3,6,9,12-tetraoxotridecanol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January to April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan-April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016 version

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Species and strain accepted by many regulatory authorities and there are ample experience and background data on them.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: 213 - 225 g for males and 179 - 200 g for females
- Fasting period before study: no
- Housing: From arrival to pairing, 5 of one sex to a cage, in polysulfone
solid bottomed cages (59.5×38×20cm). Nesting material was provided inside suitable bedding bags and changed at least 2x/week. During mating, animals house 1 of each sex, then individually for remainder of study.
twice a week.
- Diet (ad libitum except during fasting period prior to sampling for clinical pathology): laboratory rodent diet (4 RF 21, ex Mucedola S.r.l., Italy)
- Water (ad libitum): drinking water (softened by reverse osmosis)
- Acclimation period: 48 days

DETAILS OF FOOD AND WATER QUALITY:

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

IN-LIFE DATES: January to April 2021

Route of administration:

oral: drinking water

Details on route of administration:

The oral drinking water route was selected as it is a more realistic route of exposure of the test item in man than oral gavage and would allow comparison with other studies on similar substances where this route of exposure has been used previously.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Fresh solution prepared every 6 days. Stability tests previously confirmed solutions were stable for a 8 day period at 2-8 and 50hrs at room temperature..

VEHICLE
- Concentration in vehicle: Concentrations were based on findings from the range finder study and were made up as follows: 1000, 3000 and 10000 ppm for males (low, mid, hiigh dose groups respectively) and 950, 2900 and 9500 ppm for females (low, mid, hiigh dose groups respectively.)
- Purity: information held by laboratory but not included in report.

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 theoretical range from 0.8 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r >
0.99; accuracy 90-110%; precision CV <5%). 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 concentration. The method used was gas chromatography with flame ionisation detection of the analyte.

Duration of treatment / exposure:

Males: 36 days (including two weeks pre-treatment prior to mating). Females 53-65 days, including 2 week pre-treatment prior to mating).

Frequency of treatment:

Continous via drinking water

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

For actual doses received, see table in section 'any other information on methods')

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

For actual doses received, see table in section 'any other information on methods')

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

For actual doses received, see table in section 'any other information on methods')

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a range finder study with non-pregnant animals
- Fasting period before blood sampling for clinical biochemistry: yes, overnight

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, twice daily for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, incljuded as part of functional observation battery: Handling reactivity, Lachrymation, Palpebral closure, Salivation, Piloerection, Rearing, Spasms, Tonic spasms, Clonic spasms, Tonic-clonic spasms, Myoclonia, Mobility impairment, Arousal (animal activity), Vocalisation, Stereotypies, unusual respiratory pattern, Bizarre behaviour, Urination, Tremors, Gait

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: females at sacrifice.
- Anaesthetic used for blood collection: Males: Yes (isoflurane) from retro-orbital sinus except for coagulation tests where vena cava used. Females: No, abdominal vena cava used.
- Animals fasted: Yes (overnight) except for coagulation test
- How many animals: not specified
- Parameters checked: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular hemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocites, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelets, Prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
females at sacrifice.
- Anaesthetic used for blood collection: Males: Yes (isoflurane) from retro-orbital sinus except for coagulation tests where vena cava used. Females: No, abdominal vena cava used.
- Animals fasted: Yes (overnight) except for coagulation test
- How many animals:
not specified
- Parameters checked: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Total bilirubin,Total cholesterol, total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride, Inorganic phosphorus.

PLASMA/SERUM HORMONES-IMMUNOLOGY: Yes - T4 and TSH hormones
- Sampling time 14 days postpartum
- Time of blood sample collection: Termination
- Animals fasted: Yes
- How many animals: 5

URINALYSIS: Yes - 5 randomly selected males
- Time schedule for collection of urine: 24 hours before sacrifice. Immediately before collection animals gavaged with 10mL/kg drinking water
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes / No / Not specified
- Parameters checked : appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood. Sediment obtained by centrifugation (3000rpm, 10mins) examined microscopically for epithelial cells, leucocytes, crystals, spermatozoa and precursos, other abnormal components.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once, towards end of treatment
- Dose groups that were examined: 5 animals from each sex and dose group, randomly selected.
- Battery of functions tested: sensory activity to stimuli, grip strength and motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Following weighed: Adrenal glands, brain, epididymides, heart, kidney, liver, ovaries, prostate, seminal vesicles, spleen, testes, thymus, thyroid, uterus-cervix

HISTOPATHOLOGY: Yes, (5M/5F from high dose and control group randomly selected plus all abnormalities); as above plus: bone marrow, caecum, clitoral gland, colon, duodenum, eyes, femur with knee joint, ileum, jejenum incl Peyer's patches, lngs,lymph nodes (cervical & mesenteric), mammary glands (both), nasal cavity, oesophagus, parathyroid gland, penis, rectum, sciatic nerve, spinal cord, skeletal muscle, stomach, trachea, urinary bladder, vagina.

Optional endpoint(s):

Samples of blood om all parental males from all groups (40 samples) obtained at sacrifice were assayed to determine the serum levels of Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA).

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. 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. Statistical significance set at p<0.05.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two males (one each at 100 and 300mg/kg/day) died during the study and one female (dosed at 1000mg/kg/day) was sacrificed for human reason during gestation period. The former were found dead on Day 18 of the mating phase (corresponding to 32 days of treatment). These animals showed red staining on the muzzle before their death. Macroscopically, single red area of left lobe lungs and red staining of muzzle were observed in both low and mid-dose males. At microscopic observation, marked alveolar haemorrhage of the lungs was noted. The cause of death was considered to be misgavage and not treatment-related. The high dose female was sacrificed for humane reasons on Day 24 of the gestation phase. No clinical signs were recorded on this female before being sacrificed. No abnormalities were detected at gross observation. At microscopic evaluation, minimal chronic inflammation of the submucosa of the oesophagus was observed. On the basis of the in vivo signs such as prolonged time without completing the parturition, impaired parturition was considered to be the reason for sacrifice. Neither gross and microscopic evaluationdcould establish the cause of impaired parturition.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Minimal, but statiistically significant reductions in mean body weights and body weight gain were seen in mid dose group females receiving 300mg/kg/day from Day 1 to Day 7 of the post partum period. However, these changes were followed by regular increment and re-alignment to control group mean values at the end of the study. As they were not seen in the high dose group, they were not attributed to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences between control and treated animals (calcium in low dose males, reduced by 5%) and alkaline phosphatase in mid-dose females, increased by 1.8x) were not dose related, therefore they were considered to be incidental.

Endocrine findings:

no effects observed

Description (incidence and severity):

No changes were recorded in parental males and in pups on Day 14 post partum.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Single animals in each treatment group showed a slightly higher click and/or tail pinch response during the sensory reactivity evaluations. However, these alterations were not
considered of toxicological significance sincehey were randomly distributed across groups and without any correlation with the administration of the test item. Otherwise, there were no alterations in motor activity between groups at the examination performed at the end of treatment. Observation of animals at removal from the cage and in an open arena (neurotoxicity
assessment) did not reveal changes attributable to the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in terminal body weight and organ weights when compared to the controls that were considered adverse. An increase in absolute and relative mean kidney weights in high dose treated males (13% for relative kidneys weight) was not correlated to any histopathological changes, and was in the range of expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to
treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered
incidental and unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Please note that this robust study summary is based on the draft audited report of the study and not the final issued report.

Executive summary:

In a guideline and GLP OECD422 combined repeat dose and reproductive toxicity study, male and female SD rats were exposed to 3,6,9,12 -tetraoxotridecan-1 -ol (TetraEGME) by drinking water for 36 days for males and 53 -65 days for females. The average actual doses received by males were 70, 222 and 688 mg/kgbw/day and by females 126, 393 and 1249 mg/kgbw/day. During the post partum/lactation phase, high dose females were receiving 1692 mg/kgbw/day. No effects were seen in any dose group and for either sex that were deemed to be adverse and attributable to treatment.

The NOAEL in the study was determined to be the nominal limit dose of 1000 mg/kgbw/day, the maximum dose tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016 version

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,6,9,12-tetraoxotridecanol

EC Number:

245-883-5

EC Name:

3,6,9,12-tetraoxotridecanol

Cas Number:

23783-42-8

Molecular formula:

C9H20O5

IUPAC Name:

2,5,8,11-tetraoxatridecan-13-ol

impurity 1

Reference substance name:

2,2'-(ethylenedioxy)diethanol

EC Number:

203-953-2

EC Name:

2,2'-(ethylenedioxy)diethanol

Cas Number:

112-27-6

Molecular formula:

C6H14O4

IUPAC Name:

2,2'-[ethane-1,2-diylbis(oxy)]diethanol

impurity 2

Reference substance name:

3,6,9,12-tetraoxatetradecan-1-ol

EC Number:

227-090-6

EC Name:

3,6,9,12-tetraoxatetradecan-1-ol

Cas Number:

5650-20-4

Molecular formula:

C10H22O5

IUPAC Name:

3,6,9,12-tetraoxatetradecan-1-ol

impurity 3

Reference substance name:

2-(2-(2-methoxyethoxy)ethoxy)ethanol

EC Number:

203-962-1

EC Name:

2-(2-(2-methoxyethoxy)ethoxy)ethanol

Cas Number:

112-35-6

Molecular formula:

C7H16O4

IUPAC Name:

2-[2-(2-methoxyethoxy)ethoxy]ethanol

Test material form:

liquid

Details on test material:

Other identified impurities, all less than 0.1% include water and diethylene glycol methyl ether. Balance of 0.14% not identified.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Species and strain accepted by many regulatory authorities and there are ample experience and
background data on them.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: 213 - 225 g for males and 179 - 200 g for females
- Fasting period before study: no
- Housing: From arrival to pairing, 5 of one sex to a cage, in polysulfone
solid bottomed cages (59.5×38×20cm). Nesting material was provided inside suitable bedding bags
and changed at least 2x/week. During mating, animals house 1 of each sex, then individually for r
emainder of study.
twice a week.
- Diet (ad libitum except during fasting period prior to sampling for clinical pathology): laboratory
rodent diet (4 RF 21, ex Mucedola S.r.l., Italy)
- Water (ad libitum): drinking water (softened by reverse osmosis)
- Acclimation period: 48 days

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

IN-LIFE DATES: January to April 2021

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Fresh solution prepared every 6 days. Stability tests pr
eviously confirmed solutions were stable for a 7 day period.

VEHICLE
- Concentration in vehicle: Concentrations were based on findings from the range finder study and were made up as follows: 1000, 3000 and 10000 ppm for males (low, mid, hiigh dose groups respectively) and 950, 2900 and 9500 ppm for females (low, mid, hiigh dose groups respectively.)
- Purity: information held by laboratory but not included in report.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug in situ or in tray or sperm in vaginal smear (referred to as [day 0 / day 1] of pregnancy
- After14 days of unsuccessful pairing replacement the study was continued with the non-pregnant female for the repeat dose element of the study, with the female sacrified 28 days later.
- Further matings not performed (only affected one pair in high dose group.)
- After successful mating each pregnant female was caged individually.
- Any other deviations from standard protocol: no

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 theoretical range from 0.8 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 90-110%; precision CV <5%). 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 concentration. The method used was gas chromatography with flame ionisation detection of the analyte.

Duration of treatment / exposure:

Duration of treatment / exposure
Males: 36 days. Females 53-65 days.

Frequency of treatment:

Continous via drinking water

Details on study schedule:

Further information not required for a screening study

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

For actual doses received, see table in section 'any other information on methods')

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

For actual doses received, see table in section 'any other information on methods')

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

For actual doses received, see table in section 'any other information on methods')

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a range finder study with non-pregnant animals

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, twice daily for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, incljuded as part of functional observation battery: Handling reactivity,
Lachrymation, Palpebral closure, Salivation, Piloerection, Rearing, Spasms, Tonic spasms, Clonic sp
asms, Tonic-clonic spasms, Myoclonia, Mobility impairment, Arousal (animal activity), Vocalisation,
Stereotypies, unusual respiratory pattern, Bizarre behaviour, Urination, Tremors, Gait

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OTHER: A parturition check was performed from Day 20 to Day 25 post coitum. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth when the parturition was defined complete (Day 0 post partum).

Oestrous cyclicity (parental animals):

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. For 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 identification of the stages of the spermatogenic cycle was also performed in five randomly selected males of the control and high dose groups as part of the histopathology. 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. 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. The PAS- H stained sections were used to identify the spermatogenic stages. Otherwise no other sperm parameters were assessed.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, 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 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities, anogenital distance (AGD - post partum day 1 normalised to cube root of bodyweight measured on same day), presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS: Pups dying during the lactation period were weighed before the despatch to necropsy. All pups found dead in the cage were examined for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals[after the completion of the mating period on Day 36 of the study.
- Maternal animals: All surviving animals day 14 post partum

GROSS PATHOLOGY: Yes. Following weighed: Adrenal glands, brain, epididymides, heart, kidney, liver, ovaries, prostate, seminal vesicles, spleen, testes, thymus, thyroid, uterus-cervix. Parental females were examined also for the number of visible implantation sites (pregnant animals) and the number of corpora lutea (pregnant animals). Uteri of apparently non-pregnant females were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

HISTOPATHOLOGY: Yes, as above plus (5M/5F from high dose and control group randomly selected plus all abnormalities): bone marrow, caecum, clitoral gland, colon, duodenum, eyes, femur with knee joint, ileum, jejenum incl Peyer's patches, lngs,lymph nodes (cervical & mesenteric), mammary glands (both), nasal cavity, oesophagus, parathyroid gland, penis, rectum, sciatic nerve, spinal cord, skeletal muscle, stomach, trachea, urinary bladder, vagina.

Postmortem examinations (offspring):

SACRIFICE
- day 4 and 14 post partum
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- 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. 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
- not examined

Statistics:

Standard deviations were calculated as appropriate. The non-parametric Kruskal-Wallis analysis of variance was used for the non-continuous parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. Statistical significance set at p<0.05.

Reproductive indices:

Males: Copulatory index (# with confirmed mating/number cohabited), Fertility index (# which produced pregnancy/number cohabited).
Females: Copulatory index (# with confirmed mating/number cohabited), Fertility index (# pregnant/number cohabited).
Both sexes: Pre coital Interval = number of nights paired prior to detection of mating.
Pre-implantation loss, pre-natal loss, sex ratios (days 0, 4, 13)

Offspring viability indices:

Post-natal loss (days 0, 4, 13)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two males (one each at 100 and 300mg/kg/day) died during the study and one female (dosed at 1000mg/kg/day) was sacrificed for human reason during gestation period. The former were found dead on Day 18 of the mating phase (corresponding to 32 days of treatment). These animals showed
red staining on the muzzle before their death. Macroscopically, single red area of left lobe lungs and red staining of muzzle were observed in both low and mid-dose males. At microscopic observation, marked alveolar haemorrhage of the lungs was noted. The cause of death was considered to be mi
sgavage and not treatment-related. The high dose female was sacrificed for humane reasons on Day 24 of the gestation phase. No clinical signs were recorded on this female before being sacrificed. No abnormalities were detected at gross observation. At microscopic evaluation, minimal chronic infla
mmation of the submucosa of the oesophagus was observed. On the basis of the in vivo signs such as prolonged time without completing the parturition, impaired parturition was considered to be the reason for sacrifice. Neither gross and microscopic evaluationdcould establish the cause of impaired parturition.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Minimal, but statiistically significant reductions in mean body weights and body weight gain were seen in mid dose group females receiving 300mg/kg/day from Day 1 to Day 7 of the post partum period. However, these changes were followed by regular increment and re-alignment to control grou
p mean values at the end of the study. As they were not seen in the high dose group, they were not attributed to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in terminal body weight and organ weights hat were considered adverse when compared to the controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant differences between control and treated animals (calcium in low dose males, reduced by 5%) and alkaline phosphatase in mid-dose females, increased by 1.8x) were not dose related, therefore they were considered to be incidental.

Endocrine findings:

no effects observed

Description (incidence and severity):

No changes were recorded in parental males and in pups on Day 14 post partum in T4 and TSH hormones

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Single animals in each treatment group showed a slightly higher click and/or tail pinch response during the sensory reactivity evaluations. However, these alterations were not considered of toxicological significance sincehey were randomly distributed across groups and without any correlation with the administration of the test item. Otherwise, there were no alterations in motor activity between groups at the examination performed at the end of treatment. Observation of a nimals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

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):

Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Based on a lack of effects seen following histopathology of the testes and epididymides.

Reproductive performance:

no effects observed

Description (incidence and severity):

Gestation length was similar between treated and control groups. No treatment related effectswere seen in the number of implantations or in pre-implantation and pre-natal loss in all groups.

Details on results (P0)

Two females of the control group were found not pregnant at necropsy. One female dosed at 1000mg/kg/day showed unilateral total resorption. In addition, one female dosed at 1000mg/kg/day was sacrificed for humane reason due to to difficulty in delivery (dystocia). The number of females with live pups on Day 14 post partum was: 8 in the control, 10 in the low and mid-dose, 8 in the high dose group. The number of copulatory plugs were similar between groups.

Effect levels (P0)

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No adverse effects seen up to and including the limit dose of 1000 mg/kgbw/day

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Any pre-weaning clinical signs observed were comparable between low, mid-, high dose and control groups or considered incidental.

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

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:

no effects observed

Description (incidence and severity):

Sex ratios at birth, on Days 4 and 14 post partum did not show treatment-related effects, when calculated as percentage of males.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related differences were noted in thyroid weight between control and pups of treated groups. No other organs weighed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Deceased pups: No macroscopic examination could be performed, since autolysed abdominal organs were observed in the majority of the deceased pups of control and treated groups. No abnormalities were noted in the other deceased pups at necropsy examination. No findings were recorded in pups sacrificed on Day 4 post partum (culled pups) or Day 14 post partum.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Litter data of treated females (e.g. total litter size, live litter size, pup loss, litter weight and mean pup weight) did not show dose-related or treatment-related differences.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEC

Generation:

F1

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No adverse effects seen up to and including the limit dose of 1000 mg/kgbw/day

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Executive summary:

In a guideline and GLP OECD422 combined repeat dose and reproductive toxicity study, SD rats were exposed to 3,6,9,12-tetraoxatetradecan-1-ol / 3,6,9,12-tetraoxatetradecan-1-ol (TetraEGME) by drinking water for 36 days for males and 53 -65 days for females. The average actual doses received by males were 70, 222 and 688 mg/kgbw/day and by females 126, 393 and 1249 mg/kgbw/day. During the post partum/lactation phase, females were receiving 1692 mg/kgbw/day. No reproductive effects were seen in any dose group and for either sex that were deemed to be adverse and attributable to treatment. The NOAEL in the study was determined to be 1000 mg/kgbw/day (actual 688 in males, 1249 in females) , the maximum dose used.