Registration Dossier

Administrative data

Description of key information

An oral 28-day repeated toxicity study is available on rats and the data show that 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid caused stomach irritation in animals treated with doses equal of higher than 200 mg/kg/d. The NOAEL for systemic effect was considered to be 80 mg/kg/day.
A dermal 90-day studies on an analoguous substance to 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid showed a local irritation (NOAEL for local effects was 0,75 and 1,5 mg/kg/d, in rats and mice resspectively) but no systemic effects were observed in the animals treated with 12 mg/kg/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 October 2014 -- 14 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
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 Italia, Calco, Italy.
- Age at study initiation: approximately 5 weeks old.
- Mean body weight at study initiation: 174 g (range: 158 g to 193 g) for the males and 145 g (range: 126 g to 168 g) for the females.
- Fasting period before study: no
- Housing: 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: for a period of 8 days before the beginning of the treatment period.

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: 17 October 2014 to 14 November 2014
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The type of formulation (visual observation) is a solution in the vehicle.
The preparation procedure is according to Study of analyse homogeneity and stability, describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study.
The frequency of preparation (Test item dose formulations) is daily.
The delivery conditions are at room temperature, protected from light and sealed vials.

VEHICLE
- Justification for use and choice of vehicle: test item soluble in the vehicle and corn oil is commonly used for this type of study
- Concentration in vehicle: 5, 16 and 48 mg/mL
- Amount of vehicle (if gavage): 5mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations in the administered dose formulations analyzed in Weeks 1 and 4 were within the acceptable range of ± 10%.
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
Remarks:
Doses / Concentrations:
25, 80, and 240 mg/kg/day
Basis:
actual ingested
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 toxicology study.

- Rationale for animal assignment: computerized stratification procedure
Positive control:
no (not required)
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.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at the beginning of the treatment period and then once a week until the end of the study.
Each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: once before group allocation, then on the first day of treatment and 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 (see table below).

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

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.

Based upon the microscopic results of the high-dose group and after agreement of the Sponsor, the following tissues from the low- and intermediate-dose animals were examined:
- forestomach,
- stomach,
- trachea,
- cecum.
Other examinations:
no
Statistics:
yes
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
MORTALITY:
No unscheduled deaths occurred during the study.

CLINICAL SIGNS:
There were no test item-related clinical signs in animals treated at 25 mg/kg/day during the study. Reflux at dosing was noted on Day 17 in one female. This was considered incidental since it was transient and observed in one animal without relationship to dose-levels.

At 80 mg/kg/day, ptyalism was transiently observed in 3/5 males and in 2/5 females along with loud breathing in 2/5 males.

At 240 mg/kg/day, ptyalism was occasionally observed in all animals and loud breathing along with hunched posture and piloerection was noted in 1/5 males. Loud breathing was also observed in 2/5 females along with dyspnea and thin appearance in one of them.
These clinical signs were considered test item-related with a minor toxicological significance since they were transient and observed with a low incidence.
Other clinical signs (scabs and soiled around the mouth) were considered incidental since they are often observed in laboratory housed animals of this species and was observed at a low incidence.

BODY WEIGHT:
There were no toxicologically relevant test item-related changes in mean body weight and mean body weight gain during the study.
At 240 mg/kg/day, statistically significant difference in body weight change observed between Days 1 and 8 in males, was considered incidental since it was transient and isolated.

FOOD CONSUMPTION:
There were no changes in food consumption during the study.

NEUROBEHAVIOURAL EXAMINATION:
There were no toxicologically relevant test item-related effects at the Functional Observation Battery including motor activity (horizontal movements and rearing) in both genders.

Grooming was noted in 1/5 males at 80 and 240 mg/kg/day. Slight hypotonia was also observed in 1/5 males at 240 mg/kg/day. In view of the slight severity and incidence and in absence of correlating clinical signs during the study, these findings were considered to be unrelated to the test item treatment. Other changes observed at 240 mg/kg/day, limited to scabs, piloerection, hunched posture and loud breathing, were noted in 1/5 males. Loud breathing was also noted in 1/5 males at 80 mg/kg/day. These changes correlated with clinical signs observed.

HEMATOLOGY:
There were no test item-related effects on hematology parameters in males.
Changes in hematology parameters were restricted to statistically significant and not dose-related prolonged prothrombin time in females at ≥ 25 mg/kg/day, when compared to controls. In view of the minimal amplitude and the absence of dose-relationship, this difference was considered not toxicologically relevant.

CLINICAL CHEMISTRY:
There were no test item-related effects on biochemistry parameters in females.
Changes in biochemistry parameters were restricted to a slightly higher chloride level at ≥ 25 mg/kg/day (statistically significant at 25 mg/kg/day), when compared to controls. These changes were considered to be of no toxicological importance since they were of low amplitude and not dose related.
Other changes in biochemistry values (aspartate aminotransferase), including some that resulted in statistically significant differences between groups, were considered incidental since they reflected the normal inter-animal variation in this species.

URINALYSIS:
There were no test item-related effects on urinary parameters in both genders.
A very high urinary volume associated with a lower specific gravity and a pH equal to 7 was noted at 80 mg/kg/day in 1/5 males and at 240 mg/kg/day in 2/5 males, compared to controls. In view of the direction of the changes and the very high urinary volume, these differences were considered not toxicologically relevant. Therefore a contamination of urinary samples with drinking water was suspected.

ORGAN WEIGHTS:
The test item administration did not induce any changes in organ weights.

Organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, and/or were not dose-related in magnitude. The minimal, not dose related increases in the mean testis and epididymis weights in all test item-treated groups were due to low testis and epididymis weights of control male which showed severe testicular atrophy with no sperm in the epididymides.

GROSS PATHOLOGY:
Test item-related macroscopic changes were seen in the forestomach at 240 mg/kg/day.
Many white, often raised foci, with occasional thickening were noted on the wall of the forestomach in 4/5 males and 3/5 females at 240 mg/kg/day. These foci correlated with squamous hyperplasia and hyperkeratosis of the mucosa at microscopic examination.
The other macroscopic findings had no histologic correlates or correlated with common histologic findings in control rats, and were considered to be incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC (see Table 1):
The main test item-related microscopic changes were found in the forestomach at 240 mg/kg/day in both sexes, males being more affected than females. Additional changes at 240 mg/kg/day were noted in the glandular stomach in both sexes, and in the trachea and bone marrow in males.

Forestomach and stomach
Minimal to marked, multifocal to diffuse hyperplasia (i.e. increased thickness) of the squamous epithelium was observed in all animals at 240 mg/kg/day. This was associated in most animals with minimal or slight hyperkeratosis (i.e. increased thickness of the keratin layer) and slight or moderate subacute inflammation. In some males, there were minimal focal or multifocal erosions. Inflammation was mainly located in the submucosa and was characterized by mixed inflammatory cell infiltrates with eosinophils and edema.
In the glandular stomach, minimal multifocal hyperplasia of the glands was noted in a few animals with inflammation in a single animal.
There were no significant microscopic changes in the forestomach and stomach at 25 and 80 mg/kg/day.

Trachea
Minimal epithelial degeneration/regeneration characterized by loss of cilia, flattening and horizontal orientation of surface epithelial cells was noted in 2/5 males which both showed hyperplasia and inflammation in the forestomach at 240 mg/kg/day.
There were no significant microscopic changes in the trachea at 25 and 80 mg/kg/day.

Bone marrow
In 2/5 males which both showed hyperplasia and inflammation in the forestomach at 240 mg/kg/day, the myeloid cell numbers were minimally increased.
Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the rat. Among them was minimal mucosal inflammation in the cecum which was seen with higher incidence in animals given the test item at all doses (respectively 1 3 4 4 males and 1-2-0-2 females at 0-25-80-240 mg/kg/day). This inflammation consisted of mixed inflammatory cell infiltrate including eosinophils in the lamina propria of the mucosa, with occasional elongation of the crypts. In the absence of dose-relationship, given its occasional occurrence in controls, and in the absence of inflammation in the small intestine at the highest dose-level, this finding was considered as incidental and unrelated to the test item administration.
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Table 1

Incidence and severity of test item-related microscopic findings in the forestomach and stomach (n = 5)

 

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

25

80

240

0

25

80

240

Forestomach

 

 

 

 

 

 

 

 

- Hyperplasia; squamous cell

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

-

-

-

-

-

2

Slight (grade 2)

-

-

-

1

-

-

-

1

Moderate (grade 3)

-

-

-

4

-

-

-

1

Marked (grade 4)

-

-

-

-

-

-

-

1

- Hyperkeratosis

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

-

2

-

-

-

2

Slight (grade 2)

-

-

-

3

-

-

-

1

- Inflammation

 

 

 

 

 

 

 

 

Slight (grade 2)

-

-

-

4

-

-

-

2

Moderate (grade 3)

-

-

-

1

-

-

-

-

- Erosion

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

-

3

-

-

-

-

Stomach

 

 

 

 

 

 

 

 

- Hyperplasia; gland

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

-

3

-

-

-

1

- Inflammation

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

-

1

-

-

-

-

-: no findings.

 

Conclusions:
Once daily oral administration of the test item to Sprague-Dawley rats at dose-levels of 25, 80 or 240 mg/kg/day for 4 weeks, resulted in the absence of premature deaths or mortalities, no relevant adverse clinical signs, no adverse findings in in vivo parameters and in laboratory parameters at any dose-levels.
At 240 mg/kg/day, adverse inflammatory, erosive and/or hyperplasic changes in the forestomach and to a lesser extent in the stomach and trachea were recorded in the microscopic examination. These observations are considered to be adverse and were indicative of irritant properties of the test item.
Consequently, under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 80 mg/kg/day.
Executive summary:

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

 

Methods

Three groups of five male and female Sprague-Dawley rats received the test item daily by oral administration for 4 weeks at dose-levels of 25, 80 or 240 mg/kg/day. The test item was administered as a solution in the vehicle (corn oil). A control group of five male and female Sprague-Dawley rats received the vehicle alone under the same experimental conditions.

Mortality and morbidity were checked once a day during the acclimation period and at least twice a day during the treatment period. Clinical signs were recorded once a day during the study. 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 including motor activity measurement, and hematology, blood biochemistry and urinalysis investigations were performed on all animals. On completion of the treatment period, all 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 and on macroscopic lesions.

 

Results

No clinical signs were observed at 25 mg/kg/day andno toxicologically significant effects on mean body weight, mean body weight change and mean food consumption were noted at ≥ 25 mg/kg/day. In addition, there were no relevant effects at the Functional Observation Battery, on motor activity and in hematology, blood chemistry and urinary parameters at ≥ 25 mg/kg/day.

At 80 mg/kg/day, treatment-related clinical signs were limited to transient ptyalism (3 males, 2 females) and loud breathing (2 males). At 240 mg/kg/day the same clinical signs were occasionally noted in males and females along with hunched posture and piloerection in one male, dyspnea and thin appearance in one female.

The test item administration did not induce any changes in organ weights. The main histopathological changes induced by the test item administration at 240 mg/kg/day were seen in the forestomach and were indicative of irritant properties of the test item. They consisted of squamous cell hyperplasia and hyperkeratosis (correlated with macroscopic white discoloration), subacute inflammation and low grade erosions. Low grade inflammation and hyperplasia were additionally noted in the glandular stomach in a few animals at this dose-level. Minimal epithelial degeneration/regeneration was noted in the trachea in two males at this dose-level and may have been related to irritation consecutive to aspiration of the test item during the gavage procedure. Increased myeloid cell numbers seen in the bone marrow of two males at 240 mg/kg/day were considered to be related to the inflammation noted in the forestomach. There were no significant macroscopic or microscopic changes at 25 and 80 mg/kg/day.

Conclusion

Once daily oral administration of the test item to Sprague-Dawley rats at dose-levels of 25, 80 or 240 mg/kg/day for 4 weeks, resulted in the absence of premature deaths or mortalities, no relevant adverse clinical signs, no adverse findings in in vivo parameters and in laboratory parameters at any dose-levels.

At 240 mg/kg/day, adverse inflammatory, erosive and/or hyperplasic changes in the forestomach and to a lesser extent in the stomach and trachea were recorded in the microscopic examination. These observations are considered to be adverse and were indicative of irritant properties of the test item. Consequently, under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 80 mg/kg/day.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
This guideline study is considered to be reliable

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

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 16, 1996 to December 18, 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted by using method equivalent to standard guidelines in compliance with GLP. Pentaerythritol triacrylate is a constituent of PETIA.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Taconic Laboratory Animals and Services (Germantown, NY).
- Age at study initiation: 6 weeks
- Housing: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed at least once per week, rotated every 2 weeks
- Bedding: Sani-Chip® hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ), changed at least once per week. Bedding was irradiated
- Diet: NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
- Water: Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature: 72°±3° F
- Humidity: 50±15%
- Air changes: 10/h
- Photoperiod: 12 h light/dark cycle
Type of coverage:
not specified
Vehicle:
acetone
Details on exposure:
TEST MATERIAL
- Amount(s) applied: 0.5 mL/kg

VEHICLE
- Lot/batch no.: KP206 and LS0051
- Purity: Greater than 99%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed at the beginning, midpoint, and end of the study; animal room samples of these dose formulations were also analysed. Of the dose formulations analyzed, all 15 were within 10% of the target concentration, with no value greater than 104% of the target concentration; 10 of 15 animal room samples were within 10% of the target concentration.
Duration of treatment / exposure:
3 month
Frequency of treatment:
5 days per week for 14 weeks
Remarks:
Doses / Concentrations:
0, 0.75, 1.5, 3, 6, or 12 mg/kg bw
Basis:
analytical per unit body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Animals were observed twice daily and were weighed initially, weekly, and at the end of the studies. Clinical findings were recorded weekly and at the end of the studies for core study animals. Blood was collected from the retroorbital sinus on Days 4 and 23 and at the end of the studies for hematology and clinical chemistry.
Hematology: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte morphology; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials
Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids
Sacrifice and pathology:
Necropsies were performed on all animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus. Complete histopathologic examinations were performed on vehicle control and 12 mg/kg bw rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart with aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin (site of application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus. The skin at the site of application was also examined.
Other examinations:
At the end of the studies, sperm samples were collected from male animals in the vehicle control and 3, 6, and 12 mg/kg bw groups for sperm count and motility evaluations. The following parameters were evaluated: spermatid heads per testis or cauda and per gram testis or cauda and epididymal spermatozoal motility. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from females in the vehicle control and 3, 6, and 12 mg/kg bw groups for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Dermal irritation:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Details on results:
All rats survived to the end of the study. Final mean body weights and body weight gains of 12 mg/kg bw males were significantly less than those of the vehicle controls. Irritation at the site of application occurred in 12 mg/kg bw rats.

On Day 23, an increase in segmented neutrophils occurred in 12 mg/kg bw males and females. At study termination, an increased neutrophil count occurred in 6 mg/kg bw males. The neutrophilia would be consistent with skin inflammation observed microscopically. No other hematology or clinical chemistry changes were considered to be toxicologically relevant.

Thymus weights of males administered 3 mg/kg bw or greater were significantly less than those of the vehicle controls. There were no significant differences in sperm motility or vaginal cytology parameters between dosed groups and the vehicle controls.

No treatment-related lesions were observed grossly in rats except irritation at the site of application in the 12 mg/kg bw groups. Microscopically, the primary changes at the site of application consisted of epidermal hyperplasia, hyperkeratosis, epidermal degeneration and necrosis, and chronic active inflammation. The incidence and severity of hyperplasia increased with increasing dose; most animals administered 3 mg/kg bw or greater were affected. Severity was minimal to mild and characterized by focally extensive to diffuse increased thickness of the epidermis, from the normal one to three cell layers thick to four to six layers thick. Hyperplasia was accompanied by minimal to mild increased thickness of the superficial keratin layer (hyperkeratosis). Minimal hyperkeratosis without accompanying hyperplasia was present in the 0.75 and 1.5 mg/kg bw groups. Degeneration was diagnosed in many animals treated with 1.5 mg/kg bw or greater.

Degeneration was a minimal focal change consisting of intraepidermal vacuolization, presumably due to intra or intercellular fluid accumulation. Vacuoles occasionally coalesced to form small vesicles that contained a few neutrophils. Epidermal necrosis was present in some males, although a dose-related response was not clear. Necrosis consisted of partial to full-thickness coagulative change of the epidermis and was likely a pathogenic sequela of degeneration. Intraepidermal infiltration of neutrophils (suppurative inflammation) often accompanied degeneration or necrosis of the epidermis. A mixed inflammatory cell infiltrate (chronic active inflammation) was present in the dermis of animals administered 1.5 mg/kg bw or greater, with dose dependent increases in incidences and severity. Sebaceous glands at the site of application were slightly enlarged and prominent in animals with the other changes described above.
Dose descriptor:
NOAEL
Effect level:
0.75 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: Irritation
Critical effects observed:
not specified
Executive summary:

A study was conducted to assess the repeated dose dermal toxicity of the test substance in male and female rats.

Groups of 10 male and 10 female rats received dermal applications of 0, 0.75, 1.5, 3, 6, or 12 mg/kg bw of test substance in acetone, 5 days per week for 14 weeks; the dosing volume was 0.5 mL/kg bw. Animals were observed twice daily and were weighed initially, weekly, and at the end of the studies. Clinical findings were recorded weekly and at the end of the studies for core study animals. Necropsies were performed on all animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus. Blood was collected from the retroorbital sinus on Days 4, 23 and at the end of the studies for hematology and clinical chemistry.

All rats survived to the end of the study. Final mean body weights and body weight gains of 12 mg/kg bw males were significantly less than those of the vehicle controls. Irritation at the site of application occurred in 12 mg/kg bw rats. On Day 23, an increase in segmented neutrophils occurred in 12 mg/kg bw males and females. At study termination, an increased neutrophil count occurred in 6 mg/kg bw males. The neutrophilia would be consistent with skin inflammation observed microscopically. No other hematology or clinical chemistry changes were considered to be toxicologically relevant. Thymus weights of males administered 3 mg/kg bw or greater were significantly less than those of the vehicle controls. There were no significant differences in sperm motility or vaginal cytology parameters between dosed groups and the vehicle controls. No treatment-related lesions were observed grossly in rats except irritation at the site of application in the 12 mg/kg bw groups. Microscopically, the primary changes at the site of application consisted of epidermal hyperplasia, hyperkeratosis, epidermal degeneration and necrosis, and chronic active inflammation. The local NOAEL for this study could be considered to be 0.75 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 16, 1996 to December 18, 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted by using method equivalent to standard guidelines in compliance with GLP. Pentaerythritol triacrylate is a constituent of PETIA.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Taconic Laboratory Animals and Services (Germantown, NY).
- Age at study initiation: 6 weeks
- Housing: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed at least once per week, rotated every 2 weeks
- Bedding: Sani-Chip® hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ), changed at least once per week. Bedding was irradiated
- Diet: NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
- Water: Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature: 72°±3° F
- Humidity: 50±15%
- Air changes: 10/h
- Photoperiod: 12 h light/dark cycle
Type of coverage:
not specified
Vehicle:
acetone
Details on exposure:
TEST MATERIAL
- Amount(s) applied: 0.5 mL/kg

VEHICLE
- Lot/batch no.: KP206 and LS0051
- Purity: Greater than 99%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed at the beginning, midpoint, and end of the study; animal room samples of these dose formulations were also analysed. Of the dose formulations analyzed, all 15 were within 10% of the target concentration, with no value greater than 104% of the target concentration; 10 of 15 animal room samples were within 10% of the target concentration.
Duration of treatment / exposure:
3 month
Frequency of treatment:
5 days per week for 14 weeks
Remarks:
Doses / Concentrations:
0, 0.75, 1.5, 3, 6, or 12 mg/kg bw
Basis:
analytical per unit body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Animals were observed twice daily and were weighed initially, weekly, and at the end of the studies. Clinical findings were recorded weekly and at the end of the studies for core study animals. Blood was collected from the retroorbital sinus on Days 4 and 23 and at the end of the studies for hematology and clinical chemistry.
Hematology: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte morphology; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials
Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids
Sacrifice and pathology:
Necropsies were performed on all animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus. Complete histopathologic examinations were performed on vehicle control and 12 mg/kg bw rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart with aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin (site of application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus. The skin at the site of application was also examined.
Other examinations:
At the end of the studies, sperm samples were collected from male animals in the vehicle control and 3, 6, and 12 mg/kg bw groups for sperm count and motility evaluations. The following parameters were evaluated: spermatid heads per testis or cauda and per gram testis or cauda and epididymal spermatozoal motility. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from females in the vehicle control and 3, 6, and 12 mg/kg bw groups for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Dermal irritation:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Details on results:
All rats survived to the end of the study. Final mean body weights and body weight gains of 12 mg/kg bw males were significantly less than those of the vehicle controls. Irritation at the site of application occurred in 12 mg/kg bw rats.

On Day 23, an increase in segmented neutrophils occurred in 12 mg/kg bw males and females. At study termination, an increased neutrophil count occurred in 6 mg/kg bw males. The neutrophilia would be consistent with skin inflammation observed microscopically. No other hematology or clinical chemistry changes were considered to be toxicologically relevant.

Thymus weights of males administered 3 mg/kg bw or greater were significantly less than those of the vehicle controls. There were no significant differences in sperm motility or vaginal cytology parameters between dosed groups and the vehicle controls.

No treatment-related lesions were observed grossly in rats except irritation at the site of application in the 12 mg/kg bw groups. Microscopically, the primary changes at the site of application consisted of epidermal hyperplasia, hyperkeratosis, epidermal degeneration and necrosis, and chronic active inflammation. The incidence and severity of hyperplasia increased with increasing dose; most animals administered 3 mg/kg bw or greater were affected. Severity was minimal to mild and characterized by focally extensive to diffuse increased thickness of the epidermis, from the normal one to three cell layers thick to four to six layers thick. Hyperplasia was accompanied by minimal to mild increased thickness of the superficial keratin layer (hyperkeratosis). Minimal hyperkeratosis without accompanying hyperplasia was present in the 0.75 and 1.5 mg/kg bw groups. Degeneration was diagnosed in many animals treated with 1.5 mg/kg bw or greater.

Degeneration was a minimal focal change consisting of intraepidermal vacuolization, presumably due to intra or intercellular fluid accumulation. Vacuoles occasionally coalesced to form small vesicles that contained a few neutrophils. Epidermal necrosis was present in some males, although a dose-related response was not clear. Necrosis consisted of partial to full-thickness coagulative change of the epidermis and was likely a pathogenic sequela of degeneration. Intraepidermal infiltration of neutrophils (suppurative inflammation) often accompanied degeneration or necrosis of the epidermis. A mixed inflammatory cell infiltrate (chronic active inflammation) was present in the dermis of animals administered 1.5 mg/kg bw or greater, with dose dependent increases in incidences and severity. Sebaceous glands at the site of application were slightly enlarged and prominent in animals with the other changes described above.
Dose descriptor:
NOAEL
Effect level:
0.75 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: Irritation
Critical effects observed:
not specified
Executive summary:

A study was conducted to assess the repeated dose dermal toxicity of the test substance in male and female rats.

Groups of 10 male and 10 female rats received dermal applications of 0, 0.75, 1.5, 3, 6, or 12 mg/kg bw of test substance in acetone, 5 days per week for 14 weeks; the dosing volume was 0.5 mL/kg bw. Animals were observed twice daily and were weighed initially, weekly, and at the end of the studies. Clinical findings were recorded weekly and at the end of the studies for core study animals. Necropsies were performed on all animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus. Blood was collected from the retroorbital sinus on Days 4, 23 and at the end of the studies for hematology and clinical chemistry.

All rats survived to the end of the study. Final mean body weights and body weight gains of 12 mg/kg bw males were significantly less than those of the vehicle controls. Irritation at the site of application occurred in 12 mg/kg bw rats. On Day 23, an increase in segmented neutrophils occurred in 12 mg/kg bw males and females. At study termination, an increased neutrophil count occurred in 6 mg/kg bw males. The neutrophilia would be consistent with skin inflammation observed microscopically. No other hematology or clinical chemistry changes were considered to be toxicologically relevant. Thymus weights of males administered 3 mg/kg bw or greater were significantly less than those of the vehicle controls. There were no significant differences in sperm motility or vaginal cytology parameters between dosed groups and the vehicle controls. No treatment-related lesions were observed grossly in rats except irritation at the site of application in the 12 mg/kg bw groups. Microscopically, the primary changes at the site of application consisted of epidermal hyperplasia, hyperkeratosis, epidermal degeneration and necrosis, and chronic active inflammation. The local NOAEL for this study could be considered to be 0.75 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
Study duration:
subchronic
Species:
rat

Additional information

Preliminary study in rats of 14/28 days (Chevalier 2015)

The objective of this preliminary toxicity study was to evaluate the potential toxic effects of the test item, 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid following daily (or bidaily) oral administration (gavage) to male and female rats for 2 days up to 4 weeks. A total of 41 male and 41 female rats were used in this study. One group of ten male and ten female rats were treated with vehicle only (corn oil). Three groups of ten male and ten female Sprague‑Dawley rats received the test item, daily or bidaily by oral (gavage) administration as an emulsion in the vehicle, corn oil, at dose-levels of 100/200, 300 or 600/1000 mg/kg/day. The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs during the treatment period. Body weight and food consumption were recorded weekly. At the end of the treatment period, surviving animals from groups 1 and 2 were sacrificed, selected organs weights were recorded and a complete macroscopic post-mortem examination was performed. No microscopic examination was performed.

In the group 2 (100/200 mg/kg/day), no deaths were observed, however hypersalivation was recorded in all the animals during the whole dosing period. The body weight was considered to be unaffected in this group. At the necropsy, macroscopic thickening, irregular surface and/or white deposits were seen in the forestomach of males and females.

All animals of the groups 3 and 4 died or sacrificed prematurely showed numerous signs of poor health (mainly respiratory difficulties) on the day(s) preceding the death.The mortality was considered to be related to the irritant properties of the test item, with consecutive gastro-intestinal findings mainly. In the group 4, males lost weight and females gained less weight than controls. In the group 3, body weight gain of males was lower than controls. Food consumption of males was severely affected in the group 4, andlower than controls in the group 3.However,food consumption of males (group 1) and females (all groups) was considered to be comparable to that of the control group.

To conclude, all the animals treated with the test item, above 300 mg/kg/day were found dead or prematurely sacrified. These deaths were related to the irritant properties of the test item.Ante‑mortem clinical signs consisted mainly of respiratory difficulties, hypersalivation and numerous other signs of poor health. At the dose-levels above 300 mg/kg/day, body weight and food consumption were affected in males and/or females.

At 100/ 200 mg/kg/day, clinical signs were limited to hypersalivation, the body weight of animals was not affected, and the necropsy revealed the presence ofthickening, irregular surface and/or white deposits in the forestomach.

Based on the results of this study, the dose-levels above 300 mg/kg/day are considered to be exceeded the Maximum Tolerated Dose.

4 -week toxicity study (Leclere 2015)

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration to rats for 4 weeks (OECD 407). 

Three groups of five male and female Sprague-Dawley rats received the test itemdaily by oral administration for 4 weeks at dose-levels of 25, 80 or 240 mg/kg/day. The test item was administered as a solution in the vehicle (corn oil). A control group of five male and female Sprague-Dawley rats received the vehicle alone under the same experimental conditions.

No clinical signs were observed at 25 mg/kg/day andno toxicologically significant effects on mean body weight, mean body weight change and mean food consumption were noted at ≥ 25 mg/kg/day. In addition, there were no relevant effects at the Functional Observation Battery, on motor activity and in hematology, blood chemistry and urinary parameters at ≥ 25 mg/kg/day.

At 80 mg/kg/day, treatment-related clinical signs were limited to transient ptyalism (3 males, 2 females) and loud breathing (2 males). At 240 mg/kg/day the same clinical signs were occasionally noted in males and females along with hunched posture and piloerection in one male, dyspnea and thin appearance in one female.

The test item administration did not induce any changes in organ weights. The main histopathological changes induced by the test item administration at 240 mg/kg/day were seen in the forestomach and were indicative of irritant properties of the test item. They consisted of squamous cell hyperplasia and hyperkeratosis (correlated with macroscopic white discoloration), subacute inflammation and low grade erosions. Low grade inflammation and hyperplasia were additionally noted in the glandular stomach in a few animals at this dose-level. Minimal epithelial degeneration/regeneration was noted in the trachea in two males at this dose-level and may have been related to irritation consecutive to aspiration of the test item during the gavage procedure. Increased myeloid cell numbers seen in the bone marrow of two males at 240 mg/kg/day were considered to be related to the inflammation noted in the forestomach. There were no significant macroscopic or microscopic changes at 25 and 80 mg/kg/day.

To conclude, once daily oral administration of the test item to Sprague-Dawley rats at dose-levels of 25, 80 or 240 mg/kg/day for 4 weeks, resulted in the absence of premature deaths or mortalities, no relevant adverse clinical signs, no adverse findings in in vivo parameters and in laboratory parameters at any dose-levels. At 240 mg/kg/day, adverse inflammatory, erosive and/or hyperplasic changes in the forestomach and to a lesser extent in the stomach and trachea were recorded in the microscopic examination. These observations are considered to be adverse and were indicative of irritant properties of the test item. Consequently, under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 80 mg/kg/day.

90 -day repeated toxicity study by dermal route on rats and mice (read-across with analoguous substance)

A 3 month study was conducted to assess the repeated dose dermal toxicity of PETIA in mice. Groups of 10 male and 10 female mice received dermal applications of 0, 0.75, 1.5, 3, 6, or 12 mg/kg bw of test substance in acetone, 5 days per week for 14 weeks. Animals were observed twice daily and were weighed initially, weekly and at the end of the studies. Clinical findings were recorded weekly and at the end of the studies for core study animals. Blood was collected from the retroorbital sinus from core study mice at the end of the study for hematology. Necropsies were performed on all animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus. Complete histopathologic examinations were performed on vehicle control and 12 mg/kg bw mice. At the end of the studies, sperm samples were collected from male animals in the vehicle control and 3, 6, and 12 mg/kg bw groups for sperm count and motility evaluations. Mean body weights of dosed groups were similar to those of the vehicle control groups. Irritation at the site of application occurred in the 6 and 12 mg/kg bw male groups. Hematology results indicated an increased neutrophil count consistent with an inflammatory response related to the dermatitis observed histopathologically. There also was a minimal decrease in the erythron (hematocrit, hemoglobin concentration, and erythrocyte count) likely secondary to the inflammatory skin process. There were no biologically significant differences in organ weights between dosed and vehicle control groups. No lesions were observed grossly in mice except irritation at the site of application in 6 and 12 mg/kg bw males. The primary microscopic changes at the site of application were epidermal hyperplasia, degeneration, and necrosis; dermal chronic active inflammation, and sebaceous gland hyperplasia (Hejtmancik, 2005). The local NOAEL for this study could be considered to be 1.5 mg/kg bw/day.

A 3 month study was conducted to assess the repeated dose dermal toxicity of PETIA in rats. Groups of 10 male and 10 female rats received dermal applications of 0, 0.75, 1.5, 3, 6, or 12 mg/kg bw of test substance in acetone, 5 days per week for 14 weeks. Animals were observed twice daily and were weighed initially, weekly and at the end of the studies. Clinical findings were recorded weekly and at the end of the studies for core study animals. Necropsies were performed on all animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus. Blood was collected from the retroorbital sinus on Days 4, 23 and at the end of the studies for hematology and clinical chemistry. All rats survived to the end of the study. Final mean body weights and body weight gains of 12 mg/kg bw males were significantly less than those of the vehicle controls. Irritation at the site of application occurred in 12 mg/kg bw rats. On Day 23, an increase in segmented neutrophils occurred in 12 mg/kg bw males and females. At study termination, an increased neutrophil count occurred in 6 mg/kg bw males. The neutrophilia would be consistent with skin inflammation observed microscopically. No other hematology or clinical chemistry changes were considered to be toxicologically relevant. Thymus weights of males administered 3 mg/kg bw or greater were significantly less than those of the vehicle controls. There were no significant differences in sperm motility or vaginal cytology parameters between dosed groups and the vehicle controls. No treatment-related lesions were observed grossly in rats except irritation at the site of application in the 12 mg/kg bw groups. Microscopically, the primary changes at the site of application consisted of epidermal hyperplasia, hyperkeratosis, epidermal degeneration and necrosis, and chronic active inflammation (Hejtmancik, 2005). The local NOAEL for this study could be considered to be 0.75 mg/kg bw/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one repeated toxicity study is available on 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Two studies are available by dermal route on an analoguous substance of 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid; the rat study is chosen because it is the best specie to extrapolate the data to humans, and the NOAEL was lower than in the mice study.

Justification for classification or non-classification

Based on the 28 -day repeated toxicity study, no specific organ toxicity was observed, therefore no classification is required according to the Regulation EC n°1272/2008 and the Directive 67/548/EEC.