Isotridecanol, ethoxylated

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 Mar - 14 Aug 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted in 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isotridecanol, ethoxylated

EC Number:

500-241-6

EC Name:

Isotridecanol, ethoxylated

Cas Number:

69011-36-5

Molecular formula:

not available, isomeric structures

IUPAC Name:

Isotridecanol, ethoxylated

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat is the species and strain of choice because it is accepted by many
regulatory authorities and there are ample experience and background data on this species
and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone, Italy
- Females: nulliparous and non-pregnant
- Age at study initiation: 7 - 8 weeks (at arrival) + 25 days (acclimatisation)
- Weight at study initiation: (P) Males 208 - 218 g, females 182 - 194 g
- Housing: From arrival to pairing, up to 5 of one sex to a cage; during mating, one male to one female in a cage. After mating, males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation.
- Diet: Rodent diet 4 RF 21 (Mucedola srl, SettimoMilanese, Italy), ad libitum
- Water: Via water bottles, ad libitum
- Acclimatisation period: 25 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 3 Mar 2020 To: 8 May 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was dissolved in the vehicle. The preparations were made weekly (concentrations of 12.5, 37.5 and 125 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle: Corn oil had been determined previously as a suitable vehicle for the test item.
- Amount of vehicle (if gavage): 4 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Until copulation occured or 14 days as the maximum period.
- Proof of pregnancy: Sperm in vaginal smear, vaginal plug in situ or copulation plugs found in the cage tray.
- After successful mating each pregnant female was caged: individually

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. The analytical method was validated in the range from 10 to 200 mg/mL. Linearity, accuracy and precision were within the limits stated in validation protocol (r > 0.98; accuracy 85 - 115%; precision CV < 5%). In the same study, 28 h stability at room temperature and 8 day stability at 2 - 8 °C were verified in the range from 10 to 200 mg/mL. The proposed preparation procedure for the test item was checked in the same by chemical analysis (concentration) to confirm that the method was suitable.

Duration of treatment / exposure:

Males
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the pairing period and thereafter until the day before necropsy (Day 32). Males were treated for a total of 31 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, gestation and post partum periods until Day 13 post partum (for at least 51 days). One non pregnant female, females with total litter loss and those that did not mate were dosed up to the day before necropsy. Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 of gestation and on Days 1, 4, 7 and 13 post partum.

Frequency of treatment:

Daily, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a Dose Range Finding Study (ERBC Study No. E0407) and from a 13-week oral toxicity study (GLP compliant study, ERBC Study No. A3596). and with the aim to produce graded responses to the test item.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS AND MORTALITY: Yes
- Time schedule: Mortality was checked at least twice daily, clinical signs were checked at least once daily.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination, females were weighed weekly from allocation to positive identification of mating and on
Days 0, 7, 14 and 20 of gestation. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Details: The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was also measured on Days 7, 14 and 20 of gestation starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

WATER CONSUMPTION: No

SERUM HORMONES: Measurement of total triiodothyronine (total T3), total thyroxine (total T4) and thyroid stimulating hormone (TSH) was conducted for all F0-males.

Oestrous cyclicity (parental animals):

Vaginal smears were taken in the morning from Day 1 of treatment, up to and including positive identification of mating (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):

Parameters examined in F0-males: Testis weight and epididymis weight.
- Additional examination: A morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germcell layers or types, retained spermatids, multinucleated or apoptotic germcells and sloughing of spermatogenic cells into the lumen.

Litter observations:

STANDARDISATION OF LITTERS
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.

PARAMETERS EXAMINED
The following parameters were examined in F1 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. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

GROSS EXAMINATION OF DEAD PUPS
Pups found dead were examined for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.

SERUM HORMONES
Blood samples were taken and measurement of total triiodothyronine (total T3), total thyroxine (total T4) and thyroid stimulating hormone (TSH) was conducted for all pups on Day 14 post partum.

Postmortem examinations (parental animals):

SACRIFICE
Parental animals that had completed the scheduled test period, were killed by exsanguination under isoflurane anaesthesia. A detailed post mortem examination was conducted (including examination of the external surface and orifices).

-Males: surviving males were killed after the end of mating period on Day 32 of the study. Males were treated for a total of 31 days.

-Females: females with live pups were killed on Day 14 post partum. Females were dosed for a
minimum of 51 consecutive days. The females showing no evidence of copulation were killed 27 days after the last day of the mating session.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Parental females were examined also for the number of visible implantation sites and the number of corpora lutea.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathology/organ weights was conducted for the following tissues: Adrenal glands, Brain (cerebrum, cerebellum, medulla/pons), Clitoral gland, Epididymides, Kidneys, Liver, Mammary gland - Females, Mammary gland - Males, Ovaries with oviducts, Parathyroid glands, Pituitary gland, Penis, Prostate gland (dorsolateral and ventral), Sciatic nerve, Seminal vesicles with coagulating glands, Spleen, Stomach, Testes, Thymus (where present), Thyroid, Uterus – cervix, Vagina.

Postmortem examinations (offspring):

SACRIFICE
All pups found dead in the cage were examined for external and internal abnormalities. 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. Thyroid was weighed from one male and female from each litter (if possible the same pup selected for serum hormone determination) and preserved in 10% neutral buffered formalin. The thyroid weight was determined after fixation. 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 the Williams 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:
Copulation 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 females with confirmed mating/no. of females cohabitated × 100
Fertility Index (%) = no. of pregnant females/no. of females cohabitated × 100
Pre-implantation loss was calculated as a percentage from the formula: no. of corpora lutea − no. of implantations/no. of corpora lutea × 100

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

Offspring viability indices:

Pre-natal loss on Day 0 post partum, before culling, was calculated as a percentage from the formula:
no. of visible implantations − live litter size at birth/no. of visible implantations × 100

Pup 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 on 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 sizeat birth × 100

Post-natal loss on Day 13 post partum (after culling) was calculated as a percentage from the formula:
Live litter size on Day 4(after culling) − live litter size on Day 13/Live litter size on Day 4 (after culling) × 100

Sex ratios were calculated at birth, on Days 4 and 14 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In all males receiving 500 mg/kg bw/day and in 9/10 males receiving 150 mg/kg bw/day, salivation was sporadically observed during the treatment period. Also 3/10 males receiving 50 mg/kg bw/day showed salivation on three occasions. Salivation was observed sporadically during the treatment period in all females receiving 500 mg/kg bw/day and 150 mg/kg bw/day. No clinical signs were observed during the study in females receiving 50 mg/kg/day. Salivation was considered treatment-related but not adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males body weight and body weight gain recorded during the study were comparable to the control group males. In female animals, on Days 4 and 13 of the post partum period and limited to the high dose group, statistically significant slight decrease in body weight gain (-6%) was noted, when compared to the control group. Body weight gain in high dose females, increased during the first week of treatment (Day 8 of the premating phase).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption recorded in male animals during the premating period was unaffected by treatment. A statistically significant decrease (ranging from -12% to -22%) in food consumption was evident in all treated groups of female animals at the end of the post partum period only.

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

Endocrine findings:

no effects observed

Description (incidence and severity):

No differences between control and parental treated males were recorded in thyroid hormones (T3, T4 and TSH) determination performed at the end of the study.

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were present in the stomach (non-glandular region) of mid-dose and high-dose animals of both sexes. Epithelial hyperplasia was seen in the non-glandular region of the stomach (forestomach) in 9/10 males and 9/10 females of the high dose group, with minimal to moderate degree for males and minimal to mild degree for females, whereas the same lesion, of minimal degree, was noted in 1/10 males and 2/10 females of the mid-dose group. The gastric lesion was characterised by convolutions of the epithelium with downward projections of rete peg-like structures and sometimes associated with the presence of hyperkeratosis with chronic inflammation and/or oedema in the submucosa, and in one instance with mucosal erosion of non-glandular region of the stomach. Epithelial hyperplasia of the non-glandular region of the stomach (forestomach) was evaluated to be a local irritant effect relevant to the rat. Since humans do not have a forestomach, the changes are considered not relevant to human.
In one male of the high-dose group, hepatocytic necrosis of the liver associated with cortical hypertrophy of the adrenals (monolateral) was noted. As the liver change was seen only once in the high-dose group and such a change is known to occur sporadically in untreated animals, the present case was considered an incidental finding, whereas the cortical hypertrophy of the adrenals was considered secondary to the stress induced by the liver and gastric (epithelial hyperplasia) lesions and therefore not related to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The total number of oestrous cycles observed in all females before pairing (number of non-sequential days in which the females were in oestrous) were similar between control and treated groups and was of 2-3 cycles. Vaginal smears examined on the day of necropsy showed the phase of diestrous for the majority of females sacrificed on Day 14 post partum. The non-pregnant females and those not mated, showed phase of the dioestrus on the day of necropsy but the oestrous cycle during the study was regular.

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 high-dose males.

Reproductive performance:

no effects observed

Description (incidence and severity):

Copulatory index was 100% for both males and females of control and low dose groups and 90% for for mid- and high-dose groups. Fertility index both for males and females was 100% each for control and low dose groups, 90% for mid-dose group and 80% for high dose group. One mid-dose female and one high dose female did not mate. These cases were considered incidental. The number of copulatory plugs were similar between groups.
Gestation length was similar between treated and control groups and no signs of dystocia were observed. Corpora lutea, implantations and pre-implantation loss, live litter size and pre-natal loss (percentage) did not show dose-related or treatment-related differences.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects observed

2

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects observed

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No food intake (milk), small appearance, pallor and cold to touch were the main clinical signs noted in control and treated pups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Dead and/or missing pups were observed both in control and treated groups, with similar incidence.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Reductions in mean litter weight and consequently in mean pup weight were evident in all litters of treated females on Day 13 post partum. Statistical significance was present in high dose females only for the mean litter weight and in low and high dose females for the mean pup weight. However, since the mean litter and pup weight values were within the historical control data, the changes in mean values were considered incidental and not treatment-related.

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:

no effects observed

Description (incidence and severity):

No differences were recorded between control and treaded groups in thyroid hormones of pups sacrificed on Day 14 post partum.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

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. In female pups, a slight decrease, significant at statistical analysis, in the mean values was noted in low dose female pups when compared to the control value. However, since the mean value increment was not dose-related and values recorded for all treated female groups were within the historical control data, this finding was considered incidental and not treatment-related.

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No relevant differences were noted in thyroid weight of pups between control and treated groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Decedent pups: autolysis of all organs were mainly observed in pups. No milk in stomach was noted in several pups belonging to the dams of the high-dose group. These findings were considered incidental and not treatment-related due to the single occurrence. No toxicologically relevant or treatment-related findings were recorded in pups sacrificed on Day 14 post partum.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Additional data are provided under 'Attached background material' in the form of pdf documents.