Dodecane-1,12-diol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2019 to December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guideline OPPTS 870.3650

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dodecane-1,12-diol

EC Number:

227-133-9

EC Name:

Dodecane-1,12-diol

Cas Number:

5675-51-4

Molecular formula:

C12H26O2

IUPAC Name:

dodecane-1,12-diol

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL

- Solubility and stability of the test substance in the solvent: Stability for at least 24 hours at 40°C in a waterbath (formulations were stored in clear vessels, cap of waterbath was closed) is confirmed over the concentration range 1 to 200 mg/mL (low group solutions, high group suspensions), Test Facility Study No. 20191505.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 24 hours after completion of the preparation of the formulation. Formulations were heated to a maximum temperature of 60±5°C for 30 minutes to obtain visual homogeneity. Formulations were released for dosing when they have obtained a temperature of 40±1°C. Test item dosing formulations were kept at 40±1°C until and during dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle. No adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.

Test animals

Species:

rat

Strain:

other: Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: 10-11 weeks; Females: 13-14 weeks
- Weight at study initiation: Males: 270-311 g; Females: 203-245 g
A health inspection was performed before the initiation of dosing.
- Housing:On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment, bedding material, food and water.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 55-69
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were no
t performed as part of this study and these preparations were not used for dosing. Raw data of thesetrials will be retained by the Test Facility.

Frequency of treatment
Once daily at approximately the same time each day.

The dose volume for each animal was based on the most recent body weight measurement (5 mL/kg bw). The doses were given using a plastic feeding tube.
The dosing formulations were stirred continuously during dose administration.
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

Detection of mating was not confirmed in first instance for Female No. 43. Evidence of mating was obtained indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the mating in this female. The mating date of this animal animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 d ays a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 51-55 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 38-43 days.The first day of dosing was designated as Day 1.Female Nos. 45 (Group 1), 66 and 68 (Group 3) and 74 and 79 (Group 4), were not dosed on one occasion as these females were littering at the moment of dosing. Female No. 67 (Group 3) was not dosed on two consecutive days as this female was littering at the moment of dosing. The omission of one day or two days of dosing over a period of several weeks was considered not to affect thetoxicological evaluation.

Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

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 finder with oral gavage administration of the test substance in rats (Test Facility Study No. 20191502), and in an attempt to produce
graded responses to the test item. The dose levels for the main study were selected based on the results of the dose range finder with doses of 1000 and 500 mg/kg bw/day for 15 days and 3 female rats per dose. No signs of toxicity were noted at any dose level. Since no clinical signs were observed in the dose range finder, clinical observations were conducted in the main study after dosing at no specific time point, but within a
similar time period after dosing for the respective animals.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted 3 hours (±30 min) post-dose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation was in troduced as no effect was suspected.

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: Functional tests (hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity) were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 11-13).

CLINICAL PATHOLOGY: Yes
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. When non-serum samples were clotted, additional blood samples were obtained in the necropsy room (if possible). After collection all samples were transferred to the appropriate laboratory for analysis. F0-females were not fasted prior to blood sampling and necropsy. F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

- Hematology parameters: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophil (absolute), Haemoglobin, Lymphocyte (absolute), Haematocrit, Monocyte (absolute), Mean corpuscular volume (MCV), Eosinophil (absolute), Mean corpuscular haemoglobin (MCH), Basophil (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelet, Reticulocyte (absolute).
- Coagulation parameters: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
- Clinical chemistry parameters: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos).
- Thyroid hormone: Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4). Measurement of T4 was conducted for F0-males.
For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no toxicologically relevant changes were noted in T4 in F0-males, both sexes showed a similar degree of thyroid follicular hypertrophy and since thyroid weight was considered not affected by treatment.

Serum samples retained for possible future analysis were maintained by the Test Facility in the freezer (≤-75 °C). Under these storage conditions, samples are stable for 6 months.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.

Sperm parameters (parental animals):

For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Clinical observations were performed at least once daily for all pups.
Live pups were weighed individually on PND 1, 4, 7 and 13. Sex was externally determined for all pups on PND 1 and 4. Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight. All male pups in each litter were examined for the number of areola/nipples on PND 13.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Postmortem examinations (parental animals):

SACRIFICE
- Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
- Scheduled necropsies were conducted on the following days: Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration);
Females which delivered: PND 14-16; Female No. 80 which failed to deliver (with evidence of mating): Post-coitum Day 30.
All males were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0- females were not fasted.

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.
Organs Weighed at Necropsy for all selected animals: Brain, Cervixa, Epididymis, Gland adrenal, Gland coagulation, Gland parathyroid, Gland prostate, Gland seminal vesicle, Gland thyroid, Heart, Kidney, Liver, Ovaries, Spleen, Testes, Thymus, Uterus
Organs Weighed at Necropsy for all remaining animals (incl. Males that failed to sire and females that failed to deliver pups): Epididymis, Gland Coagulationa, Gland parathyroidc, Gland prostate, Gland seminal vesicle, Gland thyroid, Testes

- Representative samples of the tissues identified in the tables below were collected from all animals and preserved in 10 % neutral buffered formalin (neutral phosphate buffered 4 % formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

- Table 1: Tissue Collection and Preservation for all selected animals, and all animals that died spontaneously or were sacrificed in extremis: Animal identification, artery, aorta, body cavity, nasopharynx, bone marrow, bone (femur and sternum), brain (seven levels), cervix, epididymis, esophagus, eye, adrenal gland, coagulation gland, harderian gland, lacrimal gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (cecum, colon, rectum), larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, pancreas, skin, small intestine (duodenum, ileum, jejunum), spinal cord,spleen, stomach, testes, thymus, tongue, trachea, urinary bladder, uterus, vagina.

- Table 2: Tissue Collection and Preservation for all remaining animals (incl. males that failed to sire females that failed to deliver pups; non-pregnant, implantation sites only):
Animal identification, cervix, epididymis, coagulation gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, seminal vesicle gland, thyroid gland, gross lesions/masses, ovaries, testes, uterus, vagina.

Postmortem examinations (offspring):

METHOD OF EUTHANASIA
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20 %).
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

UNSCHEDULED AND SCHEDULED DEATHS
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.
All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (one male and one female pup) was preserved in 10 % buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

Statistics:

All statistical tests were conducted at the 5 % significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1 % and 5 % levels.

Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1 and Group 4 vs. Group 1.

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.

Incidence: An overall Fisher’s exact test was used to compare all groups at the 5 % significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating index (%): Number of females mated / Number of females paired x 100

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): Number of pregnant females / Number of females mated x 100

Gestation index (%): Number of females with living pups on Day 1 / Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%): Total number of offspring born / Total number of uterine implantation sites x 100

Live birth index (%): Number of live offspring on Day 1 after littering / Total number of offspring born x 100

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check / Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check / Number of live pups at First Litter Check x 100

Viability index (%): Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering x 100

Lactation index (%): Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Any clinical signs noted during the treatment period (e.g. swelling and dark eye, salivation, scabs, wounds, rales and alopecia) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female of the control group (No. 50) was found dead in her cage prior to dosing on Day 32 of treatment (Day 14 post-coitum). The main cause of death was marked erosion/ulceration of the trachea
epithelium indicating a gavage-related incident. Moreover, one female treated at 300 mg/kg/day (Female No. 67) was sacrificed in extremis, on Day 39 of treatment (Day 23 post-coitum), because of difficulties during delivery.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In females, the slightly increased white blood cell, neutrophil and lymphocyte counts in females at 1000 mg/kg/day (1.29x, 1.43x and 1.17x of control, not statistically significant). The mean values were
within the range of historical control data and were considered not toxicologically relevant due to the minimal magnitude of the change.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following changes distinguished treated from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
- Decreased total bilirubin at 100, 300 and 1000 mg/kg/day in males (0.87x, 0.87x and 0.83x of
control, respectively).
- Decreased bile acids at 1000 mg/kg/day in males (0.57x of control, not statistically significant).
- Decreased calcium at 100 and 1000 mg/kg in males (0.97 and 0.97x of control).
- Decreased inorganic phosphate at 1000 mg/kg/day in females (0.52x of control, not statistically significant).
These and few other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as the mean values of these parameters gen
erally remained within the range of historical control data , occurred in the absence of a dose-related response and/or as the opposite effect would be expected in case of organ toxicity.

Thyroid hormone analyses: Serum levels of T4 in F0 males were considered not affected by
treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined males up to 1000 mg/kg/day. Grip strength was similar between all groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or
histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.
All females had regular cycles of 4 days during the pre-test period. During the premating period, an irregular cycle was noted for one female at 100 mg/kg/day (No. 52), two females at 300 mg/kg/day (Nos. 66 and 67) and one female at 1000 mg/kg/day (No. 79). All these females with an irregular cycle had a normal litter, with the exception of Female No. 67 that was sacrificed in extremis on Day 23 post coitum due to difficulties during parturition. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating index not affected by treatment. All paired females showed evidence of mating.
Precoital time was considered not affected by treatment. All paired females showed evidence of mating within 4 days.
Number of implantation sites was considered not affected by treatment.
Fertility index was considered not affected by treatment. The fertility indices were 90 % for the control group and 100%, 100% and 90% for the 100, 300 and 1000 mg/kg bw/day groups, respectively.
One female of the control group (No. 48) and one female of the 1000 mg/kg group (No. 80) turned out to be not pregnant. These cases of non-pregnancy were regarded as unrelated to treatment with the test item as they occurred incidentally and without related histopathology changes in reproductive organs or decrease in number of implantation sites.

Details on results (P0)

- Gestation index and duration of gestation were considered not affected by treatment with the test item.
- All pregnant females had live offspring, except for two females of the control group, of which one female had implantation sites only (No. 42) and one female was found dead on Day 14 post-coitum (No. 50). Additionally, one female treated at 100 mg/kg/day (No. 58) appeared to have a dead fetus in the uterus at necropsy and one female treated at 300 mg/kg/day (No. 67) was sacrificed in extremis due to delivery difficulties (the uterus contained 2 dead fetuses and the cervix contained 11 dead fetuses).
- The failed pregnancies of Female Nos. 42 and 58 occurred without related histopathology changes in reproductive organs and were judged to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.
- The relatively lower gestation indices of the control and 300 mg/kg/day treated females were caused by the pregnant females that died during the post-coitum period.
- Except for one female treated at 300 mg/kg/day (No. 67), which was sacrificed in extremis due to difficulties during parturition, no other signs of difficult or prolonged parturition were noted among the pregnant females.
- Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.
- Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 77, 91, 81 and 94% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
- The relatively lower post-implantation survival indices of the control and 300 mg/kg/day treated females were caused by the pregnant females that died during the post-coitum period.
- Litter size was considered not affected by treatment with the test item up to 1000 mg/kg/day. Live litter sizes were 11.3, 11.1, 12.3 and 12.3 living fetuses/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
- The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment with the test item up to 1000 mg/kg/day.
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was 95, 99, 100 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
Four pups of the control group (Litter No. 44) and one pup at 100 mg/kg/day (Litter No. 54) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
- Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item. Viability indices were 99, 100, 98 and 99% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
One pup of the control group (Litter No. 43), two pups of the 300 mg/kg/day (Litter No. 69) and one pup of the 1000 mg/kg/day group (Litter No. 78) were missing or found dead on PND 2 or 3. Missing pups were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
- The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment with the test item.
The lactation indices were 100, 99, 100 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
One pup at 100 mg/kg/day (Litter No. 60) was found missing on PND 9, which was most likely cannibalised. No toxicological relevance was attributed to this missing pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

System:

other: not applicable

Organ:

other: not applicable

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item.
The nature and incidence of clinical signs observed remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.
The statistically significantly lower mean normalized anogenital distance of male pups at 300 mg/kg/day occurred without a dose related-trend. These variations were considered not to be related to treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 1000 mg/kg/day/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- Sex ratio
Sex ratio was considered not to be affected by treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

System:

other: not applicable

Organ:

other: not applicable

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Executive summary:

The objectives of this study were to determine the potential toxic effects of the test substance when given orally by gavage for > 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

Ten male and ten female Wistar Han rats per group were treated by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg according to OECD TG 422. The rats of the control group received the vehicle propylene glycol alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination. Females that delivered were treated for 51-55 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13 to15 days after delivery, up to and including the day before scheduled necropsy.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels for the purpose of this study.

Parental results:

There were two unscheduled deaths, both not test item-related.  One control female died on Day 32 due to a gavage-related incident and one female at 300 mg/kg/day was sacrificed in extremis on Day 39 due to parturition difficulties.

No test item-related changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption, clinical laboratory investigations (hematology, clotting parameters, clinical biochemistry (including male T4 thyroid hormone levels)), macroscopic examination, organ weights, and microscopic examination).

Reproductive results:

No test item-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results:

No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of 1,12-Dodecanediol were established:

Parental NOAEL:       at least 1000 mg/kg/day

Reproduction NOAEL:       at least 1000 mg/kg/day

Developmental NOAEL:       at least 1000 mg/kg/day