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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In the reproductive/developmental toxicity element of an OECD TG 422 screening test in rats, Oxooil LS9 elicited no evidence of impaired fertility or reproductive performance at dosages of 100, 300 or 1000 mg/kg/day. The NOAEL for fertility is 1000 mg/kg/day.


In addition, a reproductive/developmental toxicity study according to OECD TG 421 was performed as a dose-range-finder for the EOGRTS. The NOAEL for reproductive toxicity was considered to be 400 mg/kg b.w./day, since at level of 750 mg/kg b.w./day effects in fertility index and number of implants in presence of parental toxicity were seen.


 


A extended one generation study according to OECD TG 443 was performed including Cohorts 1A, 1B with extension to second mating and Cohorts 2A+2B for developmental neurotoxicity testing. 
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.


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.


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.


 


 

Link to relevant study records

Referenceopen allclose all

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
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).
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
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
Dose / conc.:
0 mg/kg bw/day
Remarks:
control
Dose / conc.:
50 mg/kg bw/day
Remarks:
low dose
Dose / conc.:
150 mg/kg bw/day
Remarks:
mid dose
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
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
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: 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.
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
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
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
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: 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.
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
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.
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:
not examined
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
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.

Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
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
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


 

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:
9 December 2009 (animal arrival) -24 March 2010 (pathology completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study undertaken according to current OECD Test Guidelines.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The toxicity subgroup for this study comprised 5 male and 5 female rats per treatment group and the reproductive subgroup comprised 5 male and 10 female rats per treatment group. This deviation was undertaken to enhance the robustness of the study.
Principles of method if other than guideline:
The toxicity subgroup for this study comprised 5 male and 5 female rats per treatment group and the reproductive subgroup comprised 5 male and 10 female rats per treatment group. The 5 male rats in each treatment group of the toxicity subgroup were used for mating with 5 female reproductive subgroup animals of the corresponding treatment group. The ten female rats in each treatment group of the reproductive subgroup were used exclusively for the reproductive/developmental toxicity phase of the study. This deviation was undertaken to enhance the robustness of the study.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: CD (SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 350-397 g males and 204-251 g females
- Fasting period before study: none
- Housing: Male: 5 animals/P2000 polycarbonate cage : singly with one female in RB3 modified polyproplyene cages during mating.
Females: 5 animals/P2000 polycarbonate cage pre-mating: singly with one male in RB3 modified polypropylene cages during mating: singly in 2154 polycarbonate cages during gestation and littering
- Diet (e.g. ad libitum): ad libitum(SDS VRF 1 certified diet)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 9 December 2009 (animal arrival) To: 1 February 2010 (necropsy completion)
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was used as supplied. All formulations were prepared freshly weekly and were stored refrigerated (2-8°C) in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 20, 60 or 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): no data
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until detection of mating (successful for all animals up to 5 days)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): singly in 2154 polycarbonate cages
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix. The stability was assessed following storage at ambient temperature (nominally 21°C) for 0 hours, 4 hours (continual stirring) and 2 days, and refrigeration (nominally 2-8°C) for 2, 8 and 15 days. Prior to initial sampling on each day, the formulation was mixed by 20-fold inversion and magnetic stirring for a minimum of 5 minutes. At each time point, single samples (nominally 1 mL) were taken for assay from the top, middle and bottom of the magnetically stirred formulation. Stability was determined from the mean concentration of the analyte in the vehicle at each sampling point. Specimen formulations (typically 400 mL) were prepared at concentrations of 2 and 200 mg/mL and equally split between four amber glass screw-capped bottles and were confirmed for 15 days when refrigerated and for 48 hours at room temperature.
Samples of each formulation prepared for administration in the first week of the dosing procedure were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed); 2 assays from each group and 1 assay. The remainder was frozen (nominally -20°C) and retained as contingency for analysis if any result required confirmation.

The GC analytical procedure was validated with respect to linearity of detector response, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision.

The homogeneity and stability was confirmed for Oxooil LS9 in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for 2 days and refrigerated storage for up to 15 days. The storage times represented the maximum time from preparation to completion of administration.

The mean concentrations of Oxooil LS9 in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation
Duration of treatment / exposure:
5 weeks for males; to day 4 of lactation for females (during week 7)
Frequency of treatment:
All animals were dosed once each day at approximately the same time each day, seven days per week.
Details on study schedule:

Age at mating of the mated animals in the study:11-12 weeks
Remarks:
Doses / Concentrations:
100, 300 or 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 10 females in the reproductive toxicity subgroup, and 5 males used for mating from the toxicity subgroup
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels of 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor with reference to previous work with this compound performed in these laboratories (Huntingdon Life Sciences Report Number: BBB0026). In that study, the CD rats received Oxooil LS9 at doses of 100, 300 or 1000 mg/kg/day for seven days, there were no findings which precluded the use of these dose levels on the subsequent 4 week general toxicity and reproductive developmental toxicity screening study.

- Rationale for animal assignment (if not random):
On arrival, the animals were removed from the transit boxes and allocated to study cages. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately. Before the start of treatment mean bodyweights were reviewed. Control males had a low mean bodyweight in comparison to treated male groups. To average the group means to similar values, two males from the Control group were changed with two males from the middle treatment (Group 3).
- Rationale for selecting satellite groups: no satellite groups used
- Post-exposure recovery period in satellite groups: no post-exposure recovery period
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

- Time schedule:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Immediately before dosing
Immediately after dosing on return of the animal to its cage
On completion of dosing of each group
Between one and two hours after completion of dosing of all groups
As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Before treatment commenced , during each week of treatment and for females on Days 0, 7, 14 and 20 after mating and Day 4 of lacation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware (as far as practically possible) of the experimental group to which the animal belonged. For females after mating, a small number of animals were subject to these observations on each Day 4 of lactation (8, 15, 4, 8 and 4 females on each Day 4 of lactation) - the observer was aware of the experimental group and this was considered acceptable.

After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.


BODY WEIGHT: Yes

- Time schedule for examinations:
All toxicity and reproductive subgroup males (10 animals per treatment group) were weighed weekly throughout the study. All toxicity and reproductive subgroup females (15 animals per treatment group) were weighed weekly for the first two weeks and the toxicity subgroup females (five animals per treatment group) were weighed during Weeks 3, 4 and 5.

FOOD CONSUMPTION: Yes

The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded during weeks 1 and 2 for all male (two cages of 5 animals per treatment group) and all female (three cages of 5 animals per treatment group) toxicity and reproductive subgroups. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

WATER CONSUMPTION : No

Oestrous cyclicity (parental animals):
For two weeks before pairing, daily vaginal smears were taken from females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed
Sperm parameters (parental animals):
Not undertaken
Litter observations:
All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter. The records maintained were as follows:

Clinical signs: Daily records were maintained for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.

Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-4 of age.

Sex ratio: The sex ratio of each litter was recorded on Days 1 and 4 of age.

Bodyweight: Individual offspring bodyweights were recorded on Days 1 and 4 of age.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: animals receiving 1000 mg/kg/day killed during weeks 3 or 4 of treatment. All remaining animals sacrificed after 5 weeks of treatment
- Maternal animals: One dam and litter were killed during gestation and one dam and litter during lactation for reasons of animal welfare. All remaining animals sacrificed on Day 4 of lactation.

GROSS NECROPSY
All animals were killed by carbon dioxide asphyxiation.

All animals were subject to a detailed necropsy. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative


ORGAN WEIGHTS

The following organs, taken from all animals, were dissected free of adjacent fat and other contiguous tissue and the weights recorded:

Epididymides (L&R) Seminal vesicles and coagulation gland
Ovaries with oviducts (L&R) Testes (L&R)
Prostate Uterus with cervix

L&R Bilateral organs weighed individually

Organ weights were also adjusted for terminal bodyweight using the weight recorded before necropsy

HISTOPATHOLOGY

The following tissues were examined for all animals of the Control and 1000 mg/kg/day treatment groups sacrificed on completion of the scheduled treatment period:

Epididymides (L&R) Seminal vesicles and coagulation gland
Mammary area - caudal† Testes (L&R)
Ovaries with oviducts Uterus with cervix
Prostate Vagina


For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted. For the assessment of the ovaries, one mid-line section of each ovary was examined for the presence of primordial follicles, growing follicles and corpora lutea
Postmortem examinations (offspring):
SACRIFICE
- F1 offspring were killed on Day 4 of age

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY

For F1 offspring surviving to scheduled termination, a full macroscopic examination of the tissues was performed, after a review of the history. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

For premature adult deaths before weaning, missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before weaning were examined as detailed above except that the cranium was not sectioned unless required to investigate a cranial abnormality. The necropsy also included an assessment for the presence of milk in the stomach, where this was possible.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not undertaken
Statistics:
See 'materials and methods' free text field below
Reproductive indices:
Mating performance and fertility
Gestation length

Offspring viability indices:
Survival indices
Sex ratio
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

Male animals receiving 1000 mg/kg/day were sacrificed on humane grounds during weeks 3 or 4 (Section 7.5.1).
.
One female receiving 1000 mg/kg/day was killed for welfare reasons on Day 20 of gestation due to general poor condition. The ante-mortem clinical signs consisted of underactivity, irregular breathing, piloerection, pale eyes and whole body pallor, abnormal black staining of the perigenital and dark ventral abdomen. Macroscopic examination revealed: Abnormal black/dark viscous fluid in the vagina and right uterine horn. This female was pregnant with eight live fetuses in the left horn and five live fetuses in the right horn with two early resorptions and one late resorption. All fetuses appeared grossly normal.
A second female receiving 1000 mg/kg/day was killed for welfare reasons on Day 3 of lactation. Ante-mortem signs consisted of thin build, whole body pallor, reduced body tone and temperature and abnormal gait. Macroscopic examination revealed the female to be thin with pale and inactive mammary tissue; all pups were of thin build and had no milk in the stomach.

Forepaw paddling was observed on Day 6 of treatment and to a lesser extent on Days 10, 14, 17, 21, 24 28 and 30 of treatment and Days 0, 7, 14 and 20 of gestation in females receiving 1000 mg/kg/day. Chin rubbing and salivation was also observed periodically after dosing for females receiving 1000 mg/kg/day, and at a low incidence for animals receiving 300 mg/kg/day. Chin rubbing, salivation and forepaw paddling was observed during gestation for females receiving 300 or 1000 mg/kg/day, and chin rubbing and salivation was observed on Day one of lactation for females receiving 1000 mg/kg/day.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

Bodyweight loss and reduced food consumption were recorded for males receiving 1000 mg/kg/day and reduced bodyweight gain for rats receiving 300 mg/kg/day and dams receiving 1000 mg/kg/day. 

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

Not applicable

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

At 1000 mg/kg/day, 2/10 females were acyclic (compared to 0/10 in the concurrent Control group). However, all females mated within 5 days, and oestrus cycles were considered unaffected by treatment.

All females mated within 4 days (i.e. at the first oestrus opportunity after pairing) with the exception of two females receiving 1000 mg/kg/day which mated 5 days after pairing.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

Not undertaken

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were considered unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)

Seminal vesicles absolute and adjusted weights were slightly low in males that received 300 mg/kg/day when compared with Controls, with adjusted weights attaining statistical significance. In the absence of any histopathological findings in this organ, the difference was considered not of toxicological importance.

Mean terminal bodyweights of lactating females that received 1000 mg/kg/day were statistically significantly lower than Controls. Unadjusted and adjusted ovary and uterus weights were unaffected by treatment.


GROSS PATHOLOGY (PARENTAL ANIMALS)

Abnormal pink coloration of the sciatic nerve, adipose tissue, seminal vesicles, prostate and urinary bladder was observed macroscopically in the treated animals given 300 or 1000 mg/kg/day(Section 7.5.1).


HISTOPATHOLOGY (PARENTAL ANIMALS)

All microscopic findings in the reproductive organs were considered to be incidental and not of toxicological importance.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted. A single case, characterized by unilateral severe atrophy of the seminiferous tubular epithelium and germ cell depletion, and was seen in the testis of one control male. Based on the single occurrence this finding was considered incidental.

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive/developmental toxicity up to a limit dosage of 1000 mg/kg/day
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)

There was no effect of treatment on pregnancy outcome. All pregnant females gave birth to a live litter except for one female receiving 1000 mg/kg/day. This female was killed for welfare reasons on Day 20 of gestation due to general poor condition. This female was pregnant with eight live fetuses in the left horn and five live fetuses in the right horn with two early resorptions and one late resorption. All fetuses appeared grossly normal.
One female receiving 1000 mg/kg/day was killed for welfare reasons on Day 3 of lactation due to general poor condition. Macroscopic examination revealed she had pale and inactive mammary tissue. All pups were of thin build and had no milk in the stomach.

There were no instances of total litter loss during the post-natal period.

The following assessment of litter responses was made based on the 10, 10, 10 and 8 litters successfully reared to termination on Day 4 post partum in the 0 (Control), 100, 300 and 1000 mg/kg/day groups, respectively. Litter data, as assessed by mean numbers of implantations, total litter size, and live litter size on Day 1and Day 4 of age, were generally similar to the Control and are considered not to be affected by treatment.

Offspring survival during the pre- and post-natal period as assessed by the post implantation survival index, live birth index, viability index was unaffected by treatment.


CLINICAL SIGNS (OFFSPRING)

There were no clinical signs observed for F1 offspring that were considered to be related to treatment with Oxooil LS9.

Two litters were observed to be cold to touch and had no milk in stomach for F0 females that received 1000 mg/kg/day.

BODY WEIGHT (OFFSPRING)

Male and female offspring mean bodyweights were slightly lower for litters of F0 dams that received 1000 mg/kg/day, and overall F1 males mean bodyweight change was low for F0 dams that received 100 or 300 mg/kg/day, when compared with Controls. F1 female mean bodyweight for F0 dams that received 100 or 300 mg/kg/day were unaffected by treatment.


SEXUAL MATURATION (OFFSPRING)

Not examined

ORGAN WEIGHTS (OFFSPRING)

Not examined

GROSS PATHOLOGY (OFFSPRING)

There were no findings at macroscopic examination of F1 offspring that were considered related to treatment.

HISTOPATHOLOGY (OFFSPRING)

Not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive/developmental toxicity up to Day 4 of lactation
Reproductive effects observed:
not specified
Conclusions:
Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive/developmental toxicity within the scope of this screening test is 1000 mg/kg/day.
Executive summary:

An OECD 422 guideline screening study was performed to GLP standards at the Laboratories of Huntingdon Life Sciences, Eye, on behalf of Evonik Oxeno GmbH., to investigate the reproductive and developmental toxicity of the test substance Oxooil LS9 when administered to rats by oral gavage. Three groups, each comprising five male and ten female rats, received Oxooil LS9 once daily at dosages of 100, 300 or 1000 mg/kg/day. An additional five males from each treatment group of the toxicity phase of this combined study were used for mating with corresponding females of the reproductive phase. The males were treated daily for a period of five complete weeks before termination. The females were treated daily for two weeks before pairing, throughout pairing and gestation and for three days of lactation, until termination on Day 4 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose, for the same duration.

Throughout the study, data were recorded on clinical condition, detailed physical and arena observations and bodyweight. Data were recorded on food consumption for all animals prior to mating, and during gestation and lactation for mated females. For females, data were recorded on oestrous cycles, mating performance and fertility and gestation length.  Organ weight and macroscopic and microscopic pathology investigations were undertaken. The clinical condition of F1 offspring, litter size and survival, sex ratio and bodyweight were assessed and macroscopic pathology investigations were undertaken.

Three of ten males treated at 1000 mg/kg/day were sacrificed on humane grounds during weeks 3 or 4 and the remaining males in this group killed prematurely in week 4 (Section 7.5.1). Two females treated at this dosage were killed for welfare reasons on Day 20 of gestation or Day 3 of lactation, respectively. Bodyweight loss and reduced food consumption were recorded for males receiving 1000 mg/kg/day and reduced bodyweight gain for males treated at 300 mg/kg/day and dams receiving 1000 mg/kg/day.

There was no effect of treatment on mating performance, fertility, reproductive performance, survival and development of the offspring, and there were no treatment-related pathological findings in the reproductive organs. Slightly lower male and female offspring mean bodyweights were recorded for the litters of females that received 1000 mg/kg/day which was considered to be a consequence of the low terminal bodyweight of females that received 1000 mg/kg/day and indicative of the toxicity in the dams at this dosage.

It is concluded that, the reproduction/developmental toxicity screening test elicited no evidence of impaired performance at 100, 300 or 1000 mg/kg/day Oxooil LS9. Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive/developmental toxicity within the scope of this screening test is 1000 mg/kg/day.

The study is considered acceptable for classification and satisfies the guideline requirements for a rat reproductive/developmental toxicity screening test.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
In-life phase: 06/05/2020 - 28/07/2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rat
Strain:
other: CD/ Crl:CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 68 days
- Weight at study initiation: Males: 369.7 g - 460.2 g; Females: 234.0 g - 284.0 g
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 21 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 10%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until pregnancy had occurred or two weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug.
Duration of treatment / exposure:
males: 6 weeks prior to mating (from test day 1 until test day 42), during the mating period (from test day 43 until test day 33 at maximum) and during the post-mating period until test day 72 to 75 (one day before sacrifice between test days 73 and 76)
females: 6 weeks prior to mating (from test day 1 until test day 42), during the mating period (from test day 43 until test day 41 at maximum) and during the lactation period until test day 68 to 83 (corresponding to lactation day 4 or shortly thereafter).
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day
Remarks:
control
Dose / conc.:
200 mg/kg bw/day
Remarks:
low
Dose / conc.:
400 mg/kg bw/day
Remarks:
mid
Dose / conc.:
750 mg/kg bw/day
Remarks:
high
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND WATER CONSUMPTION
- Food consumption for each animal per week (g/rat/week)
- Drinking water consumption monitored by visula appraisal
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, abnormal behaviour, weight

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals between TD 73 and TD 76
- Maternal animals: All surviving animals on LD 4 or shortly thereafter

GROSS NECROPSY: yes

HISTOPATHOLOGY / ORGAN WEIGHTS
Kidney, Liver
Postmortem examinations (offspring):
SACRIFICE
- on LD 4

GROSS NECROPSY
- Gross necropsy + external reproductive genitals
Statistics:
Homogeneity of variances and normality of distribution were tested using the BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT's test (p ≤ 0.01 and p ≤ 0.05).
Homogeneity of variances was test using the BARTLETT's test. In case of homogeneity, intergroup comparison was performed by the DUNNETT's test (p ≤ 0.05 and p ≤ 0.01). In case of heterogeneity of variances, a stepwise comparison of the test groups with the control group was performed using a STUDENT's t-test (p ≤ 0.05 and p ≤ 0.01).
Reproductive indices:
Gestation Index [%]= Number of litters with live pups/Number pregnant x 100
Fertility Index female [%] =Number of pregnant rats/Number of females used x 100
Offspring viability indices:

Birth Index [%] =Total number of pups born (live +dead)/Number of implantation scars x 100
Live Birth Index [%] =Number of pups born alive on day 0/1 of lactation/Total number born (live + dead) x 100
Viability Index [%]=Number of pups alive on day 4 (pre-select)/Number of pups live on day 0/1x 100
Post-implantation loss [%] =Implantations - number of pups born alive/Implantations x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight to severe salivation was noted for the male and female animals dosed with 400 or 750 mg//kg b.w./day. Additionally, the male and female high dose animals displayed piloerection, in addition ataxia was noted for one male animal.
Mortality:
mortality observed, treatment-related
Description (incidence):
No premature death was noted for the male and female animals of the low and intermediate dose
groups (200 or 400 mg Oxooil LS9/kg b.w./day). At 750 mg Oxooil LS9/kg b.w./day, two male animals had to be prematurely sacrificed on TD 8 or 65 due to their moribund condition, in addition, one male animal was found dead on TD 44.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
A reduction in the body weight was noted for the male animals dosed with 400 or 750 mg/kg b.w./day (10.8% or 18.4% below the value of the control group to the end of the study on TD 73, statistically significant at p ≤ 0.05 or 0.01). Body weight gain was accordingly reduced for the males dosed with 400 or 750 mg/kg b.w./day.
A reduced body weight at autopsy was noted at 400 and 750 mg/kg b.w./day (11.3% or 18.4% below the value of the control group, p ≤ 0.05 or 0.01).
Females:
The difference in body weight between the control and the high dose group increased during the gestation period. At the end of the gestation period the body weight of the high dosed animals was 23.2% below the value of the control group (p ≤ 0.01). During the lactation period the differences in body weight remained on the level that was noted at the end of the gestation period. The body weights of the high dosed females were 17.1% or 16.4% below the control on lactation days 1 and 4, p ≤ 0.01). Body weight gain was accordingly reduced for the females dosed with 750 mg/kg b.w./day during the pre-mating and the gestation period.

A reduced body weight at autopsy was noted at 750 mg/kg b.w./day (17.8% below the value of the control group, p ≤ 0.01).
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males: An alpha 2u-globulin nephropathy was noted for the male animals of all dose groups.
Females: No test item-related observations were noted for the female animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Fertility index: At 750 mg/kg b.w./day, a reduced fertility index was noted due to 4 non-pregnant females (all with verified insemination by their male partner) in comparison to 2 non-pregnant females in the control group (an insemination by the male partner was only verified for one of the two non-pregnant control females). At the high dose group (750 mg Oxooil LS9/kg b.w./day), small numbers of implantation sites were noted for 4 of the 5 pregnant females that survived the gestation period.
Gestation index: For the females of the high dose group (750 mg/kg b.w./day), a decrease was noted for the gestation index (60% compared to 100% in the control group) due to 2 high dose females with a total loss of implantations compared to none in the control group.
Gestation length: For the females dosed with 750 mg/kg b.w./day, an increase was noted for the gestation length (4.0% above the control group, statistically significant.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effect on pup body weight.
In the high dose group (750 mg/kg b.w./day), reduced litter weights were noted for the male and the female litters on LD 1 and 4 (between 25.9% and 43.5% below the value of the control group, statistically significant for the male and female litters combined on LD 4. This reduction was considered to be caused by the reduced number of implants. The reduced litter weight was due to the test item-related reduced number of pups per litter for 2 of 3 litters in the high dose group and due to the reduced number of implantations as secondary effect.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
The external macroscopic examination of the pups after sacrifice revealed no gross abnormalities.
Histopathological findings:
not examined
Other effects:
not examined
Birth indices and post-implantation loss:
No test item-related differences were noted for the mean number of implantation sites, the mean number of pups born (alive and dead) and the mean number of live born pups between the control group and the low and intermediate dose groups (200 or 400 mg/kg b.w./day). At the high dose group (750 mg/kg b.w./day), small numbers of implantation sites were noted for 4 of the 5 pregnant females that survived the gestation period.
Implantation sites: 14.6, 15.5, 13.3, 5.4 (control, low, mid, high dose group); live pups: 12.6, 14.2, 12.7, 4.2 (control, low, mid, high dose group)
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: reduced number of implants
Reproductive effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development based on OECD guideline 421 and in order to define dose level for the Extended One-generation Reproductive Toxicity Study based (OECD 443). The test item Oxooil LS9 was administered orally to rats at dose levels of 200, 400 or 750 mg/kg b.w./day.
On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 200 mg/kg b.w./day for males and 400 mg/kg b.w./day females, based on clinical signs observed and reductions in the body weights as well as mortality at 750 mg/kg b.w./day.; while the NOAEL for reproductive and developmental toxicity was considered to be 400 mg/kg b.w./day, since at level of 750 mg/kg b.w./day were seen effects in fertility index and number of implants in presence of parental toxicity.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the reproductive/developmental toxicity element of an OECD 421 screening test in rats, Oxooil LS9 was applied by oral gavage at dose level of 200, 400 and 750 mg/kg b.w./day.


No premature death was noted for the male and female animals of the low and intermediate dose groups. At


750 mg/kg b.w./day, two male animals had to be prematurely sacrificed on TD 8 or 65 due to their moribund condition, in addition, one male animal was found dead on TD 44.


A reduction in the body weight was noted for the male animals dosed with 400 or 750 mg/kg b.w./day (10.8% or 18.4% below the value of the control group to the end of the study on TD 73, statistically significant. Body weight gain was accordingly reduced for the males dosed with 400 or 750 mg/kg b.w./day.


Body weight gain was accordingly reduced for the females dosed with750 mg/kg b.w./day during the pre-mating and the gestation period. For the males of the intermediate and high dose group (400 or 750 mg/kg b.w./day) and for the female high dose animals, light red discolourations were noted for the sciatic nerve (males and females) and for the seminal vesicles, coagulating glands or prostate gland (males). No test item-related influence was noted on the absolute or relative organ weights of the dose groups. The histopathological examination of the kidneys and the liver of the male and female animals revealed an alpha 2u-globulin nephropathy for the male animals of all dose groups (200, 400 or 750 mg/kg b.w./day). No test item-related changes were noted for the female animals.


At 750 mg/kg b.w./day, a reduced fertility index was noted due to 4 non-pregnant females (all with verified insemination by their male partner) in comparison to 2 non-pregnant females in the control group (an insemination by the male partner was only verified for one of the two non-pregnant control females). For the females of the high dose group (750 mg/kg b.w./day), a decrease was noted for the gestation index (60% compared to 100% in the control group) due to 2 high dose females with a total loss of implantations compared to none in the control group. For the females dosed with 750 mg/kg b.w./day, an increase was noted for the gestation length (4.0% above the control group).


For the dams of the high dose group (750 mg/kg b.w./day), a marked reduction was noted for the number of implantation sites per dam (5.4 implantation sites per dam in comparison to 14.6 in the control).


No influence was noted on the number of resorptions per dam, though the post-implantation loss was increased at the high dose level due to the smaller number of implants.


No influence was noted at the live birth index. For all test groups, only one stillbirth was noted in the low dose group.


Based on the reductions in the fertility index, the gestation index and the increased gestation length at 750 mg/kg b.w./day.the NOAEL of 400 mg/kg b.w./day for reproduction was established.


 


 


An OECD 422 guideline screening study was performed to GLP standards, to investigate the reproductive and developmental toxicity of the test substance Oxooil LS9 when administered to rats by oral gavage. Three groups, each comprising five male and ten female rats, received Oxooil LS9 once daily at dosages of 100, 300 or 1000 mg/kg/day. An additional five males from each treatment group of the toxicity phase of this combined study were used for mating with corresponding females of the reproductive phase. The males were treated daily for a period of five complete weeks before termination. The females were treated daily for two weeks before pairing, throughout pairing and gestation and for three days of lactation, until termination on Day 4 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose, for the same duration.


Throughout the study, data were recorded on clinical condition, detailed physical and arena observations and bodyweight. Data were recorded on food consumption for all animals prior to mating, and during gestation and lactation for mated females. For females, data were recorded on oestrous cycles, mating performance and fertility and gestation length.  Organ weight and macroscopic and microscopic pathology investigations were undertaken. The clinical condition of F1 offspring, litter size and survival, sex ratio and bodyweight were assessed and macroscopic pathology investigations were undertaken.


Three of ten males treated at 1000 mg/kg/day were sacrificed on humane grounds during weeks 3 or 4 and the remaining males in this group killed prematurely in week 4 (Section 7.5.1). Two females treated at this dosage were killed for welfare reasons on Day 20 of gestation or Day 3 of lactation, respectively. Bodyweight loss and reduced food consumption were recorded for males receiving 1000 mg/kg/day and reduced bodyweight gain for males treated at 300 mg/kg/day and dams receiving 1000 mg/kg/day.


There was no effect of treatment on mating performance, fertility, reproductive performance, survival or development of the offspring, and there were no treatment-related pathological findings in the reproductive organs. Slightly lower male and female offspring mean bodyweights were recorded for the litters of females that received 1000 mg/kg/day, which were considered to be a consequence of the low terminal bodyweight of females that received 1000 mg/kg/day and indicative of the toxicity in the dams at this dosage.


It is concluded that, the reproduction/developmental toxicity screening test elicited no evidence of impaired performance at 100, 300 or 1000 mg/kg/day Oxooil LS9. Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) in rats for reproductive toxicity within the scope of this screening test is 1000 mg/kg/day.


 


 


An OECD TG 443 study was performed with Oxooil LS9 to evaluate the pre- and post-natal effects of Oxooil LS9 on development as well as systemic toxicity in pregnant and lactating females and young adult offspring. Specially, a possible mode of action related to endocrine disruption should be clarified. Additionally, effects on developmental neurotoxicity were evaluated. Administration of Oxoil LS9 resulted in general toxicity at the highest does level (450/300 mg/kg bw/day) in F0 and F1 generation, with male animals being more susceptible to general systemic toxicity effects. In addition, reduced fertility index and subsequently reduced gestation index were noted at the high dose level of 450 mg/kg bw/day in F0 generation. 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. The decreased number of implantation sites for F1 developmental toxicity was considered as a secondary effect of reduced fertility index of F0 females. As 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. The effects of reduced fertility were associated with general systemic toxicity of parental animals. Reduced gestation index in F1 and implantation sites for F2 generation were not reported, thus, could not be confirmed with second mating. Reduced fertility index was also evident at the highest dose level of Cohort 1B, F1 animals. The reason of lower fertility index in F1 generation could not be related to any findings in reproductive organ histopathology of Cohort 1A and all other collected data on reproductive parameters for F1 generation gave no further evidence of a test item-related reproductive toxicity effect. No effects on pre- and postnatal development were evident. Moreover, developmental neurotoxicity did not show any effects and therefore is considered of no concern. If a clear effect on reproductive performance would have been present in correlation to test item administration, all effects seen in F0 generation would have been also expected for F1 generation, presumably being much more pronounced in second generation due to critical in utero  exposure during development of gametes. However, only less marked reduced fertility was reported for F1 generation and no effects on gestation index and implantation number were present. The histopathology of male and female reproductive organs did not indicate a clear demonstration of reproductive toxicity in form of impaired fertility neigther in F0 nor in F1 generation. Thus, the reason for reduced fertility in F0 and F1 generation remains unclear and is not considered sufficient for classification and labeling according to CLP Regulation (EC) No 1272/2008.


 

Effects on developmental toxicity

Description of key information

In the reproductive/developmental toxicity element of an OECD 422 screening test in rats, Oxooil LS9 elicited no evidence of developmental toxicity at dosages of 100, 300 or 1000 mg/kg/day. The NOAEL for developmental toxicity is 1000 mg/kg/day.In the complete absence of any developmental toxicity effect at the limit dosage of 1000 mg/kg/day in the OECD 422 screening study, it is considered unlikely that Oxooil LS9 will represent a developmental risk.


A pre-natal developmental toxicity study has been conducted on Oxooil LS9. The no-observed-adverse-effect level (NOAEL) was 300 mg Oxooil LS9/kg b.w./day for the dams.None of the dams died prematurely.


Noteworthy clinical observations were noted only at the high dose level (1000 mg/kg b.w./day) in form of e.g. reduced motility, prone position and piloerection. In addition, a reduced body weight and reductions in food consumption were noted at the high dose level (1000 mg/kg b.w./day). No test item-related changes were noted during the macroscopic inspection at necropsy and for the organ weights of the heart, the kidneys and the liver.


The no-observed-adverse-effect level (NOAEL) for the fetal organism was 300 mg Oxooil LS9/kg b.w./day. At the maternal toxic dose level of 1000 mg Oxooil/kg b.w./day 2 dams were noted with a total implantation loss. Furthermore, reduced placental and fetal body weights as well as an increased incidence of delayed ossifications of the metacarpalia and the sternebra(e) were noted at the high dose level. These dose-related findings are considered to be treatment-related. No dead fetuses, no malformations and no test item-related increase in the incidence of variations were noted during the examination of the fetuses


 


In the extended one generation study reduced number of implantation sites was noted at 450 mg/kg b.w./day for prenatal development of F1 pups. However, 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. No test item-related effect was noted on the pre- and post-natal development of the F2 generation pups. The following NOAELs were derived: 


F1 generation:


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.


adverse effects on the post-natal development of the pups: NOAEL at least 450 mg Oxooil LS9/kg b.w./day


F2 generation:


adverse effects on the pre-natal development of the pups: NOAEL at least 300 mg Oxooil LS9/kg b.w./day


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

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study planned
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS
According to REACH regulation Annex IX the conduct of a prenatal developmental toxicity study in a second species is required to cover the endpoint developmental toxicity.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Octene, hydroformylation products, low-boiling (CAS number 68938-03-4)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
- Available GLP studies: A prenatal developmental toxicity study in the first species (rat) is available. In this study, the test item Oxooil LS9 was administered orally to female rats at dose levels of 100, 300 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy.
None of the dams died prematurely. Noteworthy clinical observations were noted only at the high dose level (1000 mg/kg bw/day) in form of e.g. reduced motility, prone position and piloerection. In addition, a reduced body weight and reductions in food consumption were noted at the high dose level (1000 mg/kg bw/day). No test item-related changes were noted during the macroscopic inspection at necropsy and for the organ weights of the heart, the kidneys and the liver.
At the maternal toxic dose level of 1000 mg/kg bw/day 2 dams were noted with a total implantation loss. Furthermore, reduced placental and fetal body weights as well as an increased incidence of delayed ossifications of the metacarpalia and the sternebra(e) were noted at the high dose level. These dose-related findings are considered to be treatment-related. No dead fetuses, no malformations and no test item-related increase in the incidence of variations were noted during the examination of the fetuses.
The NOAEL was 300 mg/kg bw/day for the dams as well as for the fetal organism.
Based on this study, the substance does not require classification for developmental toxicity. Thus, in accordance with REACh Regulation, Annex X, 8.7.2, testing in a second species is proposed.
- Available non-GLP studies: no studies available
- Historical human data: no data available
- (Q)SAR: No adequate QSAR model is available to fulfill this information requirement.
- In vitro methods: Currently no validated and accepted in vitro methods are available to cover this endpoint. It is currently not possible, with in-vitro models, to account for the influence of the complex processes of absorption, distribution in the body, metabolism and excretion that occur in the whole animal, which will affect the toxic properties of the test substance.
- Weight of evidence: No adequate data are available, neither for the target substance, nor for related substances, to cover this endpoint.
- Grouping and read-across: No adequate alternative substance with data suitable for read-across purposes is available.
- Substance-tailored exposure driven testing [if applicable]: Based on use conditions, exposure cannot be completely excluded.
- Approaches in addition to above [if applicable]: n.a.
- Other reasons [if applicable]: n.a.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

Column 2 of Annex X states that the reproductive toxicity studies do not need to be performed if:
- the substance is known to be a genotoxic carcinogen and appropriate risk management measures are implemented; or
- the substance is known to be a germ cell mutagen and appropriate risk management measures are implemented; or
- the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available) it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure.

If a substance is known to cause developmental toxicity, meeting the criteria for classification as toxic for reproduction category 1A or 1B: May damage the unborn child (H360D), and the available data are adequate to support a robust risk assessment, then no further testing for developmental toxicity will be necessary.

None of these conditions are met by the substance: The substance is not classified for carcinogenicity or mutagenicity. From the available subchronic repeated dose toxicity studiy as well as the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test it can be concluded, that the substance is systemically available after oral administration based on treatment related histopathological changes seen in liver and kidney in the 90 d study and clinical signs of toxicity, low body weight, macroscopic and histopathological changes seen in the mid and high dose of the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (for details see Iuclid section 7.5.1).
In addition, the substance is not classified for developmental toxicity based on the results of the PNDT conducted in rat.
Therefore, the above listed column 2 adaptions cannot be applied.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: The proposed study design is in accordance with the OECD 414 guideline (Prenatal Development Toxicity Study). The oral route of exposure is selected based on the physico-chemical properties of the substance. The rabbit is chosen as the second species as detailed in ECHA's Endpoint specific guidance R.7a.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Species:
rabbit
Route of administration:
oral: unspecified
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2 - 31, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 22, 2001
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 3001545196
- Expiration date of the lot/batch: At least one year upon receipt (December 03, 2015) of the test item, that is Dezember 03, 2016


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At + 10°C to +25°C, in a tightly closed container.
Blanketing with nitrogen before recapping is recommended.
- Stability under test conditions: At least one year upon receipt of the test item
- Solubility and stability of the test substance in the solvent/vehicle: An analysis of the test item-formulation indicated correctly prepared and homogenised test item vehicle mixtures,
which were stable for at least 24 hours.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item formulations were freshly prepared every day.The test item was diluted in the vehicle to the appropriate concentrations using a magnetic stirrer and was administered orally at a constant volume once daily from the 6th to the 20th day of gestation. Stirring of the formulations was continued until the last animal of the dose group had been dosed.

Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sulzfeld, Germany
- Age at study initiation:
- Weight at study initiation:
- Housing: The animals are kept singly in MAKROLON cages (type III plus).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature of 22°C ± 3 °C (maximum range)
- Humidity (%): relative humidity of 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle

IN-LIFE DATES: From: May 3, 2016 To:TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 59 days
- Weight at study initiation: 180.8 - 250.8 g
- Housing: Except during the mating period, the dams were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm.
- Diet (e.gad libitum): Commercial diet ssniff® R/Z V1324 ad libitum
- Water (e.g. ad libitum): Drinking water (in drinking bottles) was offered ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): The ventilation rate of the animal room was between fifteen to twenty air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle

IN-LIFE DATES: From:18 May 2016 To: 26 May 2016
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency):Commercial diet was offered daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test laboratory has extensive experience with the vehicle corn oil and also in combination with the test item oxooil. Furthermore the test item mixture with the vehicle was verified regarding stability, homogenicity and concentration.
- Concentration in vehicle: The measured actual concentrations of the test item in the test item vehicle mixtures were between 99.8% and 109.1% of the nominal concentrations, indicating correctly prepared and homogenized formulations which were stable at room temperature for at least 24h.
- Amount of vehicle (if gavage): 2 mL/kg b.w.
- Lot/batch no. (if required): 15296403
-Source: supplier: Caesar & Loretz GmbH, 40721 Hilden, Germany
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: cohoused
- Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
- M/F ratio per cage: 1 M/1 F per cage
- Length of cohabitation: Cohabitation during the dark period; If findings were negative, mating was repeated with the same partner.
- Further matings after two unsuccessful attempts: no; Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Treatment period: Day 6 to 20 of gestation
Frequency of treatment:
Once daily
Duration of test:
Start of mating May 2, 2016
First mating results May 3, 2016
First administration May 9, 2016
Termination of the in-life part May 31, 2016
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day
Remarks:
low dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
intermediate dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
high dose
No. of animals per sex per dose:
100 females in order to have at least 20 pregnant females per group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels have been selected in agreement with the Sponsor based on the results of a preliminary dose-range-finding study in rats
- Rationale for animal assignment (if not random):
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: early in in the morning and again in the afternoon of each working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Each day the body weight will be registered always at the same time

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION : Yes
- Time schedule for examinations: Daily monitoring by visual appraisal of the drinking water bottles will be maintained throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21th
- Organs examined:
Examination of the dams:
Heart, kidney and liver were weighed
Examination of the fetuses:
Macroscopic inspection (gross evaluation) and weighting of the placentae.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Weight of placentae: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: No
Statistics:
Toxicology (and Pathology, if applicable) data will be captured, whenever possible,
using the departmental computerized systems (Provantis®8 Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems will be maintained on paper according to appropriate SOPs.
Historical control data:
The relationship between 'Variations and Retardations' and 'Malformations' should be seen under the aspect of cumulative historical data.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (1000 mg Oxooil LS9/kg b.w./day) several dams were noted with signs of clinical toxicity on several test days (e.g. a reduced motility (17 of 22), salivation (21 of 22), prone position (3 of 22), piloerection (3 of 22), pale faeces (9 of 22) and an increased (4 of 22) or decreased water consumption (2 of 22)).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (1000 mg Oxooil LS9)/kg b.w./day) a statistically significantly reduced body weight was noted from gestation day 10 until laparotomy on gestation day 21 (max.: 15.0% below the value of the control group at the day of laparotomy). Body weight gain for the whole study period was 49.7% for the dams of the high dose group in comparison to 78.4% for the dams of the control group (p ≤ 0.01).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant (p ≤ 0.01) reductions in food consumption were noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day) on several gestation days after the start of treatment (max. 36.4% below the value of the control group between gestation days 6 and 7).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (1000 mg Oxooil/kg b.w./day) 4 dams with an increased water consumption and 2 other dams with a decreased water consumption were noted by visual appraisal.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Uterus and carcass weights: At the high dose level (1000 mg Oxooil LS9/kg b.w./day) statistically significant reductions were noted for the gravid uterus weight (22.4% below the value of the control group) and the carcass weight (12.5% below the value of the control group).

Further organ weights: The weighing of the heart, the kidneys and the liver revealed no test item-related differences between the dams of the control group and the treatment groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
A total resorption of all implants (a postimplantation loss of 100%) was noted for 2 of 22 dams of the high dose group (1000 mg Oxooil LS9/kg b.w./day), leading to a statistically significantly (p ≤ 0.01) increased post-implantation loss (10.9% in comparison to 4.7% in the control group).
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
pre and post implantation loss
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly (p ≤ 0.01) decreased fetal weight was noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day). The fetal weight 13.4% below the value of the control group (male and female fetuses together).
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): A statistically significantly (p ≤ 0.01) decreased fetal weight was noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day).
The fetal weight was 13.4% below the value of the control group (male and female fetuses together).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No malformation was noted during the macroscopic examination at laparotomy (external inspection and inspection of the inner organs and tissues for gross lesions).
Skeletal malformations:
no effects observed
Description (incidence and severity):
No malformation was noted during the skeletal examination according to DAWSON.
Visceral malformations:
no effects observed
Description (incidence and severity):
No malformation was noted during the soft tissue examination according to WILSON.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly (p ≤ 0.01) decreased placental weight was noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day). The placental weight at the high dose level was 9.9% below the value of the control group (male and female placentae combined).

Retardations: At the high dose level (1000 mg Oxooil LS9/kg b.w./day) the skeletal examination according to DAWSON revealed an increased incidence of delays in ossification for the metacarpalia and the sternebra(e) that are considered to be test item related.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: retardations: Increased incidence of delayed ossifications of the metacarpalia and the sternebra(e) at the high dose level
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg Oxooil LS9/kg b.w./day for the dams.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was 300 mg Oxooil LS9/kg b.w./day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a prenatal developmental toxicity study, the test item Oxooil LS9 was administered orally to female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day from the 6th to 20th day of pregnancy.


Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg Oxooil LS9/kg b.w./day for the dams.


None of the dams died prematurely.


Noteworthy clinical observations were noted only at the high dose level (1000 mg/kg b.w./day) in form of e.g. reduced motility, prone position and piloerection.


In addition, a reduced body weight and reductions in food consumption were noted at the high dose level (1000 mg/kg b.w./day).


No test item-related changes were noted during the macroscopic inspection at necropsy and for the organ weights of the heart, the kidneys and the liver.


 


The no-observed-adverse-effect level (NOAEL) for the fetal organism was 300 mg Oxooil LS9/kg b.w./day.


At the maternal toxic dose level of 1000 mg Oxooil/kg b.w./day 2 dams were noted with a total implantation loss. Furthermore, reduced placental and fetal body weights as well as an increased incidence of delayed ossifications of the metacarpalia and the sternebra(e) were noted at the high dose level. These dose-related findings are considered to be treatment-related.


No dead fetuses, no malformations and no test item-related increase in the incidence of variations were noted during the examination of the fetuses.


 


Pre- and postnatal effects of Oxooil Ls9 on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of rats were analysed in this EOGRTS. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. The target dose levels in this study were selected to be 0, 50, 150, 450 mg/kg b.w./day via oral administration.


At 450 mg Oxooil LS9/kg b.w./day, a decreased number of implantation sites was noted. 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. 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. Adverse effects on the pre-natal development of the pups were seen at 450 mg Oxooil LS9/kg b.w./day due to a reduced number of implantation sites triggered by a reduced fertility index and gestation index seen in F0 females at 450 mg/kg. The post-implantation loss of 100 % that was noted for these 7 females was a physiological effect due to only 1 or 2 implantation sites or a problem with littering. Hence, if we exclude these 7 females with a post-implantation loss of 100 % from the calculation, 11 females with a percentage of post-implantation loss between 0.0 % and 50.0 % were left (mean value 12.3 %). Hence, the post-implantation loss of these 11 females were within the range of the control group (12.80 ± 25.08 %).The observed increased implantation loss and also the decreased birth index that were noted at the high dose level were due to a physiological effect of a low number of implantation sites and problems with littering. A test item-related increase in the number of resorptions at the high dose level could not be postulated. This was considered as secondary effect of reduced maternal fertility index.


For F2 generation after second mating, 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.


In addition, all other routinely examined endpoint for pre- and postnatal development within OECD TG 443 were not considered of toxicological concern as no test item-related adverse effects were reported. Moreover, developmental neurotoxicity did not show any effects and therefore is considered of no concern. 


F1 generation:


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.


adverse effects on the post-natal development of the pups: NOAEL at least 450 mg Oxooil LS9/kg b.w./day


F2 generation:


adverse effects on the pre-natal development of the pups: NOAEL at least 300 mg Oxooil LS9/kg b.w./day


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


 

Justification for classification or non-classification

Within the scope of an OECD TG 422 reproductive/developmental screening study, a prenatal developmental toxicity study according to OECD TG 414 and a extended one generation study according to OECD TG 443, no classification for reproductive or developmental toxicity is indicated for Oxooil LS9 according to the general classification and labelling requirements for dangerous substances and preparations (Directive 67-548-EEC) or the classification, labelling and packaging (CLP) regulation (EC) No 1272/2008.

Additional information