Glycerol, propoxylated, 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:

16 June 2015 -- 28 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine

EC Number:

500-744-0

EC Name:

Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine

Cas Number:

162492-10-6

Molecular formula:

(C3H8O3) (C3H6O)m(C3H2O)n ( C4H11N)o ; m (1-6) ; n (1-3) , o (1-3)

IUPAC Name:

Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine

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: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: approximately 10 weeks old for the the males and 9 weeks old for the females on the first day of treatment
- Mean body weight at study initiation: for the males 398 g (range: 376 g to 417 g) and for the females 255 g (range: 236 g to 281 g).
- Housing: polycarbonate cages (Tecniplast 2154, 940 cm²)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 30 June 2015 to 28 August 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Type of formulation
(visual observation): - solution in the vehicle.

Preparation procedure: - according to analysis homogeneity/stability study describing the preparation procedure for a range of concentrations covering the lowest and highest doses used in this study.

Frequency of preparation: - test item dose formulations
on the days of treatment

- vehicle dose formulations
based on the in-house procedures.

Delivery conditions: - at room temperature.
- the test item dose formulations were administered within 5 hours after the end of their preparation.

VEHICLE
- Justification for use and choice of vehicle: suitable formulation in corn oil

- 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: remained within an acceptable range of variation compared to nominal values except for group 3 formulation in week 1 (but formulation given only once to the animals, formulation concentration was checked on week 2 and was correct).

Duration of treatment / exposure:

About 5 weeks for males or 6 weeks for females

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 (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of a previous 2 week toxicity study where groups of five male and five female Sprague-Dawley rats were given the test item in corn oil daily at 100, 300 or 1000 mg/kg/day.
During the second week of treatment, ptyalism was noted in both sexes at 300 mg/kg/day and at 1000 mg/kg/day and, dyspnea but mainly loud breathing were observed at 1000 mg/kg/day. Thin appearance, abdominal breathing and/or hunched posture were also noted during the second week of the study in one male at 300 and at 1000 mg/kg/day.
There were no adverse effects on body weight changes, food consumption or organ weights. There were no test item-treatment-related macroscopic findings.

- Rationale for animal assignment: computerized stratification procedure

Positive control:

no (not required)

Examinations

Observations and examinations performed and frequency:

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

CLINICAL OBSERVATIONS:
Time schedule: once a day.

DETAILED CLINICAL SIGNS:
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: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating (or until sacrifice), on Days 0, 7, 14 and 20 post-coitum and Days 1 and 5 post-partum.

FOOD CONSUMPTION:
Time schedule: the quantity of food consumed by each male was measured once a week, from the first day of treatment until the start of the mating period. The quantity of food consumed by each female was measured once a week, from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-5 p.p..

HEMATOLOGY:
- Time schedule: on the day of sacrifice

CLINICAL CHEMISTRY:
- Time schedule: on the day of sacrifice

Sacrifice and pathology:

SACRIFICE
Male animals: all surviving animals after the end of the mating period
Female animals: all surviving animals = Day 6 post-partum (p.p.).
Females which did not deliver: on Day 26 post-coitum (p.c.),
Female with no evidence of mating: 25 days after the end of the mating period as no delivery occurred.

ORGAN WEIGHTS: see table below


GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all parent animals

HISTOPATHOLOGY:
- on all tissues listed in the Tissue Procedure Table from the first five sacrificed as scheduled males and the first five females sacrificed on Day 6 p.p. of the control and high-dose groups (groups 1 and 4),
- forestomach from the first five sacrificed as scheduled males and the first five females sacrificed on Day 6 p.p. of the mid-dose group (group 3),
- on all macroscopic lesions of all groups,
- on all tissues listed in the tissue Procedure Table from the two animals (group 4) that died or were sacrificed prematurely.

Other examinations:

no

Statistics:

yes

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, ptyalism was observed during most of the treatment period, and loud breathing was also generally noted for a large part the treatment period. Loud breathing was considered to be adverse (serious clinical sign in 14/18 surviving animals for a long time generally) and ptyalism of minor toxicological importance.
One male and one female also experienced piloerection, round back, eyes half-closed, dyspnea and/or abdominal breathing for a few days at the end or at the beginning of the study, respectively.
One male was found to be emaciated in Week 2. This was also considered to be test item treatment-related but of minor toxicological relevance (single animal with this clinical sign).
At 300 mg/kg/day, ptyalism and loud breathing were recorded in 7 and 1 animals, respectively, and were considered to be related to the test item treatment and of minor toxicological importance in view of the absence of severity (ptyalism: not serious clinical sign) or of the limited incidence (loud breathing in 1/20 animals only).
There were no test item treatment-related clinical signs at 100 mg/kg/day.
The other clinical signs recorded in the study (chromorhinoirrhea, chromodacryorrhea, cutaneous lesion, area of hair loss) were not considered to be test item treatment-related: there were in limited incidences and are of common background of laboratory rats.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 1000 mg/kg/day, there were two deaths:
- one male prematurely sacrificed on Day 11 due to poor clinical condition [pallor of extremities (Day 11), cold to the touch (Day 11), loud breathing (from Day 2), abdominal breathing (from Day 10), dyspnea (from Day 10) and ptyalism (from Day 3) were recorded before death],
- one female found dead during pregnancy on Day 19 p.c.. Loud and abdominal breathing and ptyalism were observed for at least 12 days prior to its death.
Both deaths were considered to be related to the test item treatment.

There were no other premature deaths in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day:
- in males, when compared with controls, statistically significant lower mean body weight was noted from the first week of treatment. Mean body weight gain was indeed lower for the first 2 weeks of the study (mainly in the first week correlating with the decrease in mean food consumption). From the third week of treatment, mean body weight gain increased and the statistical difference in mean body weight disappeared. Mean body weight gain decreased again at the end of the treatment period,
- in females, when compared with controls, mean body weight gain had a tendency to be lower than in controls in the first week of treatment (correlating with the decrease in mean food consumption) but was higher than in controls in the second week of treatment. Mean body weight was not affected throughout the study.
These variations from controls were considered to be test item treatment-related but non-adverse (because of the low amplitude of body weight changes).
At 100 and 300 mg/kg/day, there were no test item treatment-related effects on mean body weights and mean body weight changes. The statistically significant higher mean body weight gain in males at both doses from Days 15 to 29 were considered to be not relevant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, mean food consumption was toxicologically significantly lower during the first week of treatment in both sexes. Mean food consumption had also a tendency to be lower than in controls during gestation but this was considered to be of no toxicological significance in the absence of statistical significance and because of the low amplitude of variation.
There were no effects on mean food consumption at 100 and 300 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test item treatment-related effects on mean hematology data (no dose-relationship and no relevant statistically relevant differences from controls).
At 300 mg/kg/day, one female had a moderately elevated white blood cells count (30.04 G/L vs. mean of 16.38 in controls) related to a mild increase in neutrophils, lymphocytes and monocytes counts. But this was not observed in the high-dose and one control male also had a high white blood cell count (35.79 G/L). It was thus considered to be incidental.
At 1000 mg/kg/day, when compared with controls, males had minimally prolonged mean prothrombin and activated partial thromboplastin times. These findings were considered to be test item treatment-related but non-adverse (as the difference were mild without any correlates at microscopy and biochemistry).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

At 1000 mg/kg/day, when compared with controls, males and females had statistically significantly, higher mean urea or creatinine concentration, respectively. As the amplitude of variations from controls was mild, these findings were considered to be of no toxicologically relevance.
The high creatinine level noted in females at 100 mg/kg/day was due to one animal (73.2 µmol/L, with also a high urea level) and considered to be incidental. This animal also had high-levels of ionized phosphorus which indicate a decrease in glomerular filtration rate when associated to high urea and creatinine levels.
Statistically significant variations in potassium and triglycerides levels in males at 100 or 300 mg/kg/day were considered to be incidental (not dose-related).
At 1000 mg/kg/day, there were one male and one female with a high bile acids level and one female with a high glucose concentration. As they were noted in limited incidence and as bile acids level can be highly variable, they were considered to be of no toxicologically significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related mean organ weight differences.
The final mean body weight was considered to be unaffected by the test item administration.
There were higher absolute and relative-to-body mean adrenal weights in males treated at 1000 mg/kg/day (+34% for the relative-to-body weights; p<0.05). These differences correlated with microscopic adreno-cortical hypertrophy seen in one male and which was considered to be mostly the consequence of stress, as noted in the found dead female.
The other absolute and relative-to-body mean weight changes were not considered to be test item-related because they were of insufficient magnitude, not dose-related and uncorrelated with microscopic findings, including the increases in relative-to-body testes and epididymides weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled deaths: There were no test item-related gross findings in unscheduled deaths with the exception of many, up to 0.2 cm in diameter, white discolorations in the forestomach from one high-dose female which correlated with test item-related minimal erosion/ulcer and slight hyperplasia/hyperkeratosis.
Terminal sacrifice: At the end of the treatment period, there were no test item-related gross findings.
The few gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age because they were non dose-related, isolated and/or correlated with common background lesions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled deaths : One male sacrificed on Day 11 had a focal slight hyperplasia of forestomach accompanied by hyperkeratosis probably related to the test item administration. One female found dead on Day 19 p.c. had minimal erosion/ulcer and slight hyperplasia/hyperkeratosis in the forestomach, marked lymphoid atrophy and slight adreno-cortical hypertrophy. The finding in the forestomach was related with the test item which may have irritant properties and the findings in the thymus and adrenals were related with stress.
Terminal sacrifice:
- Forestomach : At the end of the treatment period, slight focal erosion/ulcer, with squamous cell hyperplasia and hyperkeratosis were noted in 1/5 females treated at 1000 mg/kg/day and were considered to be related with the test item administration, although of low incidence and severity, and thus to be non-adverse in absence of macroscopic lesions. The forestomach from males and females treated at 300 mg/kg/day were also examined and no lesions were seen in the forestomach of these animals.
- Other organs : The minimal cortical hypertrophy seen in one male treated at 1000 mg/kg/day was considered to be secondary to the stress.
There were a few other microscopic observations seen only in males or females treated with test item at 1000 mg/kg/day. These findings were seen at low incidence, at low severity, and correlated with background lesions seen in the rats of these strain and age.

Histopathological findings: neoplastic:

no effects observed

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:

clinical signs

mortality

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1

Sex	Male				Female
Dose-level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Number of animals	10	10	10	9	10	10	10	9
Ptyalism			6	9			1 (P, G)	9 (P) 8 (G) 9 (L)
Loud breathing			1	8				6 (P) 4 (G, L)
Abdominal breathing				1				1 (P, G)
Dyspnea				1
Emaciated appearance				1
Piloerection				1				1 (P)
Round back				1				1 (P)
Eyes half-closed								1 (P)

P: premating and mating periods, G: gestation period, L: lactation period.

Table 2

Sex	Male				Female
Dose-level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Pre-mating (females) or whole study (males)
Day 1	392	399	404	398	254	253	256	257
		(+2)	(+3%)	(+2%)		(0)	(+1%)	(+1%)
Day 8	431	440	440	407*	266	266	269	262
		(+2%)	(+2%)	(-6%)		(0)	(+1%)	(-2%)
Day 15	462	475	472	428**	278	276	284	282
		(+3%)	(+2%)	(-7%)		(-1%)	(+2%)	(+1%)
Day 22	471	489	488	449	/	/	/	/
		(+4%)	(+4%)	(-5%)
Day 29	492	518	515	481	/	/	/	/
		(+5%)	(+5%)	(-2%)
Day 36	511	538	529	481	/	/	/	/
		(+5%)	(+4%)	(-6%)
Days 1 - 8	+40	+40	+37	9#	+13	+13	+12	+5
Days 8 - 15	+31	+35	+32	+19*	+11	+10	+15	+20*
Days 15 - 29	+30	+43*	+43*	+53#	/	/	/	/
Days 29 - 36	+19	+21	+14	0*	/	/	/	/
Days 1 - 36	+119	+139	+125	+83*	/	/	/	/
Gestation
Day 0 p.c.	/	/	/	/	279	274	288	284
Day 20 p.c.	/	/	/	/	437	439	463	431
Days 0 - 20 p.c.	/	/	/	/	+158	+166	+175	+146
Lactation
Day 1 p.p.	/	/	/	/	345	347	365	335
Day 5 p.p.	/	/	/	/	354	363	384	350
Days 1 - 5 p.p.					+8	+17	+19	+16

p.c.:post-coitum,p.p.:post-partum;/: not applicable; in brackets: differences from control values.

*: p<0.05, **: p<0.01, #: p<0.001.

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions and results of this study the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 300 mg/kg/day, based on the two deaths and the loud breathing present in most animals at 1000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats the possible health hazards (including immunological effects) likely to arise from repeated exposure over a relative limited period of time.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 5p.p..The test item was administered at dose-levels of 100, 300 and 1000 mg/kg/day in the vehicle (corn oil). Another group of ten males and ten females received the vehicle, alone, under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used.

The concentration of the dose formulation was checked in study Weeks 1, 3 and 6 and one more time (in Week 2) for groups 1 and 3.

 

The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recordedonce a week during premating, mating (food consumption not during mating) and gestation (0, 7, 14 and 20p.c.) periods and during lactation on Days 1 and 5p.p..

Prior to sacrifice, blood samples were taken from five males and five females per group for analysis of hematology and blood biochemistry parameters.

The males were sacrificed after at least 5 weeks of treatment and the females on Day 6p.p..Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopicpost-mortemexamination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on several organs from five males and five females in the control and high-dose groups and from the dead animals, on forestomach from five males and five females in the mid-dose group and on all macroscopic lesions.

Results

 

Chemical analyses

There was no test item found in control formulation samples and the test item concentrations in the administered dose formulations analyzed were within an acceptable range of variations except in Week 1 for group 3 dose formulation which was found at +12.6%. However, the formulation was given only once to the animals and thus did not have an impact on the study conclusion.

 

Mortality

At 1000 mg/kg/day, there were two deaths considered to be test item treatment-related: one male prematurely sacrificed on Day 11 for poor clinical conditions and one female found dead on Day 19 p.c.. Both rats had at least loud breathing for several days before death. At pathology,there were no clear causes of death/moribundity. The dead animals had hyperplasia of forestomach with hyperkeratosis (grossly described as white discoloration in the female) probably related to the test item administration, and findings in the thymus and adrenals (female) which were stress-related.

There were no premature deaths at 100 and 300 mg/kg/day.

 

Clinical signs

At 1000 mg/kg/day, loud breathing and ptyalism were observed for a long time during the treatment period. Loud breathing was considered as adverse (serious clinical sign recorded in 14/18 surviving animals generally for a long time). Two animals also experienced piloerection, round back, eyes half-closed, dyspnea and/or abdominal breathing for a few days, and one other male was emaciated in Week 2.

At 300 mg/kg/day, ptyalism (7/20 animals) and loud breathing (1/20 animal) were considered to be of minor toxicological significance.

There were no test item-related clinical signs at 100 mg/kg/day.

Body weight

At 1000 mg/kg/day,when compared with controls, mean body weight was lower in males for the first half of the treatment period (down to -7%, p<0.01) along with a low mean body weight gain (mainly during the first treatment week: +9 gvs.+40 g in controls, p<0.001).From the third week of treatment, mean body weight gain increased (+53 gvs+30, p<0.001) but was nul in the last week of treatment (vs.+19 g in controls). In females, at the same dose, mean body weight was not affected; there was a tendency towards a low mean body weight gain in the first week of treatment (+5 gvs.+13 g), followed by a higher mean body weight gain the week after (+20 gvs.+11 g, p<0.05). These variations of low magnitude were considered to be non-adverse.

There were no test item-related effects at 100 and 300 mg/kg/day.

Food consumption

At 1000 mg/kg/day, when compared with controls, mean food consumption was lower during the first week of treatment in both sexes (-21/-22%, p<0.001/0.01) correlating with the concurrent decreased in mean body weight gain.

There were no test item-related effects at 100 and 300 mg/kg/day.

 

Hematology/Coagulation

There were no test item treatment-related effects at hematology.

At 1000 mg/kg/day, when compared with controls, males had minimally non-adverse prolonged mean prothrombin (23.4svs.20.1, p<0.05) and activated partial thromboplastin (15.7svs.13.3, p<0.05) times.

 

Biochemistry

There were no toxicologically significant findings at any dose-level.

Pathology

At terminal sacrifice, there were no test item-related mean organ weight differences and no test item-related gross findings.

At microscopy, slight non-adverse test item-related erosion/ulcer, with squamous cell hyperplasia and hyperkeratosis, were noted in 1/5 females treated at 1000 mg/kg/day.

Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until Day 5 p.p. for females, at dose-levels of 100, 300 and 1000 mg/kg/day.

Based on the experimental conditions and results of this study:

- the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 300 mg/kg/day, based on the two deaths and the loud breathing present in most animals at 1000 mg/kg/day.