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Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 9, 2021 to May 9, 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD TG compliant GLP study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2018
Deviations:
no
Remarks:
There were no deviations from the OECD TG. However, between Day 60-63, slightly higher volume of doses were administered. The deviations were minor. No effect on study interpretation.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Sponsor
- lot/batch number of test material: 1B25AB
- Purity: 99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool and dry (+2 to +8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: dose formulations at the dose of 5 mg/mL and 100 mg/mL are stable for 6 hours and 48 hours at room temperature (reference: Study No.: BIO-ANM 1807)


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is one of the recommended species by regulatory agencies for conducting toxicity assessment studies among rodents.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals (Bioneeds)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Housing: Two animals of same sex were housed in a standard polysulphonate cage (size: L 43 x B 28 x H 21 cms) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The contaminant analysis test report for food, water and bedding material nearest to the experiment period are included in the report.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.6°C to 22.9°C
- Humidity: 47% to 65%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered through oral (gavage) route using stainless steel cannula as it is the probable route in human.
Vehicle:
water
Remarks:
Distilled
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed in a beaker and small quantity of vehicle was added and mixed with a glass rod. Thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the beaker and this was transferred into the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.


VEHICLE
- Concentration in vehicle: 0, 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): 7-23, JA072
- Purity: not relevant
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis for concentration verification was done by Analytical Chemistry Department of Bioneeds India Private Limited as per validated analytical methods detailed in the Study No. BIO-ANM 1807.

Formulation analysis for concentration verification was performed for all the dose formulations on day 1 (or prior to first dosing) and during month 2 and month 3 of the treatment. The prepared formulations were sampled in duplicate sets from each dose level of prepared formulations including vehicle control.. Exact volumes of test item formulation samples were included in study report.

The collected samples were transferred to Analytical Chemistry Department of Bioneeds India Private Limited for dose formulation analysis at established stability conditions. One set of aliquot of each formulation was analysed.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once/day
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Formulations are considered acceptable, mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is ≤10.0%.
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Formulations are considered acceptable, mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is ≤10.0%.
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Formulations are considered acceptable, mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is ≤10.0%.
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Formulations are considered acceptable, mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is ≤10.0%.
No. of animals per sex per dose:
10 (Recovery groups (5/sex additional))
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A dose range finding study (study no. BIO-CTX 401) was conducted with the dose levels of 100, 300 and 1000 mg/kg/day. The results of the study indicated that there were no adverse changes in any of the parameters up to the dose levels of 1000 mg/kg/day. Hence, in the current study, same dose levels of 100, 300 and 1000 mg/kg/day were selected as low (G2), mid (G3) and high (G4/G4R) doses.

- Fasting period before blood sampling for clinical biochemistry:
10-12 hours before blood collection


- Rationale for selecting satellite groups: recovery groups


- Post-exposure recovery period in satellite groups: 28 days post exposure


- Dose range finding studies:
A dose range finding study (study no. BIO-CTX 401) was conducted with the dose levels of 100, 300 and 1000 mg/kg/day. The results of the study indicated that there were no adverse changes in any of the parameters up to the dose levels of 1000 mg/kg/day.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before initiation of the treatment and at weekly intervals thereafter during the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 before test item administration and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during Week 13 for vehicle control and high dose main group animals and during Week 17 for recovery group animals. No treatment related ocular changes were observed in high dose group animals. Hence, the examination was not extended to lower dose group animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 91 and 119 for main and recovery groups, respectively.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- Parameters checked: See attachments

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: same as hematology
- Animals fasted: same as hematolog
- Parameters checked: See attachments

PLASMA/SERUM HORMONES/LIPIDS: Yes (T4, T3, TSH)
- Time of blood sample collection: at termination
- Animals fasted: Not specific

URINALYSIS: Yes
- Time schedule for collection of urine: Day 91 and 119 (main and recovery, respectively)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided)
- Parameters checked: see attachment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 13 for vehicle and high dose main group; Week 17 for recovery group.
- Dose groups that were examined: See above
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
The data was subjected to statistical analysis. The computer printout of the data (in the form of appendix) was verified with the original raw data. After verification, the data was subjected to statistical analysis using SPSS software version 27. Body weight, percent change in body weight (with respect to Day 1 body weight), feed consumption, organ weights and ratios, hematological and clinical chemistry estimations and urine analysis parameters (pH, specific gravity and urobilinogen), FOB parameters were subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment and recovery groups comparing with the respective vehicle control group data. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). Statistically significant changes obtained from the aforementioned tests was designated by the superscripts throughout the report as stated below:
*: Statistically significant (P<0.05)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
statistically significant lower percent change in body weight with respect to day 1 was noted during days 1-8 (G4RF). The noted variation is considered incidental in the absence of change in body weights and no concomitant variation in feed consumption was noted.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
statistically (p<0.05) significant higher feed consumption during week 4 to week 6 in G2 females was noted. The noted variation is considered incidental as the noted changes were inconsistent and there were no concomitant variations in body weights.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
In main group males, increase in RBC (Erythrocyte count) in G2 (+6.4%), percentage eosinophils in G3: (+90.2%) and decrease in MCHC (Mean corpuscular hemoglobin concentration) in G4: (-3.9%), decrease in absolute neutrophils in
G4: (-51.6%) and decrease in APTT (Activated Partial Thromboplastin Time) in
G2: (-20.5%); G3: (-23.0%); G4: (-27.7%); In females decrease in percent reticulocyte count in G4: (-29.3%), absolute reticulocyte count in G4: (-29.6%) and increase in percentage monocytes in G2: (+54.9%); G4: (+58.0%) and increase in APTT (Activated Partial Thromboplastin Time) in G2: (+19.8%) were noted.
Decrease in APTT in males is toxicologically not significant. All the other noted variations are considered incidental as they lack dose dependency and/or the significance were minimal in nature and random in occurance.
In recovery males, decrease in absolute neutrophils in G4R: (-29.3%) was noted and in recovery females, increase in percentage basophils in G4R: (+46.2%) and decrease in MCV (Mean corpuscular volume) in G4R: (-7.9%) and decrease in MCH (Mean corpuscular haemoglobin) in G4R:(-8.1%) was noted. All the noted variations in recovery groups are also considered incidental as similar incidences were not noted at the end of treatment period and differ between sexes. Further, the variations noted could be due to random biological variation.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
In main group males, increase in calcium G4: (+5.1%), albumin G4: (+6.6%), albumin globulin ratio G3: (+11.3%), chloride G3: (+1.6%); G4: (+1.7%) and potassium G4: (+7.4%); in females increase in total protein G2: (+5.7%); G3: (+6.9%), albumin G3: (+6.8%), globulin G2:(+9.2%), urea in G3: (+19.8%), LDL (Low Density Lipoprotein) in G4: (+34.3%) and BUN (Blood Urea Nitrogen) in G3: (+19.8%) and decrease in creatinine in G4: (-8.2%) and calcium in G4: (-5.3%) was noted. All changes are considered incidental because of their minimal nature and also few varaitions lack dose dependency and some were not associated with any microscopic changes in associated organs.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
in main group males statistically significant increase in specific gravity in (G4) and decrease in pH in G4 was noted which is considered incidental in the absence of any microscopic changes in kidneys. In females decrease in pH in (G3), increase in specific gravity in (G3), was noted which is considered incidental as it lacks dose dependency.
In recovery males, statistically significant increase in specific gravity in (G4R), was noted which is considered incidental because of its isolated occurrence and no such variation was noted in other sex.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
statistically significant increase in movement count in (G4 males) was noted. In the absence of any adverse changes in any of the other neuromuscular parameters, the noted changes are considered incidental and also there were no clinical signs observed during the experimental period that could be attributed to the variations noted.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Relative organ weight: In main group females, decrease in weight of lungs in G3 was noted.
Absolute organ weight: In recovery females, increase in absolute weight of brain in G4R was noted.
Relative organ weight: In recovery males, increase in weight of brain in G4R and increase in weight of liver in G4R was noted and in recovery females increase in weight of brain in G4R was noted.
The variations noted were not associated with any macroscopic changes and hence considered incidental.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Few microscopic findings observed in the study such as ultimobranchial cyst/s in thyroid gland, epithelial cyst/s in thymus, ectopic adrenocortical tissue in adrenal gland and all other findings were considered incidental as they occurred randomly across the groups including concurrent controls and/or were expected for laboratory rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No treatment related changes in thyroid hormones (T3, T4 and TSH) were noted. In females decrease in T3 in G4F was noted. In recovery males decrease in T3 in G4RM and in recovery females increase in TSH in G4RF were noted. These variations are considered incidental as there were no macroscopic changes in noted groups and also no microscopic changes in high dose main groups.


Vaginal smear examination revealed normal stages of estrous cyclicity both in main and recovery females.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No adverse effects observed at the highest dose tested.
Key result
Critical effects observed:
no
System:
other: No adverse effects were observed in this study

See attachment titled 'Summary_Tables' for all results in a summary form.

Conclusions:
Based on the observed results and experimental conditions used in the study, the repeated oral administration of test item, DI ETHYL DI GLYCOL (DEDG) to Sprague Dawley rats for 90 consecutive days, did not result in any changes in growth, feed consumption, ocular changes, organ weights and gross or histopathological changes. Hence, the No Observed Adverse Effect Level (NOAEL) of the test item, DI ETHYL DI GLYCOL was found to be 1000 mg/kg/day when administered for a period of 90 consecutive days repeatedly by oral (gavage) route to Sprague Dawley rats under the experimental conditions and the doses employed.
Executive summary:

The objective of this study was to assess the toxic potential of the test item, DI ETHYL DI GLYCOL (DEDG) when administered for a period of 90 consecutive days repeatedly by oral (gavage) to Sprague Dawley rats. This study provides information on major toxic effects, target organs, possibility of cumulative effects and also to assess the reversibility of effects (after 28 days recovery period) and an estimate of the No Observed Adverse Effect Level (NOAEL).


A total of 100 Sprague Dawley rats (50 males + 50 females) were distributed to 4 main and 2 recovery groups. Each main group (G1 and G4) consisted of 10 rats/sex and each recovery group (G1R and G4R) consisted of 5 rats/sex. The animals allocated to groups G2, G3, G4/G4R were administered with oral doses of 100, 300 and 1000 mg/kg body weight/day of DI ETHYL DI GLYCOL. The animals allocated to group G1/G1R received vehicle (distilled water) alone. The vehicle/test item formulations were administered by oral (gavage) route at an equivolume of 10 mL/kg body weight.


The test item formulations were stable for 6 hours and 48 hours at room temperature. Formulation analysis for concentration verification was performed for all the dose formulations prior to initiation of treatment, month 2 and month 3 of the treatment period. As per the results, the dose formulations were within the acceptable limits of ±10% to the nominal concentrations with less than 10%. All animals were observed once daily for clinical signs and twice daily for mortality; weekly for detailed clinical examination, weekly body weight and feed consumption; ophthalmoscopic examination during week 13 for control and high dose main groups (G1 and G4) and during week 17 for recovery groups (G1R and G4R). Neurological/Functional observational battery during week 13 for control and high dose main group animals (G1 and G4) and during week 17 for recovery group animals (G1R and G4R).


At the end of treatment and recovery period (day 91 for main groups and day 119 for recovery group), blood and urine samples were collected and analysed. Subsequently, the animals were sacrificed and subjected to gross pathological examination and the organs were collected, weighed and preserved. The tissues/organs in vehicle control (G1) and high dose (G4) group animals were subjected to histopathological examination.


No treatment related clinical signs of toxicity or mortality was observed in any of the doses tested. No treatment related changes in body weight, percent change in body weight with respect to day 1, feed consumption, functional/neurological examination and ophthalmoscopic examination were noted. No adverse treatment related changes were noted in haematology, clinical chemistry, coagulation and urinalysis parameters. There were no test item related significant variations noted in any of the thyroid hormones assessed (T3, T4 and TSH) when compared with vehicle control group.


There were no gross pathological changes noted in any of the rats. There were no test item related histopathological changes noted.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August 2012 - 11 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted in accordance with OECD guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 53 to 60 days
- Weight at study initiation: 259 to 322 g for males and 190 to 250 g for females
- Fasting period before study:
- Housing: Animals were housed inside a restricted access rodent facility. The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. Before the study the room was cleaned and disinfected.
The animals were housed five of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid. Softwood bark-free fibre was used as bedding and was sterilised by autoclaving and changed at appropriate intervals each week. Cages, food
hoppers and water bottles were changed at appropriate intervals.
- Diet (e.g. ad libitum): The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1
Maintenance Diet), except overnight before routine blood sampling and during exposure. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes, except during exposure.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40-70
- Air changes (per hr): Each animal room was supplied with filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light):12/12

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each exposure system consisted of a stainless steel concentric jet atomiser and a snout only ADG exposure chamber. Different doses of DEDG were achieved by varying the concentrations of the test article in the system whilst keeping the duration of exposure constant.
- System of generating particulates/aerosols: Aerosol produced by a stainless steel concentric jet atomiser A narrow bore needle was used
The atomiser was mounted vertically down into the top of the prechamber
- Temperature: The chamber temperatures were similar for each group on each day of the study. The observed temperatures for all groups remained within the normally accepted range for inhalation exposure of rats, as per OECD Guidelines for the Testing of Chemicals Number 412 (22±3°C)
- Air flow rate: Inlet: From in-house compressed air system – breathing quality
Atomiser – 8 L/minute
Diluent – 10L/min
- Extract Airflow:
Drawn by in-house vacuum system
Filtered locally
Flow – 20 L/minute




Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours a day, 5 days a week
Remarks:
Doses / Concentrations:
0.25 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.8 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2.5 mg/L
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: The exposure concentrations (0, 0.25, 0.80 or 2.5 mg/L) were selected following completion of a one week inhalation dose range finding study in rats (Huntingdon Life Sciences Study Number: OOE0008), in which no effects were noted at a target exposure concentration of
2.5 mg/L.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Pre-exposure observation
During exposure however, observation severely restricted due to tube restraint
As each animal is returned to its home cage
As late as possible in the working day

In addition, a more detailed weekly physical examination was performed on each animal to monitor general health.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly throughout the treatment and on the day of necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data but the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1), and each week throughout the
treatment period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 4 of treatment (before dosing), blood samples were obtained from all animals
after overnight withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein.
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes (overnight)
- How many animals: all study animals

CLINICAL CHEMISTRY: Yes
At the same time and using the same animals as for peripheral haematology, further blood samples (nominally 0.7 mL) were collected into tubes containing lithium heparin as anticoagulant. All tubes were mechanically agitated for at least two minutes and the sample subsequently centrifuged at 2000 g for 10 minutes in order to separate the plasma. After separation, the plasma was examined in respect of:
Using a Roche P Modular Analyser:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)


URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data


Other examinations:
sperm analysis and sperm morphology
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
blood chemistry
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths on this study.
There were no treatment-related clinical signs observed during the weekly detailed physical examination.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain of males exposed to 2.49 mg/L was lower than the concurrent controls over the 4 weeks of treatment (Days 1-29, 0.85X Control). Review of the individual twice weekly bodyweights, however, did not show any consistent effects and as there were no similar effects on the females and the difference was not statistically significant; the lower bodyweight gain in males is considered unrelated to DEDG.
There were no effects of DEDG in males exposed to 0.257 or 0.686 mg/L or any females given DEDG.

FOOD CONSUMPTION
There were no effects of DEDG on food consumption.

HAEMATOLOGY, BONE MARROW
There were no treatment-related effects on the cellularity and composition of the marrow or the myeloid to erythroid ratio.

BLOOD CHEMISTRY
Group mean cholesterol and triglyceride levels were higher than the concurrent controls in all treated male groups. A similar effect was not apparent in females and there was no dose response. There were no corroborating pathological changes to explain these higher levels,
and as there were no similar effects in females and no dose response, these intergroup differences are considered to be of no toxicological significance.
There were no treatment-related effects on the other blood chemistry parameters investigated.

ORGAN WEIGHTS
Group mean adrenal weights (adjusted for bodyweight) of males exposed to 2.49 mg/L were statistically significantly higher than the concurrent control. There was no similar effect in females. Pathological changes in the adrenals correlated with the increased weights, but the adrenal changes were considered likely to be related to the stress of restraint during the inhalation procedure, unlikely to be related to treatment and not adverse.

Group mean liver weight (adjusted for bodyweight) of males exposed to 2.49 mg/L was statistically significantly higher than the concurrent control, but as there was intergroup variability in this parameter, no changes noted for females, and no corroborating pathological changes in the liver this change is considered to be unrelated to treatment.
Other intergroup differences seen were small and largely inconsistent between groups and sexes. Therefore, these were considered to be due to individual variation in results and not to be related to treatment

GROSS PATHOLOGY
The macroscopic examination performed after 4 weeks of treatment revealed no intergroup differences of note.
The nature and incidence of all findings were consistent with the commonly seen background of macroscopic changes in Sprague Dawley rats at these laboratories

HISTOPATHOLOGY: NON-NEOPLASTIC
No findings were seen that were clearly related to treatment with DEDG.
Minimal or slight generalised hypertrophy of the zona fasciculata was seen in three male animals exposed to 2.49 mg/L. This finding correlated with the higher bodyweight-adjusted group mean adrenal weight, compared with male controls, for males exposed to 2.49 mg/L.

In the right testis, seminiferous tubular vacuolation and or cellular debris, and in the right epididymis, degenerative spermatogenic cells and or cellular debris in the ducts, were seen at higher incidences in control males than in males exposed to 2.49 mg/L. Therefore, these
findings were not related to treatment, but were considered possibly related to the stress associated with the inhalation procedure (Dodd, 1999; Lee, 1993) and correlated with the atypical values reported at sperm analysis for control animals.



Dose descriptor:
NOAEL
Effect level:
2.49 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects up to the highest dose tested..
Critical effects observed:
no

There was a very low level of DEDG detected at analysis of the samples collected from the control group. In 18/23 occasions the identified peak was below the limit of detection, and in the other 5 samples was very low (approximately 25% that of the low dose levels). It was established that there was a low level of DEDG vapour within the animal room, which could have contaminated the external surfaces of the sampler and possibly absorbed into the trapping solvent used in analysis. Animals of the control group were exposed to clean external fresh air in a sealed system and were considered to have no direct contact with this low level of contamination, and this is considered to have no impact on the study integrity.

Conclusions:
DEDG was administered to Sprague Dawley rats by snout only inhalation exposure for 6 hours a day, 5 days a week for 4 weeks at estimated exposure concentrations of 0.257, 0.686 or 2.49 mg/L.
No adverse treatment related effects were apparent in any of the parameters investigated and DEDG was well tolerated over the 4 week treatment period.
The No Observed Adverse Effect Level (NOAEL) was 2.49 mg/L.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 490 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August 2012 - 11 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted in accordance with OECD guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 53 to 60 days
- Weight at study initiation: 259 to 322 g for males and 190 to 250 g for females
- Fasting period before study:
- Housing: Animals were housed inside a restricted access rodent facility. The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. Before the study the room was cleaned and disinfected.
The animals were housed five of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid. Softwood bark-free fibre was used as bedding and was sterilised by autoclaving and changed at appropriate intervals each week. Cages, food
hoppers and water bottles were changed at appropriate intervals.
- Diet (e.g. ad libitum): The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1
Maintenance Diet), except overnight before routine blood sampling and during exposure. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes, except during exposure.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40-70
- Air changes (per hr): Each animal room was supplied with filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light):12/12

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each exposure system consisted of a stainless steel concentric jet atomiser and a snout only ADG exposure chamber. Different doses of DEDG were achieved by varying the concentrations of the test article in the system whilst keeping the duration of exposure constant.
- System of generating particulates/aerosols: Aerosol produced by a stainless steel concentric jet atomiser A narrow bore needle was used
The atomiser was mounted vertically down into the top of the prechamber
- Temperature: The chamber temperatures were similar for each group on each day of the study. The observed temperatures for all groups remained within the normally accepted range for inhalation exposure of rats, as per OECD Guidelines for the Testing of Chemicals Number 412 (22±3°C)
- Air flow rate: Inlet: From in-house compressed air system – breathing quality
Atomiser – 8 L/minute
Diluent – 10L/min
- Extract Airflow:
Drawn by in-house vacuum system
Filtered locally
Flow – 20 L/minute




Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours a day, 5 days a week
Remarks:
Doses / Concentrations:
0.25 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.8 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2.5 mg/L
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: The exposure concentrations (0, 0.25, 0.80 or 2.5 mg/L) were selected following completion of a one week inhalation dose range finding study in rats (Huntingdon Life Sciences Study Number: OOE0008), in which no effects were noted at a target exposure concentration of
2.5 mg/L.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Pre-exposure observation
During exposure however, observation severely restricted due to tube restraint
As each animal is returned to its home cage
As late as possible in the working day

In addition, a more detailed weekly physical examination was performed on each animal to monitor general health.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly throughout the treatment and on the day of necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data but the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1), and each week throughout the
treatment period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 4 of treatment (before dosing), blood samples were obtained from all animals
after overnight withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein.
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes (overnight)
- How many animals: all study animals

CLINICAL CHEMISTRY: Yes
At the same time and using the same animals as for peripheral haematology, further blood samples (nominally 0.7 mL) were collected into tubes containing lithium heparin as anticoagulant. All tubes were mechanically agitated for at least two minutes and the sample subsequently centrifuged at 2000 g for 10 minutes in order to separate the plasma. After separation, the plasma was examined in respect of:
Using a Roche P Modular Analyser:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)


URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data


Other examinations:
sperm analysis and sperm morphology
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
blood chemistry
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths on this study.
There were no treatment-related clinical signs observed during the weekly detailed physical examination.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain of males exposed to 2.49 mg/L was lower than the concurrent controls over the 4 weeks of treatment (Days 1-29, 0.85X Control). Review of the individual twice weekly bodyweights, however, did not show any consistent effects and as there were no similar effects on the females and the difference was not statistically significant; the lower bodyweight gain in males is considered unrelated to DEDG.
There were no effects of DEDG in males exposed to 0.257 or 0.686 mg/L or any females given DEDG.

FOOD CONSUMPTION
There were no effects of DEDG on food consumption.

HAEMATOLOGY, BONE MARROW
There were no treatment-related effects on the cellularity and composition of the marrow or the myeloid to erythroid ratio.

BLOOD CHEMISTRY
Group mean cholesterol and triglyceride levels were higher than the concurrent controls in all treated male groups. A similar effect was not apparent in females and there was no dose response. There were no corroborating pathological changes to explain these higher levels,
and as there were no similar effects in females and no dose response, these intergroup differences are considered to be of no toxicological significance.
There were no treatment-related effects on the other blood chemistry parameters investigated.

ORGAN WEIGHTS
Group mean adrenal weights (adjusted for bodyweight) of males exposed to 2.49 mg/L were statistically significantly higher than the concurrent control. There was no similar effect in females. Pathological changes in the adrenals correlated with the increased weights, but the adrenal changes were considered likely to be related to the stress of restraint during the inhalation procedure, unlikely to be related to treatment and not adverse.

Group mean liver weight (adjusted for bodyweight) of males exposed to 2.49 mg/L was statistically significantly higher than the concurrent control, but as there was intergroup variability in this parameter, no changes noted for females, and no corroborating pathological changes in the liver this change is considered to be unrelated to treatment.
Other intergroup differences seen were small and largely inconsistent between groups and sexes. Therefore, these were considered to be due to individual variation in results and not to be related to treatment

GROSS PATHOLOGY
The macroscopic examination performed after 4 weeks of treatment revealed no intergroup differences of note.
The nature and incidence of all findings were consistent with the commonly seen background of macroscopic changes in Sprague Dawley rats at these laboratories

HISTOPATHOLOGY: NON-NEOPLASTIC
No findings were seen that were clearly related to treatment with DEDG.
Minimal or slight generalised hypertrophy of the zona fasciculata was seen in three male animals exposed to 2.49 mg/L. This finding correlated with the higher bodyweight-adjusted group mean adrenal weight, compared with male controls, for males exposed to 2.49 mg/L.

In the right testis, seminiferous tubular vacuolation and or cellular debris, and in the right epididymis, degenerative spermatogenic cells and or cellular debris in the ducts, were seen at higher incidences in control males than in males exposed to 2.49 mg/L. Therefore, these
findings were not related to treatment, but were considered possibly related to the stress associated with the inhalation procedure (Dodd, 1999; Lee, 1993) and correlated with the atypical values reported at sperm analysis for control animals.



Dose descriptor:
NOAEL
Effect level:
2.49 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects up to the highest dose tested..
Critical effects observed:
no

There was a very low level of DEDG detected at analysis of the samples collected from the control group. In 18/23 occasions the identified peak was below the limit of detection, and in the other 5 samples was very low (approximately 25% that of the low dose levels). It was established that there was a low level of DEDG vapour within the animal room, which could have contaminated the external surfaces of the sampler and possibly absorbed into the trapping solvent used in analysis. Animals of the control group were exposed to clean external fresh air in a sealed system and were considered to have no direct contact with this low level of contamination, and this is considered to have no impact on the study integrity.

Conclusions:
DEDG was administered to Sprague Dawley rats by snout only inhalation exposure for 6 hours a day, 5 days a week for 4 weeks at estimated exposure concentrations of 0.257, 0.686 or 2.49 mg/L.
No adverse treatment related effects were apparent in any of the parameters investigated and DEDG was well tolerated over the 4 week treatment period.
The No Observed Adverse Effect Level (NOAEL) was 2.49 mg/L.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 490 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

The repeated dose toxicity of the notified substance, DEGDEE, and category members, DEGME, DEGDME, DEGEE and DEGBE has been investigated in a variety of laboratory animal species, by different routes of exposure and exposure durations. Generally, the target organs and toxicity profiles of glycol ethers are detectable shortly after administration. Many effects are notable after single exposure. There is minimal difference between the effects following subacute (up to 28 days) and subchronic (up to 90 days) exposure, either in qualitative or in quantitative terms. Administration routes are of minor importance for the effects observed (ECETOC TR95, 2005). These studies indicated that the targets of toxicity for the diethylene glycol monoalkyl and dialkyl ethers category members include the peripheral blood, bone marrow, lymphoid organs, liver, kidney and the germinal epithelium of the testes. At high, sublethal doses, a reversible CNS depression is sometimes seen; this is a general feature of solvent toxicity. The target organs and the severity of the toxic effects are depending on the metabolites produced from the glycol ethers, the biotransformation of DEGME, DEGDME, DEGEE, DEGDEE, and DEGBE resulting in the ether cleavage and formation of alkoxyacetic acids such as methoxyacetic acid (MAA), ethoxyacetic acid (EAA) and butoxyacetic acid (BAA), respectively.

The metabolite BAA has been shown to be responsible for pre-haemolytic effects on the peripheral blood (although not biologically significant for human blood), the metabolites MAA and EAA to a lesser extent produce toxic effects in reproduction (testes). MAA has also an effect on haematopoetic system. However high doses of this category members are needed to produce these effects. This is because less alkoxyacetic acids is produced during the metabolism of diethylene glycol alkyl ethers compare with ethylene glycol alkyl ethers. Moreover the metabolites MAA and EAA have different clearance rate. Clearance of EAA was significantly higher than of MAA in the male and female rats (Aasmoe et al,1999). This would explain why results of studies performed with DEGDEE suggested that the notified substance does not pose significant toxic risk associated with repeated exposure. Repeated exposure (7 hr/d, 5 d/wk, 17 x 7h, whole body) of Wistar rats to saturated DEGDEE vapour at 2.6mg/L (equivalent to 1774mg/kg/day) resulted in restlessness but no remarkable findings at necropsy (Gage, 1970). This was confirmed by a new 28 day inhalation study (6 hr/d, 5 d/wk, 4 weeks, nose-only) in rat. DEGDEE caused no treatment related effects at doses of up to 2.49 mg/l (1671.8mg/kg/day) (HLS report No OOE0005, 2013).

Justification for classification or non-classification

Two subacute day repeat dose toxicity studies performed with DEGDEE by the inhalation route had no other effects than CNS clinical signs and potential reduced weight gain. Therefore DEGDEE is not classified according to CLP criteria (Regulation (EC) No 1272/2008).