Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with diethylamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August 2012 - 29 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with diethylamine

EC Number:

500-425-6

EC Name:

Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with diethylamine

Cas Number:

159034-91-0

Molecular formula:

Not available for this UVCB

IUPAC Name:

Reaction product of poly(oxy-1,2-ethanediyl), .alpha.-hydro-.omega.-[(1-oxo-2-propenyl)oxy]-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1) and N-ethylethanamine

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories France, l’Arbresle, France
- Age at study initiation: approximately 6 weeks old
- Mean body weight at study initiation: the males had a mean body weight of 185 g (range: 169 g to 200 g) and the females had a mean body weight of 171 g (range: 148 g to 184 g)
- Fasting period before study: no
- Housing: the animals were housed by five, in polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: a period of 8 days before the beginning of the treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 07 September 2012 to 05 October 2012.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. It was mixed with the required quantity of vehicle. The dose formulations were kept under magnetic stirring at least 30 minutes before delivery to the study room.
No correction factor was applied to the calculation of dose-levels.
The frequency of dose formulation preparation was on a daily basis. The dose formulations were delivered to the study room at room temperature and protected from light.

VEHICLE
- Justification for use and choice of vehicle: soluble in the vehicle and corn oil is commonly used for this type of study.
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: GC-FID
Test item concentrations: within the acceptance criteria (± 10%), except for formulations prepared in week 4 for groups 3 and 4 which were respectively at +10.9% and +11.3%. This was considered to have no impact on the study
Homogeneity: not assessed, dose formulation is a solution
Stability: not assessed, dose formulation prepared daily

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor based on the results of a previous 2 week toxicity study performed in the same species and strain. In this study, three groups of three males and three females received the test item by gavage at 100, 300 or 1000 mg/kg/day as a suspension in corn oil. Another group of three males and three females received the vehicle only. The only test item-related effect seen in this study was ptyalism at 300 and 1000 mg/kg/day.

Therefore, 1000 mg/kg/day was selected as the high dose-level for this 4-week study. Mid and low dose-levels were selected in order to cover approximately 3-fold intervals.

- Rationale for animal assignment: computerized randomization procedure.

Positive control:

no (not required)

Examinations

Observations and examinations performed and frequency:

MORTALITY/MORBIDITY:
- Time schedule: once a day during the acclimation period and at least twice a day during the treatment period.

- CLINICAL SIGNS:
- Time schedule: once a day, at approximately the same time.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once before the beginning of the treatment period and then at least once a week until the end of the study.

BODY WEIGHT:
- Time schedule: once before the beginning of the treatment period, on the first day of treatment and then at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: once a week until the end of the study.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: once in week 4.

HEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS:
- Time schedule: at the end of the treatment period.

Sacrifice and pathology:

ORGAN WEIGHTS: at the end of the treatment period.

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination of all study animals.

HISTOPATHOLOGY:
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.

Other examinations:

no

Statistics:

Citox software (version D.6) was used to perform the statistical analyses of body weight, hematology, blood biochemistry and urinalysis data. PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ptyalism, breathing difficulties, thin appearance and hunched posture observed at 1000 mg/kg/day and ptyalism noted at 300 mg/kg/day were considered to be test item-related and, with the exception of ptyalism, adverse. The treatment had to be stopped for 1 day for one female following poor health condition.
Ptyalism was observed generally from the first week of the treatment, breathing problems in the second half of the treatment period and hunched posture towards the end of the treatment period. Thin appearance was observed following body weight loss recorded in the affected males.

Incidental findings included scabs, chromodacryorrhea, thinning of hair, eye opacity, lacrimation, half-closed eyes, soiled muzzle, piloerection and bent head.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated at 1000 mg/kg/day had a mean terminal body weight and mean body weight gains lower than controls, particularly in the second half of the treatment period. This was considered to be test item-related but non adverse as the mean body weight at the end of the study was affected by -10% only when compared with controls.
There was no dose-relationship in female mean terminal body weight and mean body weight gain over the treatment period; the statistical differences observed were considered to be fortuitous.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males treated at 1000 mg/kg/day had a mean food consumption toxicologically significantly lower than controls during the last week of treatment (18.7 g/animal/day vs. 23.5 in controls, i.e. -20%). This non adverse effect was considered to be test item-related.
In females, the non dose-related differences from controls in mean food consumption were considered to be fortuitous.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects. Mean values from test item-treated groups were comparable to those of the controls.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The statistical differences in blood biochemistry (sodium and chloride) seen in males treated at 100 or 300 mg/kg/day were considered to be fortuitous as no effects at 1000 mg/kg/day thus no dose-relationship and included in historical control data.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, there were 3/5 males and 2/5 females with 2 to 4 of the followings findings: hunched posture, dyspnea, piloerection and/or hypoactivity. The males had a trend toward a slight increase of the mean rectal temperature at this dose-level (38.4°C vs. 37.7 in controls). These findings were considered to be test item-related and the clinical signs observed at FOB reminded those observed throughout the study.
At 300 mg/kg/day, one male had dyspnea, piloerection and hypoactivity. As this was observed in one isolated animal, the other animals from this group showing either no findings or only one finding (hunched posture or piloerection), this was considered to be of no toxicological importance at this dose-level.
There were no effects on motor activity considered as test item-related at any dose.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No organ weight differences were attributed to Diethylamine modified ethoxylated trimethylolpropane triacrylate.
Statistically higher mean relative-to-body brain and epididymis weights, and lower mean absolute liver weights were observed in males treated at 1000 mg/kg/day. These weight differences were within the range of historical control data and were most likely related to the statistically lower final mean body weights observed in this group.
As other mean weight changes were minimal or of opposing trends between the male and female groups, a relationship to test item treatment was excluded.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were attributed to treatment with Diethylamine modified ethoxylated trimethylolpropane triacrylate.
The few gross findings reported were of those commonly observed in the rat of this strain and age and therefore a relationship to test item treatment was excluded.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Moderate (grade 3) multifocal chronic bronchioalveolar inflammation was observed in the lung of one out of five males treated at 1000 mg/kg/day. This was characterized by peribronchial fibrosis foci, minimal hyperplasia of the bronchial epithelium and mixed inflammatory cell infiltrates. Clinical signs were observed in this male: loud breathing on days 20 and 21, along with dyspnea from day 27. Breathing difficulties were also observed in two other males and two females treated at 1000 mg/kg/day and no microscopic findings were noted in the lungs.
This respiratory finding was considered to be possibly test item treatment-related.
Minimal or slight mucification of the vagina epithelium were seen in two out of five females treated at 1000 mg/kg/day (vs. none in controls). As this isolated finding may occur spontaneously in the rat, a relationship to test item treatment was considered to be unlikely.

Other microscopic findings were of those that are commonly observed in the rat of this strain and age and therefore none was considered to be test item treatment-related.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

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 signs

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The test item was administered daily for 4 weeks by oral route to male and female Sprague-Dawley rats at dose-levels of 100, 300 or 1000 mg/kg/day in corn oil. The No Observed Adverse Effect Level (NOAEL) was considered to be at 300 mg/kg/day based on the adverse clinical signs observed at 1000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 4 weeks.

The study was performed according to the international guidelines (OECD No. 407 of 03 October 2008 and Commission Regulation No. 440/2008 of 30 May 2008, Part B7) and in compliance with the principles of Good Laboratory Practice.

 

Methods

Three groups of five male and five female Sprague-Dawley rats received the test item by daily oral administration for 28 days at dose-levels of 100, 300 or 1000 mg/kg/day. The test item was administered as a solution in the vehicle (corn oil) at a constant dosage-volume of 5 mL/kg/day. A control group of five males and five females received the vehicle alone under the same experimental conditions.

Test item concentrations were checked on formulations used in weeks 1 and 4.

The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. In addition, detailed clinical examinations were performed at least once weekly. Body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as food consumption. Towards the end of the dosing period, a Functional Observation Battery (FOB), motor activity measurement, hematology, blood biochemistry and urinalysis were performed on all animals.

On completion of the treatment period, the animals were euthanized and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues from control- and high-dose animals sacrificed at the end of the treatment period and on all macroscopic lesions.

 

Results

The test item concentrations in the administered dose formulations analyzed in weeks 1 and 4 were within the acceptance criteria (± 10%), except for formulations prepared in week 4 for groups 3 and 4 which were respectively at +10.9% and +11.3%. This was considered to have no impact on the study.

At 100 mg/kg/day, there were no toxicologically relevant effects.

 

There were no unscheduled deaths during the study and no test item-related effects on motor activity at any dose.

 

The following clinical signs were observed at 1000 mg/kg/day: ptyalism (10/10 animals), breathing difficulties (5/10), thin appearance (2/10) and hunched posture (5/10). Ptyalism was also noted at 300 mg/kg/day in 5/10 animals. Half of the 1000 mg/kg/day animals had hunched posture, dyspnea, piloerection and/or hypoactivity at FOB. The males also showed a trend toward a slight increase of the mean rectal temperature (38.4°C vs. 37.7 in controls). The clinical signs (except ptyalism) observed in the animals treated at 1000 mg/kg/day were considered to be adverse.

 

At 1000 mg/kg/day, males also had a mean body weight at the end of the study (vs. 359, p<0.05) and mean body weight gains (days 1 to 28: +vs.+170) lower than controls, particularly in the second half of the treatment period, and a mean food consumption toxicologically significantly lower than in controls during the last week of treatment (18.7 g/animal/day vs. controls, i.e. -20%). These findings were considered to be test item-related but not adverse. There were no test item-related effects on mean body weight and mean food consumption in females at 1000 mg/kg/day and in both sexes at 300 mg/kg/day.

At 300 and 1000 mg/kg/day, there were no effects on hematology, blood biochemistry and urinalysis parameters.No mean organ weight differences and no macroscopic post-mortem findings were attributed to the test item at any dose. Moderate (grade 3) multifocal chronic bronchioalveolar inflammation was observed in the lung of one out of five males treated at 1000 mg/kg/day, which could be a major contributing factor to breathing difficulties. This respiratory finding was considered to be possibly test item-related.

 

Conclusion

The test item was administered daily for 4 weeks by oral route to male and female Sprague-Dawley rats at dose-levels of 100, 300 or 1000 mg/kg/day in corn oil.

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) was considered to be at 300 mg/kg/day based on the adverse clinical signs observed at 1000 mg/kg/day.