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Description of key information

2 current guideline, reliability 1 studies for acute oral toxicity and dermal toxicity, 3 supporting studies for oral, dermal and inhalation and supporting data on the analogue phenoxy ethanol

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 30, 2008 - February 20, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG425 and in accordance with the principles of GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)
Version / remarks:
U.S. EPA Health Effects Test Guidelines, OPPTS 870.1100 (2002), JMAFF 12-Nousan-8147, November 2000.
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Test type:
up-and-down procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxicology Department of Safety Assessment, Advinus Therapeutics Private Limited, Bangalore 560 058, India
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 174 - 187 g
- Fasting period before study: Fasted overnightprior to dosing
- Housing: Rats were housed individually in standard polysulfone cages (Size: approximately L 425 x B 266 x H 175 mm), with stainless steel top grill having facilities for pelletted food and drinking water.
- Diet (e.g. ad libitum): Ssniff rats/mice pellet food - maintenance meal - low in germs manufactured by Ssniff Spezialdiäten GmbH., Ferdinand- Gabriel-Weg 16, D-59494 SÖest, Germany, was provided to the animals.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cumpurifier manufactured by Eureka Forbes Ltd., Mumbai - 400 001, India, was provided to animals in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: Seven, ten, thirteen, seventeen, twenty and twenty four days under laboratory conditions after physical examination for treatment steps.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light):12 hours light and 12 hours dark cycle
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
not applicable
Doses:
Animal 1: 5000 mg/kg
Animal 2: 2800 mg/kg
Animal 3: 1750 mg/kg
Animal 4: 2800 mg/kg
Animal 5: 5000 mg/kg
Animal 6: 5000 mg/kg
No. of animals per sex per dose:
not applicable
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed five times on test day 1 (day of administration) i.e. at 30 minutes and four times at hourly intervals (post administration) and once daily during days 2 – 15.
- Necropsy of survivors performed: At the decedents were subjected to gross necropsy as soon as possible after death. All surviving and moribund animals were necropsied at the end of the observation period. Surviving rats were euthanised with isoflurane anaesthesia. External surface of the body, all orifices, tissues and organs of the thoracic and abdominal cavities of all animals were examined. All gross findings were recorded.
Statistics:
Dose progression and stopping criteria was calculated using a dedicated software program (Acute Oral Toxicity (Guideline 425) Statistical Program) provided by the EPA. A Sigma of 0.2 was used to establish the starting dose and dose progression.
Preliminary study:
not applicable
Sex:
female
Dose descriptor:
LD50
Effect level:
3 526 mg/kg bw
Based on:
test mat.
Mortality:
Animal 6: 5000 mg/kg body weight (4.5 ml/kg B.wt ) - Coma on day 2 and died on day 2.
Clinical signs:
See below
Body weight:
Dead rats had lost body weight and all surviving rats had gained body weight through the observation period. in all treatment steps.
Gross pathology:
Animal 1, 3, 4 and 5: No abnormality detected at necropsy
Animal 2 and 6: At necropsy the findings were petechial haemorrhages in the gastric mucosa and intestine and the urinary bladder was filled with blood tinged urine.
Other findings:
None

CLINICAL SIGNS AND PRE-TERMINAL DEATHS

Step 1 : 5000 mg/kg body weight (4.5 ml/kg B.wt).

• Clinical signs : Recumbency and coma. No response to touch, sound and toe pressing. Onset of clinical signs at 30 minutes post treatment and persisted till 7 hours.

• The rat was considered as moribund and euthanized. There was no abnormality detected at necropsy.

Step 2 : 2800 mg/kg body weight (2.52 ml/kg B.wt)

• Clinical signs : Recumbency and coma. No response to touch, sound and toe pressing. Onset of clinical signs at 30 minutes post treatment and persisted till 4 hours. At 6 hours post treatment the animal showed signs of recovery and had regained consciousness, but it was dull and lethargic and continued to be dull, lethargic associated with piloerection till 24 hours post dosing. The rat showed symptoms of recumbency and urine dribbling with blood tinged urine and the rat died on day 3 post dosing. At necropsy, the findings were petechial haemorrhages in the gastric mucosa and intestine and the urinary bladder was filled with blood tinged urine.

Step 3 : 1750 mg/ kg body weight (1.58 ml/kg B.wt )

• Clinical signs : There were no clinical signs till day 15 (termination) and the rat was euthanized. There was no abnormality detected at necropsy.

Step 4 : 2800 mg/kg body weight (2.52 ml/kg B.wt)

• Clinical signs: There were no clinical signs till day 15 (termination) and the rat was euthanised. There was no abnormality detected at necropsy.

Step 5: 5000 mg/kg body weight (4.5 ml/kg B.wt)

• Clinical signs : Recumbency at 30 minutes post treatment, ataxia of limbs was observed from 1 hour to 4th hour post treatment. There were no clinical signs from day 2 till day 15 termination. The rat was euthanised. There was no abnormality detected at necropsy.

Step 6: 5000 mg/kg body weight (4.5 ml/kg B.wt )

• Clinical signs : Recumbency at 30 minutes to 4th hour post treatment on day one . Coma on day 2 and died on day 2. At necropsy the findings were petechial haemorrhages in the gastric mucosa and intestine and the urinary bladder filled with blood tinged urine.

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the present study results, the estimated acute oral LD50 of the test item Diethylene glycol mono phenyl ether is 3526 mg/kg body weight and with a 95% PL.
Executive summary:

The test item Diethylene glycol mono phenyl ether was tested for acute oral toxicity in female Wistar rats. The test item was administered as received (undiluted) as a single oral dose to fasted (16 - 18 hours) rats. The treatment was initiated as follows, and the dose for the subsequent steps was arrived at by using the AOT425 StatPgm by using a sigma of 0.2.

Step 1: 5000 mg/kg body weight (4.5 ml/kg B.wt) was administered to one female rat. Note: The first animal was inadvertently administered a dose of 5000 mg/kg B.wt. This deviation had no impact on the study.

• Clinical signs: Recumbency and coma. No response to touch, sound and toe pressing. Onset of clinical signs at 30 minutes post treatment and persisted till 7 hours.

• The rat was considered as moribund and euthanised, no abnormality was detected at necropsy.

Step 2: 2800 mg/kg body weight (2.52 ml/kg B.wt) was administered to the second female rat.

• Clinical signs: Recumbency and coma. No response to touch, sound and toe pressing. Onset of clinical signs at 30 minutes post treatment and persisted till 4 hours. At 6 hours post treatment the animal showed signs of recovery and had regained consciousness, but it was dull and lethargic and continued to be dull, lethargic associated with piloerection till 24 hours post dosing. The rat showed symptoms of recumbency and urine dribbling with blood tinged urine and the rat died on day 3 post dosing. At necropsy the findings were petechial haemorrhages in the gastric mucosa and intestine and the urinary bladder was filled with blood tinged urine.

Step 3: 1750 mg/kg body weight (1.58 ml/kg body weight) was administered to the third female rat.

• Clinical signs: There were no clinical signs till day 15 (termination) and the rat was euthanised. There was no abnormality detected at necropsy.

Step 4: 2800 mg/kg body weight (2.52 ml/kg body weight) was administered to fourth female rat.

• Clinical signs: There were no clinical signs till day 15 (termination) and the rat was euthanised. There was no abnormality detected at necropsy.

Step 5: 5000 mg/kg body weight (4.5 ml/kg body weight) was administered to the fifth female rat.

• Clinical signs: Recumbency at 30 minutes post treatment, ataxia of limbs was observed from the 1st hour to 4th hour post treatment. There were no clinical signs from day 2 till day 15 termination. The rat was euthanised. There was no abnormality detected at necropsy.

Step 6: 5000 mg/kg body weight (4.5 ml/kg body weight) was administered to the sixth female rat.

• Clinical signs: Recumbency at 30 minutes to 4th hour post treatment on day one. Coma on day 2 and died on day 2. At necropsy the findings were petechial haemorrhages in the gastric mucosa and intestine and the urinary bladder filled with blood tinged urine.

As per the AOT425StatPgm the stopping criteria was met and the surviving rats were observed for 14 days post treatment and the LD50 arrived at after the 14 day observation period.

Based on the present study results, the estimated acute oral LD50 of the test item Diethylene glycol mono phenyl ether is 3526 mg/kg body weight and with a 95% PL. Confidence interval is 0 to greater than 20000, as per the agency developed Software Program AOT425 StatPgm.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
3 526 mg/kg bw
Quality of whole database:
Good

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 30, 2008 - February 20, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 402 and in accordance with the principles of GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
U.S. EPA Health Effects Test Guidelines, OPPTS 870.1200 (1998), JMAFF 12-Nouan-8147, November 2000, Official Journal of the European Communities. Methods for the Determination of Toxicity, Part B.3 (Acute Toxicity Dermal), Directive 2004/73/EC, 29 April 2
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxicology, Department of Safety Assessment, Advinus Therapeutics Private Limited, Bangalore 560 058, India
- Age at study initiation: 10 – 11 weeks
- Weight at study initiation: Males : 272 to 279 g, Females: 209 to 214g
- Housing: Rats were housed individually in standard polysulfone cages (Size: approximately L 425 x B 266 x H 175 mm), with stainless steel top grill having facilities for pelletted food and drinking water. Bedding: steam sterilized clean paddy husk was used and changed along with the cage twice a week.
- Diet (e.g. ad libitum): Ssniff rats/mice pellet food - maintenance meal - low in germs manufactured by Ssniff Spezialdiäten GmbH., Ferdinand- Gabriel-Weg 16, D-59494 SÖest, Germany , was provided to the animals.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cumpurifier manufactured by Eureka Forbes Ltd., Mumbai - 400 001, India, was provided to animals in polycarbonate bottles with stainless steel sipper tubes.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
Approximately 24 hours prior to treatment, the hair on the dorsolateral thoracic surface of the skin was clipped (approximately 8 x 10 cm) with an electric clipper (Aesculap® - Germany).

Doses were calculated based on individual body weight, the undiluted test item at the dose of 5000 mg/kg body weight (A volume of 4.5 ml/kg bodyweight) was applied directly to the prepared skin of the animal, taking care to spread the substance evenly over the entire dose area, and covered with a cotton gauze (size: 9 x 5 cm of 6 ply for males and 8 x 5 cm of 6 ply for females) and secured in position by adhesive tape wound around torso. The test item contact period with the skin was for 24 hours.

After the 24-hour contact period, the patches were removed and the application site was wiped with water using clean towels to remove any residual test substance.
Duration of exposure:
24 hours
Doses:
5000 mg/kg body weight
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations: four times on the test day one (at hourly intervals after application) and once daily during days 2 - 15.
Weighing: Individual body weights of animals were recorded on test days 1 (Pre-application), 8 (7 days post application) and 15 (14 days post application).
- Necropsy of survivors performed: yes
Statistics:
None
Preliminary study:
An initial dose of 5000 mg/kg was applied. This dose level did not produce test substance related mortality, hence, no additional dose levels were tested.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
There was no pre-terminal deaths in any of the treated rats.
Clinical signs:
There was no Clinical/ toxic signs observed in any of the treated rats
Body weight:
All rats had gained bodyweight through the observation period.
Gross pathology:
There were no local skin reactions and no abnormality detected at necropsy.

None

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results, the acute dermal LD50 of Diethylene glycol mono phenyl ether in Wistar rats is greater than 5000 mg/kg body weight.
Executive summary:

The acute dermal toxicity of Diethylene glycol mono phenyl ether was tested in male and female Wistar rats.

Based on the individual body weight, the undiluted test item at the dose of 5000 mg/kg body weight (a volume of 4.5 ml/kg bodyweight) was applied directly to the prepared skin of the animal, taking care to spread the substance evenly over the entire dose area, and covered with a cotton gauze (size: 9 x 5 cm of 6 ply for males and 8 x 5 cm of 6 ply for females) and secured in position by adhesive tape wound around torso. The test item contact period with the skin was for 24 hours. After 24 hour contact period with the skin, the unabsorbed test item at the site of application was removed by wiping with water and the rats were observed for 15 days. There were no toxic signs, local skin reactions and pre-terminal deaths. There were no abnormalities detected at necropsy.

Based on the above results it is concluded that the LD50 of Diethylene glycol mono phenyl ether is greater than 5000 mg/kg bodyweight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
good

Additional information

Acute Oral toxicity:

Data on Di-EPh: In a well conducted acute oral toxicity study (Up Down Procedure) the LD50 of the substance was found to be 3526 mg/kg bw/day in female rats.

Dermal Toxicity:

Data on Di-EPh: In a well conducted acute dermal toxicity study the LD50 in rats was found to be >5000 mg/kg bw.

Inhalation Toxicity:

Data on Di-EPh: In the acute inhalation study conducted in 1952 in male rats, there was no mortality following 8 hours whole body exposure to a saturated air concentration at room temperature (equivalent to approximately 10 -15 ppm Di-EPh based on vapour pressure). The nature of the test material is unclear and the methods and results are minimally reported in the study report. Therefore, although the study indicates a low order of acute toxicity via the inhalation route, the data are considered as unreliable and disregarded. Given the low vapour pressure of this compound it is not considered necessary to perform a new study to assess the acute inhalation toxicity of the test material since acute inhalation exposures to the material are unlikely.

Data on Phenoxy Ethanol: This data is provided here for reference only, since the valid study on Di-EPh is sufficient to characterise the hazards of the substance. The oral LD50 of phenoxy ethanol in rats is 1850 mg/kg bw/day. The dermal and inhalation LD50 (LC50) values are more consistent with those of Di-EPh, with values greater than the cut off for classification.


Justification for selection of acute toxicity – oral endpoint
Reliability 1 OECD guideline study

Justification for selection of acute toxicity – dermal endpoint
Reliability 1 OECD guideline study

Justification for classification or non-classification

In all studies the acute toxicity values are in excess of the thresholds for classification. Therefore no classification for acute oral, dermal or inhalation toxicity is required.