Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The test item, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and until sacrifice for males, or through gestation and until Day 14 p.p. for females, at dose-levels of 100, 300 and 1000 mg/kg/day. Based on the results, the NOAEL (No Observed Adverse Effect Level) was considered 300 mg/kg/day for systemic toxicity due to clinical signs observed at the high dose-level and 100 mg/kg/day for local toxicity due to microscopic findings noted in the forestomach of animals of the mid- and high-dose groups. The NOAEL for reproductive toxicity of females was 300 mg/kg/day due to the effects on oestrous cycle noted in the high dose females. The NOAEL for reproductive toxicity of males was 1000 mg/kg/day. The NOAEL for pups development was 100 mg/kg/day considering the observed pup loss and reduced litter/pup weights noted in mid- and high dose levels.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
ordered from Charles River Italia S.p.A., Calco (Lecco), Italy and supplied by Charles River Laboratories, Domaine des Oncins, 69210 Saint Germain Nuelles, France.
-Age/Weight: The animals were approximately 7 to 8 weeks old and weighing 200-225 g for males and 175-200 g for females. After arrival, the weight range for each sex was determined and found to be 216 - 233 g for males and 199 - 209 g for females.
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Housing:
in polysulfone solid bottomed cages
- Diet (e.g. ad libitum):
4 RF 21,Mucedola S.r.l., Via G. Galilei, 4,20019 SettimoMilanese (MI), Italy
- Water (e.g. ad libitum):
drinking water
- Acclimation period:
An acclimatisation period of 33 days was allowed before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 2°C
- Humidity (%):
55 ± 15%
- Air changes (per hr):
approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light):
12 h/12 h

IN-LIFE DATES: 14 May 2019 to 24 July 2019
Route of administration:
oral: gavage
Vehicle:
other: 2% aqueous solution of carboxymethylcellulose.
Details on exposure:
The required amount of test item was suspended in the vehicle. The preparationswere made daily/weekly (concentrations of 10, 30 and 100 mg/mL) based on stability data obtained.
Concentrations were calculated and expressed in terms of test item as supplied.
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight.
Control animals received the vehicle alone at the same dose volume.
Details on mating procedure:
Mating was monogamous (one male to one female). Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (spermidentification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification of copulation occurred.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate both the analytical method and the preparation procedure and to verify the stability of the preparations (ERBC StudyNo. A3524). The analytical method was validated in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in ERBC SOPs for suspensions (r >0.98; accuracy 85-115%; precision CV <10%). In the same study, a 28 hour stability at room temperature and a 32 day stability at 2-8°C were verified in the range from 10 to 100 mg/mL.
The proposed preparation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirmthat the method was suitable. All results for all levels were within the acceptability limits stated in ERBC SOPs for suspensions (85-115%) and homogeneity (CV <10%). In the present study, samples of the preparations made on two occasions during the study (Day 1 and again towards the end of the study) were analysed to check the homogeneity and concentration. Chemical analysis was carried out by the Analytical Chemistry Department using a gas chromatography (GC) with flame-ionisation detection (FID). The software used for this activity was Chromeleon 7, version 7.2.2.6394 (Thermo Scientific). Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (85-115% for concentration and CV <10% for homogeneity).
Duration of treatment / exposure:
In the males (30/31 days in total):
- 2 weeks before mating,
- during the mating period,
- until sacrifice,
In the females (7 to 8 weeks of treatment for at least 51 days):
- 2 weeks before mating
- during the mating period,
- during gestation,
- during lactation until Day 14 p.p. inclusive for lactating females,
- or until sacrifice for females with no delivery.
Frequency of treatment:
daily
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose-levels were selected in agreement with the Sponsor, and are based on the results of a previous study. In this study, five male and five female Sprague Dawley rats were given the test item at 100, 300 or 1000 mg/kg/day for 2 weeks. One high dose female animal was sacrificed for humane reasons on Day 14. On the day of sacrifice, the animal appeared moribund. No clinical signs were noted in this animal in the days prior to sacrifice. At necropsy examination, the finding observed was limited to a slightly distended stomach. Due to the absence of signs of toxicity and the single mortality occurred, it was not possibile to correlate the mortality to the toxicity of the test item. No relevant clinical signs were observed during the treatment. No effects on body weight were observed during the study. No effects on food consumption were observed in males and females receiving dose levels= 300 mg/kg, during the study. Food consumption in females of the high dose group appeared reduced during the second week of treatment. At post mortem examination performed at the end of the study, no treatment-related changes were noted in the animals.
Based on the results obtained in this study, the same dose-levels were used in the present study.
- Rationale for animal assignment: computerized stratification procedure
Positive control:
no (not required)
Parental animals: Observations and examinations:
MORTALITY/MORBIDITY:
- Time schedule: at least twice a day during the treatment period.
CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.
DETAILED CLINICAL SIGNS
- Time schedule: once before the beginning of the treatment period and then at least once a week until the end of the study.
BODY WEIGHT:
- Time schedule: Males will be weighed weekly from allocation to termination. Females will be weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams will also be weighed on Days 1, 4, 7, 13 post partum and just before to necropsy.
FOOD CONSUMPTION:
- Time schedule: The weight of food consumed by each cage of males and females will be recorded weekly (whenever possible) during the pre-mating period starting from Day 1 of dosing up to mating.
Individual food consumption for mated females will be measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.
HEMATOLOGY
- Time schedule: on the day of scheduled sacrifice.
CLINICAL CHEMISTRY
- Time schedule: on the day of scheduled sacrifice.
URYNALYSIS
-Time schedule : during the last week of treatment for males only.
EUTHANASIA:
Parental animals in extremis or killed for humane reasons will be killed under carbon dioxide asphyxiation. Parental animals that have completed the scheduled test period, will be killed by exsanguination under isoflurane anaesthesia.
Parental males
The males will be killed after the mating of all females or after at least 28 days of treatment period.
THYROID HORMONES:
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using Luminex Magpix system and the MILLIPLEX MAP Rat Thyroid Magnetic Bead Panel kit (MerkMillipore, cat. no. RTHYMAG-30K).
The determination was restricted as detailed below:
– Samples from all parental males from all groups
– Samples from pups on Day 14 post partum
Since no treatment related effects were seen in the determination performed in parental males and in pups on Day 14 post partum, samples obtained from females and from pups on Day 4 post partum were not analysed;
Parental females
The females with live pups will be killed on Day 14 post partum. The females with total litter loss will be killed on the day of the occurrence of total litter loss or shortly after.
ORGAN WEIGHTS
Parental animals
From all animals completing the scheduled test period, the organs indicated in Annex 1 will be dissected free of fat and weighed. The ratios of organ weight to body weight will be calculated for each animal. At the discretion of the pathologist, organs may be weighed from animals dying or killed prior to terminal kill.
PRESERVATION AND FIXATION
Samples of all the tissues listed in Annex 1 will be fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which will be fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).
HISTOPATHOLOGY
The tissues required for histopathological examination are listed in Annex 1. In the first instance, the examination will be restricted as detailed below:
a) Tissues specified in Annex 1 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term;
b) Tissues specified in Annex 1 from all animals killed or dying during the treatment period.
c) All abnormalities in all groups.
Since differences were observed in stomach and liver between control and high dose animals, the examination was extended to:
– Stomach and liver in all males and females of Groups 2 and 3 and in the remaining animals of control and high dose groups.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with
Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
Oestrous cyclicity (parental animals):
Stock females:
Oestrous cycle was monitored by vaginal smears in all stock females for 2 weeks before allocation in order to exclude from the study females with irregular cycle. These data are not tabulated in this report, but will be archived with all raw data. No replacement occurred after allocation of the animals.
Females allocated to groups:
Vaginal smears were taken in the morning from Day 1 of treatment, up to positive identification of mating including not less than 2 weeks before the pairing.
The vaginal smear data were examined to determine the following:
– anomalies of the oestrous cycle;
– pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were also taken from all females, before despatch to necropsy.
Sperm parameters (parental animals):
The testes and epididymides will be cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) will be performed.
Litter observations:
Pups identification, weight and observations
As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1, 4 and 13 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters. After culling, all pups were sacrificed with the dams on Day 14 post partum.

Culling - Day 4 post partum
On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection.
Two pups (males or females, selected for culling) were sacrificed for blood collection. Two male pups were selected for dam no. X1140067 (Group 4), whereas, two female pups were selected for dam no. X1140033 (Group 2) and dam nos. X1140061, X1140063 and X1140071 (Group 4).

Anogenital distance
The anogenital distance (AGD) of each pup was measured on Day 1 post partum. The measure of AGD was normalized to the cube root of the pup’s body weight measured on Day 1 post partum.

Nipple count
The presence of nipples/areolae in male pups was checked on Day 13 post partum (a daybefore despatch to necropsy).

Clinical pathology investigations
Blood samples for haematology, clinical chemistry and coagulation were collected, at the end of treatment period, by random selection from 5 males and 5 females (females with viable litters) of each group, under condition of food deprivation.
Males
Blood samples for haematological investigations, biochemical tests and hormone determination were collected under isoflurane anaesthesia from the retro-orbital sinus. Blood samples for coagulation test (food available) were collected at necropsy from the vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.
Females
As a part of the sacrificial procedure, blood samples for all determinations were withdrawn from the abdominal vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.
Postmortem examinations (parental animals):
Sacrifice and pathology
EUTHANASIA:
Parental animals in extremis or killed for humane reasons will be killed under carbon dioxide asphyxiation. Parental animals that have completed the scheduled test period, will be killed by exsanguination under isoflurane anaesthesia.
Parental males
The males will be killed after the mating of all females or after at least 28 days of treatment period.
Parental females
The females with live pups will be killed on Day 14 post partum. The females with total litter loss will be killed on the day of the occurrence of total litter loss or shortly after.
ORGAN WEIGHTS
Parental animals
From all animals completing the scheduled test period, the organs indicated in Annex 1 will be dissected free of fat and weighed. The ratios of organ weight to body weight will be calculated for each animal. At the discretion of the pathologist, organs may be weighed from animals dying or killed
prior to terminal kill.
PRESERVATION AND FIXATION
Samples of all the tissues listed in Annex 1 will be fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which will be fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).
HISTOPATHOLOGY
The tissues required for histopathological examination are listed in Annex 1. In the first instance, the examination will be restricted as detailed below:
a) Tissues specified in Annex 1 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term;
b) Tissues specified in Annex 1 from all animals killed or dying during the treatment period.
c) All abnormalities in all groups.
Since differences were observed in stomach and liver between control and high dose animals, the examination was extended to:
– Stomach and liver in all males and females of Groups 2 and 3 and in the remaining animals of control and high dose groups.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
STATISTICS
Standard deviations were calculated as appropriate. For variables, e.g. body weight, food consumption, clinical pathology parameters and organ weights, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means or the nonparametric Kolmogorov-Smirnov test. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of theWilliams test. The criterion for statistical significance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Postmortem examinations (offspring):
Pups that had completed the scheduled test period (Day 4 or Day 14 post partum) were euthanised by intraperitoneal injection of Thiopenthal.
Pups selected for blood collection for hormone determination were killed on the day of blood sampling.
All pups found dead in the cage were examined for external and internal abnormalities.

Organ weights
Pups at Day 14 post partum
Thyroids were weighed from one male and one female pup selected for blood collection for hormones determination and preserved in 10% neutral buffered formalin.The thyroid weights were determined after fixation.
All culled pups sacrificed on Day 4 post partum were subjected to an external examination.
Sex was determined by internal gonads inspection.
All live pups sacrificed on Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Pups with abnormalities were retained in 10% neutral buffered formalin.
Statistics:
Standard deviations were calculated as appropriate. For variables, e.g. body weight, food consumption, clinical pathology parameters and organ weights, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test,
depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the nonparametric version of theWilliams test. The criterion for statistical significance was p<0.05.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Reproductive indices:
- Males
Copulatory Index(%) = (no. of males with confirmed mating/no. of males cohabitated) ×100
Fertility Index (%) = (no. of males which induced pregnancy/no. of males cohabitated) ×100

- Females
Copulatory Index(%) = (no. of femaleswith confirmed mating/no. of females cohabitated) ×100
Fertility Index (%) = (no. of pregnant females/no. of females cohabitated) ×100

- Males and females
Pre coital Interval = The number of nights paired prior to the detection of mating

- Females
*Pre-implantation loss(a) was calculated as a percentage from the formula:
(no. of corpora lutea-no. of implantations/no. of corpora lutea) ×100
*Pre-natal loss(b) was calculated as a percentage from the formula:
[(no. of visible implantations-live litter size at birth)/no. of visible implantations] ×100
*Post natal loss at Day 0 post partum was calculated as a percentage from the formula:
[(Total litter size-live litter size)/Total litter size] ×100
*Post natal loss(b) at Day 4 post partum (before culling) was calculated as a percentage from the formula:
[(Live litter size at birth-live litter size at Day 4(before culling))/Live litter size at birth] ×100
*Post natal loss(b) at Day 13 post partum (after culling) was calculated as a percentage from the formula:
[(Live litter size onDay 4(after culling)-live litter size onDay 13)/Live litter size onDay 4(after culling)] ×100
Sex ratios were calculated at birth, on Day 4 and on Day 14 post partum and were presented as the percentage of males per litter.

a Reference: Developmental and reproductive toxicology: A practical Approach (second edition)
b As reported in the OECD 422 guideline
Offspring viability indices:
not calculated.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See 7.5.1
Mortality:
mortality observed, non-treatment-related
Description (incidence):
See 7.5.1
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See 7.5.1
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See 7.5.1
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See 7.5.1
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
See 7.5.1
Urinalysis findings:
no effects observed
Description (incidence and severity):
See 7.5.1
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
See 7.5.1
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):
See 7.5.1
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroids hormone
Parental males
Triiodothyronine was increased in one male dosed at 1000 mg/kg/day. Due to the minimal incidence and the absence of other related findings (e.g. changes of the other hormones), this change was not considered to be of toxicological significance.
The statistically significant increase of thyroxine recorded in males dosed at 300 mg/kg/day was not dose-related, therefore it was considered to be incidental.
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction in the number of non sequential days in which the females were in oestrous was observed in all groups of treated females. However, Groups 2 and 3 are within the historical control data (mean of days 2.78).
Vaginal smears examined on the day of necropsy to determine the stage of the oestrous cycle showed the phase of dioestrous for the not pregnant females of control and low dose group (Group 2). For the two non pregnant females of the mid-dose group (Group 3), dioestrous and oestrous were observed (no. X1140055 and X1140059, respectively). Dioestrous was recorded for all females sacrificed on Day 14 post partum.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in
the germinal epithelium was noted in all control and treated males.
1) Toxicologic Pathology, 39: 337-347, 2011 - SIEGRID DAMSCH
2) Toxicologic Pathology, 38: 5S-81S, 2010 BOB THOLEN
Reproductive performance:
no effects observed
Description (incidence and severity):
The number of copulatory plugs found in the cage was from 3 to 5 (as a mean value). The majority of females mated in 1-4 days of cohabitation with the exception of three females that mated after 12-14 days. In particular, two females of the control group, nos. X1140009 and X1140019, mated after 12 and 14 days respectively and one of the low dose group (no. X1100037) that mated after 14 days. The mean pre-coital intervals (number of days paired up to spermpositive day) in order of ascending dose levels were: 5 (control), 3, 3 and 2. The difference between groups (statistically significant for Group 4 only) was considered not toxicologically relevant since the high value in the control was due to two females that conceived after 12 and 14 days of pairing. Therefore, this finding was considered to be incidental.
Copulatory index was 100% for both males and females of all treated groups. One control male (no. X1140010), one low dose male (no. X1140028) and two mid-dose males (nos. X1140056 and X1140060) failed to induce pregnancy. These cases were considered incidental. Fertility index both for males and females was 100% for high dose group, 90% for control and low dose groups and 80% for the mid-dose group.
See 7.5.1.
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive function (oestrous cycle)
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No compound-related effects were observed (isolated findings and/or of common background). Pre-weaning clinical signs were comparable between treated and control groups or considered incidental.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
An increased incidence of pup loss on Day 4 post partum was observed in females of the mid- and high dose groups when compared to control.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases in litter weight, and consequently in mean pup weight, were observed on Day 13 post partum in the mid- and high dose groups.
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):
no effects observed
Description (incidence and severity):
No differences in the anogenital distance (normalised value) to the cube root of the body weight, performed on Day 1 post partum, were seen between control and treated groups for male pups.
A slight increase in the anogenital distance values (mean) was noted in high dose female pups when compared to the control value. However this change was dose-unrelated and therefore cannot be conclusively attributed to treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were found in male pups on Day 13 post partum.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex ratios at birth, on Days 4 and 14 post partum did not show relevant differences between groups, when calculated as the percentage of males.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Remarks:
pups development
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Critical effects observed:
no
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The test item, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and until sacrifice for males, or through gestation and until Day 14 p.p. for females, at dose-levels of 100, 300 and 1000 mg/kg/day. Based on the results, the NOAEL (No Observed Adverse Effect Level) was considered to be 300 mg/kg/day for systemic toxicity due to clinical signs observed at the high dose-level and 100 mg/kg/day for local toxicity due to microscopic findings noted in the forestomach of animals of the mid- and high-dose groups. The NOAEL for reproductive toxicity of females was 300 mg/kg/day due to the effects on oestrous cycle noted in the high dose females. The NOAEL for reproductive toxicity of males was 1000 mg/kg/day. The NOAEL for pups development was 100 mg/kg/day considering the observed pup loss and reduced litter/pup weights noted in mid- and high dose levels.
Executive summary:

The test item,2-OCTANOL, was administered daily to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and until sacrifice for males, or through gestation and until Day 14 p.p. for females, at dose-levels of 100, 300 and 1000 mg/kg/day

All doses (100, 300 and 1000 mg/kg/day) were administered orally, by gavage. The control group received 2% aqueous solution of carboxymethylcellulose. The dose volume was 10mL/kg body weight. Males were treated daily for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 30/31 days. Females were treated for 2 weeks prior to pairing, during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days). Four non pregnant females were dosed up to the day before necropsy.

 

Mortality observed in one female of the high dose group could not to be clearly related to treatment due to the absence of clear pathological findings.

A number of clinical signs indicating toxicity (i.e. salivation, ataxia, hunched and/or prone posture, lethargy) were observed in both males and females treated at 1000 mg/kg/day. Salivation was generally observed in the animals treated at 300 mg/kg/day.

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

No differences of toxicological significance were noted throughout the study in the body weight and body weight gain of treated males. In females of the high dose group, a slight decrease in body weight was noted during the post coitum and post partum periods. An increase in body weight gain in females of the high dose group was recorded at the end of the premating period.

No effects on food consumption were observed in males. In females of the high dose group, food consumption was slightly reduced during the first week of treatment and during the post coitum and post partum periods.

Motor activity, grip strength and sensory reactivity to stimuli performed at the end of the treatment period did not reveal changes attributable to the test item.

Change in neutrophilis (increase) recorded in males treated at dose levels 300 mg/kg/day was considered to be not adverse. No changes were recorded in prothrombin time measured at the end of the study.

Fluctuations of some biochemical parameters (albumin, bile acids, calcium, sodium and bilirubin) in males and/or females were considered to be unrelated to treatment, incidental or considered to be not adverse. No relevant changes were observed in urine analysis performed in males at the end of the treatment period.

Changes observed in triiodothyronine in a single male treated at 1000 mg/kg/day was not considered to be of toxicological significance due to the minimal incidence and the absence of other related findings (e.g. changes of the other hormones).

Changes observed in thyroid hormones measured in pups, were considered not adverse or not related to the test item.

Statistically significant reduction in the number of non sequential days in which the females were in oestrous was observed in high dose group of treated females.

The pre-coital interval, copulatory and fertility indices did not show any differences that could be related to treatment.

No differences were noted in implantation sites, pre-natal loss data and gestation length of females between treated and control groups, as well as in sex ratios.

An increased incidence of pup loss on Day 4post partumwas observed in females of the mid- and high dose groups.

Decreases in litter weight, and consequently in mean pup weight, were observed on Day 13post partumin the mid- and high dose groups.

Pre-weaning clinical signs were comparable between treated and control groups or considered incidental. Also thyroid weights recorded in treated pups were comparable to the control mean value. No nipples were observed in male pups on Day 13post partum.

No negative effects in anogenital distance were seen in male pups of treated groups. A slight increase in the anogenital distance values (mean) was noted in female pups of high dose dams. However, this change was dose-unrelated and therefore cannot be conclusively attributed to treatment.

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14post partumdid not reveal any treatment-related effect.

No changes were observed on terminal body weight of study animals of both sexes, when compared to the control group. A statistically significant increase was observed in mean liver weight of high dose animals of both sexes.

At macroscopic observation performed at post mortem, the most relevant change was thickening of the non glandular region of the stomach observed in most high dose rats of both sexes sacrificed at the end of treatment period, whereas in a single instance, it was observed in one mid-dose animal of both sexes and in one control male.

Treatment-related changes were present in the non glandular region of the stomach (forestomach) of mid- and high dose animals of both sexes and consisted of squamous epithelial hyperplasia associated with hyperkeratosis (orthokeratotic), chronic inflammation and/or oedema in the submucosa, in some instances associated with mucosal ulceration/erosion.

Treatment-related changes were also noted in the liver of high dose males and consisted of minimal centrilobular hepatocellular hypertrophy indicating a possible microsomal enzyme induction as an adaptive response to the exposure to the test item."

Based on the results, the parental NOAEL (No Observed Adverse Effect Level) was considered 300 mg/kg /day for systemic toxicity due to clinical signs observed at the high dose-level and 100 mg/kg/day for local toxicity due to microscopic findings noted in the forestomach of animals of the mid- and high-dose groups.

The NOAEL for reproductive toxicity of females was 300 mg/kg/day due to the effects on oestrous cycle noted in the high dose females. The NOAEL for reproductive toxicity of males was 1000 mg/kg/day. The NOAEL for pups development was 100 mg/kg/day considering the observed pup loss and reduced litter/pup weights noted in mid- and high dose levels.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Good; compliant to GLP and testing guidelines;
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The toxic effects on Sprague Dawley rats of 2-OCTANOL, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and early lactation of the offspring were investigated in this study.

All doses (100, 300 and 1000 mg/kg/day) were administered orally, by gavage. The control group received 2% aqueous solution of carboxymethylcellulose. The dose volume was 10 mL/kg body weight. Males were treated daily for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 30/31 days. Females were treated for 2 weeks prior to pairing, during pairing, post coitum and post partumperiods until Day 13 post partum(for at least 51 days). Four non pregnant femaleswere dosed up to the day before necropsy.

Mortality observed in one female of the high dose group could not to be clearly related to treatment due to the absence of clear pathological findings. A number of clinical signs indicating toxicity (i.e. salivation, ataxia, hunched and/or prone posture, lethargy) were observed in both males and females treated at 1000 mg/kg/day. Salivation was generally observed in the animals treated at 300 mg/kg/day. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. No differences of toxicological significance were noted throughout the study in the body weight and body weight gain of treated males. In females of the high dose group, a slight decrease in body weight was noted during the post coitum and post partumperiods. An increase in body weight gain in females of the high dose group was recorded at the end of the premating period. No effects on food consumption were observed in males. In females of the high dose group, food consumption was slightly reduced during the first week of treatment and during the post coitum and post partum periods.

Motor activity, grip strength and sensory reactivity tostimuliperformed at the end of the treatment period did not reveal changes attributable to the test item.

Change in neutrophilis (increase) recorded in males treated at dose levels 300 mg/kg/day was considered to be not adverse. No changes were recorded in prothrombin time measured at the end of the study. Fluctuations of some biochemical parameters (albumin, bile acids, calcium, sodium and bilirubin) in males and/or females were considered to be unrelated to treatment, incidental or considered to be not adverse.No relevant changes were observed in urine analysis performed in males.

Changes observed in triiodothyronine in a single male treated at 1000 mg/kg/day was not considered to be of toxicological significance due to the minimal incidence and the absence of other related findings (e.g. changes of the other hormones).Changes observed in thyroid hormones measured in pups, were considered not adverse or not related to the test item.

Statistically significant reduction in the number of non sequential days in which the females were in oestrous was observed in high dose group of treated females.

The pre-coital interval, copulatory and fertility indices did not show any differences that could be related to treatment.

No differences were noted in implantation sites, pre-natal loss data and gestation length of females between treated and control groups, as well as in sex ratios.

An increased incidence of pup loss on Day 4 post partum was observed in females of the mid- and high dose groups.

Decreases in litter weight, and consequently in mean pup weight, were observed on Day 13 post partum in the mid- and high dose groups.

Pre-weaning clinical signs were comparable between treated and control groups or considered incidental. Also, thyroid weights recorded in treated pups were comparable to the control mean value. No nipples were observed in male pups on Day 13 post partum.

No negative effects in anogenital distance were seen in male pups of treated groups. A slight increase in the anogenital distance values (mean) was noted in female pups of high dose dams. However, this change was dose-unrelated and therefore cannot be conclusively attributed to treatment.

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.

No changes were observed on terminal body weight of study animals of both sexes, when compared to the control group. A statistically significant increase was observed in mean liver weight of high dose animals of both sexes.

At macroscopic observation performed atpost mortem, the most relevant change was thickening of the non glandular region of the stomach observed in most high dose rats of both sexes sacrificed at the end of treatment period, whereas in a single instance, it was observed in one mid-dose animal of both sexes and in one control male.

Treatment-related changes were present in the non glandular region of the stomach (forestomach) of mid- and high dose animals of both sexes and consisted of squamous epithelial hyperplasia associated with hyperkeratosis (orthokeratotic), chronic inflammation and/or oedema in the submucosa, in some instances associated with mucosal ulceration/erosion. Treatment-related changes were also noted in the liver of high dose males and consisted of minimal centrilobular hepatocellular hypertrophy indicating a possible microsomal enzyme induction as an adaptive response to the exposure to the test item."

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Based on the results, the NOAEL (No Observed Adverse Effect Level) was considered to be 300 mg/kg/day for systemic toxicity due to clinical signs observed at the high dose-level and 100 mg/kg/day for local toxicity due to microscopic findings noted in the forestomach of animals of the mid- and high-dose groups.

The NOAEL for reproductive toxicity of females was 300 mg/kg/day due to the effects on oestrous cycle noted in the high dose females. The NOAEL for reproductive toxicity of males was 1000 mg/kg/day. The NOAEL for pups development was 100 mg/kg/day considering the observed pup loss and reduced litter/pup weights noted in mid- and high dose levels.

There is no available evidence suggesting that the registered substance is a human reproductive toxicant. Indeed, the statistically significant reduction in the number of non sequential days in which the females were in oestrous was observed at a dose-level (1000 mg/kg/day) inducing clinical signs and effects on body weight gain.In addition, the statistically significant decreases in litter weight and mean pup weight, observed on Day 13 post partum in the mid- and high dose groups were slight, unspecific and not dose-related.

Effects on developmental toxicity

Description of key information

No study available.

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

Justification for classification or non-classification

According to the available data and CLP criteria, no classification for toxicity for reproduction is warranted.