2-(2-(2-ethoxyethoxy)ethoxy)ethanol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 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:

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: 210 - 224 g for males and 179 - 199 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: 27 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: February 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 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.

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: 35/36 days. Females 53-65 days.

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: 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

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: Not specified
- How many animals: Not specified

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- 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.

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):

One high dose female was sacrificed for humane reasons on the day of start of parturition (Day 23 post coitum) due to the difficulties with delivery. Pale appearance and red staining of the vagina were observed prior to sacrifice. Macroscopically, pale color of liver and red staining of the urogenital area were observed. At microscopic observation, inflammation and congestion of uterus and atrophy of thymus were noted. The factors contributory to the illness status of this animal were ascribed to difficulty in delivery and unrelated to treatment.

Body weight and weight changes:

no effects observed

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):

A statistically significant increase of monocytes recorded in mid dose males (300mg/kg/day) was not seen in the top dose group and, since ithre was no dose response relationship, was considered to be incidental.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cholesterol was higher than controls in females dosed at 1000 mg/kg/day (44%). Since data within groups were homogeneous and a minimal increase of cholesterol was also recorded in females dosed at 300 mg/kg/day (26% above controls, but not reaching statistical significance), a correlation with treatment cannot be excluded. However, due to the slight severity and in the absence of other related changes (histopathological findings and/or other clinical chemistry parameters), the cholesterol increase was not considered to be adverse. An increase of bile acids recorded in females dosed at 1000 mg/kg/day was due to the high value of one animal ). Due to the minimal incidence, this finding was considered to be unrelated to treatment. The only other statistically significant differences between control and treated animals (decrease of alkaline phosphatase and bilirubin and increase of cholesterol in males, increase of globulin in females) were not dose related and/or of minimal severity, therefore they were considered to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant increase in motor activity in the high dose males. However, despite there being a dose response, this outcome was driven by two animals with high activity whilst the comparatively low values seen in the concurrent control were driven by 3 animals. (Counter units: concurrent control 671 (SD 174), historic control 745 (5 year data), group 4: 927 (SD=141). The effects was not seen in females. No treatment-related changes were seen in the sensory reaction to stimuli (including grip strength and landing footsplay) measured at the end of the study in all treated groups of both sexes. The findings were not considered as a result of treatment and not adverse. See table below in 'other information' for individual animal scores.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

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

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No treatment-related changes were recorded in parental males and in pups on Day 14 post partum on the levels of T4 and TSH. Thyroid stimulating hormone (TSH) was increased in two parental males dosed at 300 mg/kg/day. Compared with mean control, changes were 2.4 and 2.5 fold, respectively. Due to the absence of dose-relation and the minimal incidence, this change was considered to be unrelated to treatment

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

Motor activity values recorded for males:

Animal #	Concurrent control	Group 4
1	855	1085
2	456	746
3	840	819
4	638	1024
5	565	959

Executive summary:

In a guideline and GLP OECD422 combined repeat dose and reproductive toxicity study, SD rats were exposed to 2 -(2 -(2 -ethoxyethoxy)ethoxy)ethanol (TEGEE) by drinking water for 35/36 days for males and 53 -65 days for females. The average actual doses received by males were 83, 262 and 901 mg/kgbw/day and by females 122, 376 and 1296 mg/kgbw/day. During the post partum/lactation phase, females were receiving 1714 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.

A treatment-related increase in cholesterol was observed in females dosed at nominal 300 and 1000 mg/kg/day, which only reached statistical significance in the high dose group. This increase was not considered to be adverse due to the slight severity and the absence of other related changes (histopathological findings and/or other clinical chemistry parameters. The NOAEL in the study was determined to be 1000 mg/kgbw/day (actual 906 in males, 1296 in females) , the maximum dose used.

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:

2-(2-(2-ethoxyethoxy)ethoxy)ethanol

EC Number:

203-978-9

EC Name:

2-(2-(2-ethoxyethoxy)ethoxy)ethanol

Cas Number:

112-50-5

Molecular formula:

C8H18O4

IUPAC Name:

2-[2-(2-ethoxyethoxy)ethoxy]ethan-1-ol

impurity 1

Reference substance name:

2,2'-oxydiethanol

EC Number:

203-872-2

EC Name:

2,2'-oxydiethanol

Cas Number:

111-46-6

Molecular formula:

C4H10O3

IUPAC Name:

2,2'-oxydiethanol

Test material form:

liquid

Details on test material:

Other identified impurities, all less than 0.1% include water, triethylene glycol methyl ether and diethylene glycol ethyl ether. Balance of 0.08% 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: 210 - 224 g for males and 179 - 199 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: 27 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: February to April 2021

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity: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 respec
tively) 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: 35/36 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:

Males: 35/36 days. Females 53-65 days.

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 Days 35 and 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):

One high dose female was sacrificed for humane reasons on the day of start of parturition (Day 23 post coitum) due to the difficulties with delivery. Pale appearance and red staining of the vagina were observed prior to sacrifice. Macroscopically, pale color of liver and red staining of the urogenital area were observed. At microscopic observation, inflammation and congestion of uterus and atrophy of thymus were noted. The factors contributory to the illness status of this animal were ascribed to difficulty in delivery and unrelated to treatment.

Body weight and weight changes:

no effects observed

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):

Cholesterol was higher than controls in females dosed at 1000 mg/kg/day (44%). Since data within groups were homogeneous and a minimal increase of cholesterol was also recorded in females dosed at 300 mg/kg/day (26% above controls, but not reaching statistical significance), a correlation with treatment cannot be excluded. However, due to the slight severity and in the absence of other related changes (histopathological findings and/or other clinical chemistry parameters), the cholesterol increase was not considered to be adverse. An increase of bile acids recorded in females dosed at 1000 mg/kg/day was due to the high value of one animal ). Due to the minimal incidence,
this finding was considered to be unrelated to treatment. The only other statistically significant differences between control and treated animals (decrease of alkaline phosphatase and bilirubin and increase of cholesterol in males, increase of globulin in females) were not dose related and/or of minimal severity, therefore they were considered to be incidental.

Urinalysis findings:

not examined

Behaviour (functional 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 concurrent and historic controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

T4 and TSH hormones

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.

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.
Pups sacrificed on Days 4 and 14 post partum: No findings were recorded in pups sacrificed for humane reason due to the sacrifice occurred
in the relative dam. 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 2 -(2 -(2 -ethoxyethoxy)ethoxy)ethanol (TEGEE) by drinking water for 35/36 days for males and 53 -65 days for females. The average actual doses received by males were 83, 262 and 901 mg/kgbw/day and by females 122, 376 and 1296 mg/kgbw/day. During the post partum/lactation phase, females were receiving 1714 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 906 in males, 1296 in females) , the maximum dose used.