Registration Dossier

Administrative data

Description of key information

The substance is not toxic after oral repeated exposure.

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:
24/09/2016-29/11/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Monument Chemical Kentucky,LLC/Batch n° 15DR198795
- Expiration date of the lot/batch:27/07/2016


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility in water: 13.1g/L at 21°C
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat: Crl: (WI(Han) (outbref, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Females: nulliparous and non-pregnant
- Weight at study initiation: 150 g
- Housing: group housing of 5 animals per sex in Macrolon cages with sterlized sawdust as bedding material and paper as cage-enrichment
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before the start of treatment under laboratory conditons

DETAILS OF FOOD AND WATER QUALITY:
Diet and water evaluation for contaminants and/or nutrients was performed according to facility standard procedures.
There were no finding that could interfere with the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C):19.7-20.8°C
- Relative humidity (%):45-69 %
- Air changes (per hr): a least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hours dark cycle

IN-LIFE DATES: From:11/11/2015 To:11/02/2016
Route of administration:
oral: gavage
Details on route of administration:
Oral gavage using a plastic feeding tube. Formulation were placed on a magnetic stirrer during dosing
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0, 100, 300, 1000 mg/kg bw
- Amount of vehicle (if gavage): 5mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis of dose preparations:
1. Sample (0.5 mL) were taken and weighed.
2. During the sampling, formulations were placed on a magnetic stirrer.
3. Immediately after sampling or 5 hours at room temperature under normal laboratory light conditions or after 8 days in the refrigerator in the dark, sample were store on dry ice

Samples formulation were analysed for homogeneity (highest and lowest concentration) and accuracy preparation (all concentrations).
Stability in vehicle over 5 hours at room temperature under normal laboratory ligh conditions and over 8 days in the refrigerator in the dark was determined.

Accuracy preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration.
Homogeneity was demonstrated if coefficient of variation was ≤ 10%.
Formulation were considered stable if the relative difference before and after storage was maximally 10%.

Results:
No test item was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).
The formulation of Group 2 and Group 4 prepared were homogeneous (i.e. coefficient of variation ≤ 10%).
Formulations at the entire range were stable when stored at room temperature at normal laboratory light for at least 5 hours and for 8 days in the refrigerator in the dark (i.e. relative
difference ≤ 10%)
Duration of treatment / exposure:
90 days (animals were dosed up to the day prior to necropsy
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with maximum of 6 hours difference between the earliest and latest dose.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels based on results of a 14-day oral range finding study with 2,2'-p-phenylenedioxydiethanol (HQEE) by daily gavage in rat.
- Randomization: by computer-generated random algorithm according to body weight, with all animals within +/- 20% of the sex mean.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: yes
- Time schedule: weekly

WATER CONSUMPTION: yes
- Time schedule: subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pre-test and at 13 weeks
- Dose groups that were examined: all animals at pre-test and group 1 and 4 at week 13
Since no treatment-related ophtalmologic findings were noted in week 13, the eyes of the rats of groups 2 and 3 were not examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment
- Anaesthetic used for blood collection: Yes: isoflurane
- Animals fasted: Yes: animals were deprived of food overnight (for a maximum 24 hours)
- How many animals: all animals
- Parameters checked in table [No.1] under section "Any other information on materials and methods"] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment
- Animals fasted: Yes, animals were deprived of food overnight (for a maximum 24 hours)
- How many animals: all animals
- Parameters checked in table [No.2] under section "Any other information on materials and methods"] were examined. were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:No

IMMUNOLOGY: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes see table n° 3 under section "Any other information on materials and methods"

HISTOPATHOLOGY: Yes see table n° 3 under section "Any other information on materials and methods"
- all tissues collected at the scheduled sacrifice from all group 1 and 4 animals
- all tissues from control male no. 9 which was terminated in extremis.
- all gross lesions were eximined

ORGAN WEIGHTS:
- the organ weights were recorded from the surviving animals in the schedule day of necropsy:
- Adrenal glands - Spleen
- Brain - Testes
- Epididymides - Thymus
- Heart - Uterus
- Kidneys - Protaste
- Liver - Seminal vesicles including coagulating glands
- Ovaries - Thyroid including parathyroid
Other examinations:
Other observations:
- Mortality/viability: at least twice daily
- Functional observations:During week 12 of treatment the following test were performed on the first 5 animals/sex of Group 1 and Group 4 after dosing at no specific time point, but within similar time period after dosing for the respective animals:
- hearing ability, pupillary reflex and static righting reflex
- Fore- and hind-limb grip strength
- locomotor activity: total movements and ambulations are reported
Statistics:
- Dunnett-test bases on a pooled variance estimate was applied for the comparison of the treated groups and the conrol groups for each sex, if variable could be assumed to follow a normal distribution
- Steel-test was applied of the data could not be assumed to follow a normal distribution
- Fisher Exact-test was applied to frequency data
- Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
- Wilcoxon test was applied in case intergroup difference were seen to compare the threated groups to the control groups.

All test were two-sided and in all cases p <0.05 was accepted as the lowest level of significance. Test statistics were calculated on the basis of exact values for means and pooled variances.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed after dosing in all animals of the highest dose group and one control female. The onset of this effect started at low incidence at week 3 and became more frequent during the further course of the study in males and females at the high dose level until the end of the dosing period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A lower body weight and body weight gain was shown by males at 1000 mg/kg primarily during the second half of the treatment period (reaching 10% reduction in body weight compared to controls).
Although the female at 1000 mg/kg bw suggested a slighly higher body weight compared to controls, the body weight and body weight gain of female animals were considered to remain in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before and after correction for body weight remained similar to control levels over the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The nature and incidence of ophtalmology finding noted during pretest and in Week 13 was similar among the groups, and occured within the range considered normal for rats of this age and strain.
These findings were threfore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A slightly lower reticulocyte level was noted in males at 300 mg/kg and 1000 mg/kg. Since this level was considered within the normal range for these rats of this age and strain and in absence of any other corroborative finding in haematological parameters and morphological examination, this findings was considered no toxicologically relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Slightly higher potassium was noted in males and females in a dose related reponses, achieving statistically significance in females at 1000 mg/kg only.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all eximaned animals.
Grip strength and motor activity was similar between control and high dose animals.
All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically relevant alterations in organ weights.
A slightly higher liver weight was noted in females at 1000 mg/kg. Since this finding occured in absence of findings in clinical biochemistry and histopathology and all individual values were within the range expected for rats of this age and this strain, this finding was not considered toxicologically relevant.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
Critical effects observed:
no

Summary dose range-finding study:

1. Materials and methods:

Same as those used during the main study

2. Test system:

      - 3 males* (group-housed, allocated at random)

- approximately 6 weeks old at start of treatment

*Males were used as the most sensitive sex in 28 -day study

3. Treatment:

- Duration of treatment: 14 days

- Dose levels: 500 and 1000 mg/kg body weight

- Dose volume: 5 mL/kg bw

- Vehicle: water

4. Observations:

- Clinical signs: Daily at 0 -15 min, 1 hour and 3 hours after dosing

- Mortality: at least twice daily

- Body weight: weekly

- Food consumption: weekly

5. Pathology:

All animals were subjected to an external, thoracic and abdominal examination on Day 15 after the last observation of clinical signs. No organs were fixed. Animals were not deprived of food prior to necropsy.

6. Results:

- No mortality for the 2 groups

- Clinical signs: hunched posture was noted in one animal only on day 3 only for 2 groups

- Body weight: normal

- Food consumption: normal

- Macroscopy observation: Thymus in one animal of 500 mg/kg group and Kidney (pelvic dilation) in one animal of 1000 mg/kg group.

7. Conclusion:

Based on the results of this range finding study, the dose levels for the main study were established at 100, 300 and 1000 mg/kg body weight.

Moreover, no clear peak effect of occurence of clinical signs was observed in the range findings study. Therefore, clinical observation in the main study were conducted immediately after dosing, and functional observation tests were conducted after dosing at no specific time point, but whithin a similar time period after dosing for the respective animals.

Conclusions:
Based on the absence of systemic toxicity at all dose levels the NOAEL for 2,2'p-pphenylenedioxydiethanol (HQEE) was established at least 1000 mg/kg.
In addition, based on the slight treatment related effects a NOEL was established at 300 mg/kg.
Executive summary:

The repeated dose 90 -day oral toxicity study with 2 -2'-p-phenylenedioxydiethanol by daily gavage in Wistar rat has been performed according to OECD 408 guideline without significant deviations.

Based on the results of a 14 -day range finding study, the dose level for the main study were selected to be 0, 100, 300 and 1000 mg/kg.

The test item formulated in water, was administrated daily for at least 90 days by oral gavage to Wistar rats. One control group and three groups were tested, each consisting of 10 males and 10 females.

Chemical analyses of formulation were performed to assess accuracy, homogeneity and stability over 5 hours at room temperature under normal laboratory light conditions and over 8 days in the refrigerator in the dark.

The results of the formulation analyses confirmed that the test item were prepared accurately and homogenously and were stable for the both conditions mentioned here above.

The animals were dosed for 90 days and the following parameters were evaluated: the clinical signs daily, the functional observation test in week 12, the body weight and the food consumption weekly, opthalmoscopy at pre-test and at week 13, clinical pathology and macroscopy at termination of the study, the organ weight and the histopathology on selected tissues.

Detailed daily clinical observations revealed salivation from week 3 onwards in both sexes at 1000 mg/kg with increasing frequency during the study.

Based on the occurence at the high dose it is suggested to be dose related, however, on the minor severity the effect is considered not to be adverse.

A lower body weight statistically significant (up to 10% loss versus control) and reduced weigh gain was observed in male animals at 1000 mg/kg. The effects were very slight and the incidental statistical significance was probably due to relatively high control values in this period. Therefore, these changes were considered not adverse in nature.

Slightly higher potassium values were noted in males and females reaching statistical significance in females dosed at 1000 mg/kg. Based on the dose response distribution this slight change was considered treatment related, however, not considered adverse.

In conclusion, based on the absence of systemic toxicity at all dose levels the NOAEL was established at least 1000 mg/kg. In addition, based on the slight treatment related effects a NOEL was established at 300 mg/kg.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study was performed according to internationaly accepted guideline and GLP

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

not applicable

Additional information

For the repeated dose toxicity endpoint there are 2 studies available assessing toxic effects of the test item after repeated dose via oral route. No studies are available for the inhalation or dermal route. However, the data available is sufficient for classification purposes and the oral route is the most relevant route of administration for exposure in humans.

- The key study (C.G.M. Beerens-Heijnen, 2016) is a Repeated dose 90 -Day Oral Toxicity Study in rats, performed to generate information concerning the effects of the test item on males and females during a repeated dose exposure. This study was performed according to OECD Guideline 408.

The test substance was administered daily by oral gavage in Wistar rat at dose levels of 100, 300 and 1000 mg/kg for at least 90 days.

One control group and three test item groups were tested, each consisting of 10 males and 10 females.

All animals were observed for clinical signs, functional observation test, body weight, food consumption, opthalmoscopy, clinical pathology and macroscopy, organ weight and the histopathology on selected tissues.

Detailed daily clinical observations revealed salivation from week 3 onwards in both sexes at 1000 mg/kg with increasing frequency during the study.

Based on the occurence at the high dose it is suggested to be dose related, however, the effect is considered not to be adverse.

A statistically significant lower body weight statistically significant (up to 10% loss versus control) and reduced weigh gain was observed in male animals at 1000 mg/kg. The effects were very slight and the incidental statistical significance was probably due to relatively high control values in this period. Therefore, these changes were considered not adverse in nature.

Slightly higher potassium values were noted in males and females reaching statistical significance in females dosed at 1000 mg/kg. Based on the dose response distribution this slight change was considered treatment related, however, not considered adverse.

In conclusion, based on the absence of systemic toxicity at all dose levels the NOAEL was established at at least 1000 mg/kg.

- The supporting study (Hosenfeld and Hankinson, 1988) is a subacute oral study in the rat. This study was performed according to OECD Guideline 407. However, only limited information is available.

The only observed effect of toxicological significance was a decreased mean platelet count in the high dose males.

Consequently, the NOAEL is 0.3% in diet, equivalent to 249 mg/kg/day and 262 mg/kg/day in males and females respectively.

Justification for classification or non-classification

The substance did not induce any adverse effects after 28 days or 90 days of repeated dosing.

According to the criteria set out in the CLP Regulation No 1272/2008 in section 3.9. and specific values set out in Table 3.9.2 the substance should not be classified.

The substance is therefore not toxic after a repeated exposure.