Hexamethylene diacrylate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun - Sep 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 424 (Neurotoxicity Study in Rodents)

Principles of method if other than guideline:

The subchronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic effects.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

EC Number:

256-032-2

EC Name:

(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

Cas Number:

42978-66-5

Molecular formula:

C15 H24 O6

IUPAC Name:

(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): C-178
- Physical state: liquid
- Analytical purity: 100% active ingredient
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 41 days (age: 28 days at receipt)
- Weight at study initiation: week 0 males: 157-163 g; week 0 females: 133-139 g
- Housing: individually in elevated stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:

other: not occluded

Vehicle:

corn oil

Details on exposure:

TEST SITE
- Area of exposure: the back of the rats
- % coverage: no data
- Type of wrap if used: not occluded no further data
- Time intervals for shavings or clipplings: all animals were clipped ca. 23 h prior to initial dose. The animals were reclipped when necessary.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.077 ml/kg
- Concentration (if solution): 1.0, 3.33 and 10.0 %
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): immiscible with water
- Amount(s) applied (volume or weight with unit): 2.077 ml 7kg of the test substance in corn oil
- Concentration (if solution): 1.0, 3.33 and 10.0 % of the test substance in corn oil

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

the remaining samples of weekly dosing solutions for each dose were returned to the sponsor for analysis, no further data

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

66.666 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: for mortality and gross signs of toxicologic or pharmacologic effects

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly including signs of local or systemic toxicity, pharmacologic effects and palpation for tissue masses

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest, weekly during treatment and terminally (after fasting)


OTHER:
- blood was obtained from over night fasted rats via venipuncture of the orbital sinus under light ether anestehsia, the same animals were used that were intended for formalin-fixation (5/sex/dose).
- hematology upon termination: hemoglobin, hematocrit, erythrocytes, clotting time, total and differential, leukocytes, erythrocytes morphology
- clinical chemistry upon termination: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, blood urea nitrogen, fasting glucose, total protein, total bilirubin, sodium, potassium, calcium, inorganic phosphorus
- urinanalysis 6 days before termination: gross appearance, specific gravity, pH, protein, glucose, occult blood

Sacrifice and pathology:

One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed
in formalin. Organ and organ-body weight ratios (adrenals, brain, liver, kidney, heart, spleen, testes with epididymides, ovaries) were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues (liver, kidney, lung, heart, stomach, adrenal, pituitary, testes, ovaries,
spleen and skeletal muscle) were conducted on all formalin-fixed control animals and the high dose animals. The remaining animals were perfused
intravenously with glutaraldehyde under sodium pentobarbital anesthesia . Quantitative assessments of teased tibial nerve preparations were
performed on all glutaraldehyde-perfused animals in control animals and the high dose animals. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast blue staining) from these same animals.

Other examinations:

- neurologic functions were evaluated monthly
- Parameters examined according to a scoring system: posture, gait, muscular tone, reflexes (corneal), righting and toe-pinch
- no further data

Statistics:

Body weight, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of
all dose groups were compared to control at each time interval.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Weekly physical examinations of all animals failed to indicate any toxic effects of the test material other than irritation at the application site. Alopecia observed on the forepaws and legs was the most common finding in both sexes; however, the incidence was spread over all test groups, including control.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

The test substance produced moderate levels of irritation, in a dose-related manner, beginning the first week of the study. Males were generally more susceptible than females to the dermal effects of the test substance throughout the study. Dermal effects (erythema and eschar formation) were only scored as present or absent therefore, the number of times per week the effects were noted was used as a general indication of the severity of the dermal observations.
- 20 mg/kg/day: erythema noted occassionally during the final 2 months; more frequently in the initial 3 weeks; exfoliation observed in approximately one-half during weeks 2 and 3 with diminishing frequency
- 66.6 mg/kg/day: erythema noted with a somewhat higher frequency than 20 mg/kg/day, frequency in females comparable to control. Exfoliation and eschar recorded for most animals by week 3.
- 200 mg/kg/day: erythema, exfoliation, and eschar seen in most animals of both sexes beginning in week 1. Atonia was observed in one males and one female, fissures present in one female and four males. A persistant fissuring was observed in one male rat from week 2 through week 7.
Males appeared somewhat more sensitive than females to erythema and eschar formation.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled termination of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly reduced body weights in high dose male rats (versus control male rats) were noted from weeks 3 through 12, except during weeks 10 and 11. Male rat weights of the low and mid dose groups were also reduced in dose-related fashion, but the differences were not statistically significant in comparison to control values. No statistically significant effects on weight were seen in treated female rats, however, high dose female animals never exceeded 95% of the mean weights of control females after 2 weeks on test.

Food consumption and compound intake (if feeding study):

not examined

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):

None of the hematologic parameters evaluated differed significantly from control values.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Of the clinical chemical parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Protein (2+, 100 mg/dl) was confirmed to be present in the urine of one mid-dose male and female, in two high-dose males and one high-dose female. A large amount of occult blood was also present in the urine of this one high-dose female. The specific gravity of this high-dose female and one mid-dose female was also high (>1.090). These findings suggested a possible effect of the test substance on the kidneys, but no alterations were observed in the histopathological examination to support this conclusion. Additionally, without relation to urinary volumes and creatinine, the relevance of in urinary protein concentration is questionable. All in all, the singular changes are not considered adverse by the registrant.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was a significant downward trend in male liver weights; however, this was not evident in the liver/body weight ratio and is therefore of doubtful significance. Other organ weights and organ/body weight ratios were comparable across all groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Formalin-fixed rats:
No changes, gross or microscopic, were evident which could be attributed to a systemic toxic effect of the test substance. The most common spontaneous gross necropsy findings, occurring across all groups, were inflammations around the ear tags, and slight hair loss on the extremities. No unusual microscopic pathological findings were evident which could be attributed to the topical administration of the test substance.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Month 1 neurological function tests showed two high-dose males with slightly reduced corneal response. All other evaluations were normal.
Month 2 neurological function tests were unremarkable in control, low and mid dose animals (both sexes). Four high dose males and three high dose females showed slightly abnormal gait described as "stilted". A slight decreased corneal reflex was observed in four males and one female. A moderately decreased toe pinch response (hindtoes only) was also present in one male rat.
Month 3 neurological function tests showed a slightly stilted gait and altered righting reflex in one male control rat and a slightly relaxed body tone in one mid dose male rat. One male high dose animal continued to exhibit a moderately decreased toe-pinch response (hindtoes only) . All neurological observations were normal in both the control and treated female rats at Month 3.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Glutaraldehyde-perfused rats:
Histopathological examinations of hematoxylin and eosin and Luxol-fast Blue stained slides of brain, spinal cord and sciatic nerve from ten rats (5/sex) treated with the high dose of the test substance failed to reveal any treatment-related lesions when these tissues were compared to similar ones from ten control rats (5/sex). In addition, microscopic examination of 50 teased nerve fibers from the tibial nerve of ten high-dose animals (5/sex) were comparable to those of the controls. When quantitative measurements were taken of myelinated nerve fibers in cross-section of the distal sciatic nerve, a slight shift to larger diameters could be detected in high-dose males when compared to the controls. However, there was also a slight decrease in fiber diameters in treated females. The relatively large standard deviation in data from both males and females suggest that these slight changes in fiber diameters are not significant. Moreover, the absence of lesions by more conventional histopathological examinations substantiate this conclusion.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

LOAEL

Remarks:

skin irritation

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

2

Dose descriptor:

NOAEL

Remarks:

for systemic toxicity

Effect level:

66.66 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

3

Dose descriptor:

NOAEL

Remarks:

for systemic toxicity

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

20 mg/kg bw/day (nominal)

System:

integumentary

Organ:

skin

Treatment related:

yes

Dose response relationship:

yes

Applicant's summary and conclusion

Conclusions:

The test substance did not induce neurotoxicity and no sub-chronic toxicity other than dermal irritation and decreased body weight was observed in animals, under the conditions tested. Thus, the NOAEL for local effects was considered to be 20 mg/kg/day and the NOAEL for systemic effects (based on body weight) was concluded to be 66.6 mg/kg/day.

Executive summary:

The sub-chronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic toxic effects. Three groups of 20 rats each (10/sex/level) were treated topically with 20, 66 2/3, and 200 mg/kg/day 5 days per week for 3 months. Twenty control animals (10/sex) were treated with corn oil. Solutions at appropriate levels were prepared in corn oil and a constant dose volume (2.077 ml/kg) was applied to all animals. Treatment sites were not occluded. Dermal observations were performed pretest and 5 times/week throughout the study. Clinical laboratory studies were performed at termination. Neurological function evaluations were performed at months 1, 2, and 3. At three months, all animals were terminated. One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed in formalin. Organ and organ body weight ratios were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues were conducted on all formalin-fixed Group 1 and 4 animals. The remaining animals were perfused intravenously with glutaraldehyde under sodium pentobarbital aesthesia. Quantitative assessments of teased tibial nerve preparations were performed on all glutaraldehyde-perfused animals in Group 1 and 4. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast Blue staining) from these same animals. Erythema, eschar and exfoliation were recorded during the initial week of the study and maximum frequency of these effects were seen during Weeks 2 and 3. A dose-response was noted, with males being slightly more susceptible. In the last 2 months of the study most treated animals developed an apparent tolerance to the irritant effects of the test substance. Significantly lower body weights were recorded throughout the study for Group IV males (200 mg/kg/day). Body weights for female rats and Group II and III male rats were not significantly affected. Routine toxicologic and pharmacologic signs were unremarkable throughout the study. Month 2 neurologic evaluations showed an effect on gait in 4 of 10 male and 3 of 10 female Group IV rats. Reduced corneal reflex was also seen in some rats in this group; however, Month 3 evaluations failed to show these effects. Hematological and clinical chemistry parameters appeared unaffected by treatment with the test substance. However, there was a dose-related increase in urinary protein values in both sexes. Organ weights, gross necropsy observations and microscopic studies did not reveal any systemic toxic effects. Using tibial nerve teasing techniques, no morphometric differences were found between Group land Group IV glutaraldehyde perfused rats. Quantitative assessment of tibial nerve fiber cross-sections showed slightly increased diameters for males and slightly decreased diameters for females. These changes were not considered to be treatment-related.