 450 mg/kg.

1. c) adverse effects on the post-natal development of the pups:

NOAEL at least 450 mg Oxooil LS9/kg b.w./day

 

F1 Generation:

General toxicity during post-weaning development (Cohorts 1A and 1B, 2A)

NOAEL 150 mg Oxooil LS9/kg b.w./day

Due to a reduced body weight that was noted for the male animals at 300 mg/kg (Cohort 1B) and 450 mg/kg (Cohort 1A).

Reproductive developmental toxicity (Cohort 1B)

1. a) adverse effects on the reproductive parameters of the parental females:

NOAEL 150 mg Oxooil LS9/kg b.w./day

Due to a reduced fertility index at 300 mg/kg.

1. b) adverse effects on the pre-natal development of the pups:

NOAEL at least 300 mg Oxooil LS9/kg b.w./day

1. c) adverse effects on the post-natal development of the pups:

NOAEL at least 300 mg Oxooil LS9/kg b.w./day

 

Neurological developmental toxicity (Cohorts 2A and 2B, Group 4 from Cohort 1A)

NOAEL at least 450 mg Oxooil LS9/kg b.w./day