Registration Dossier

Administrative data

Description of key information

Sub-chronic oral toxicity studies are available for amyl cinnamic aldehyde; both sub-acute and sub-chronic dermal toxicity studies are available for the read-across substance hexyl cinnamic aldehyde.

Systemic oral toxicity was evident in the subchronic dietary study for rats treated at 4000 ppm, the effects included enlarged liver and kidneys.  Reduced stomach weights, males, and small intestine weights, females, were evident after six weeks of treatment.  No treatment related microscopic changes were evident.

Systemic dermal effects observed following subchronic exposure included mortalities at the higher dose levels (1000 and 500 mg/kg bw/day), reduced bodyweights and increased food consumption trends at 250, 500 and 1000 mg/kg bw/day. Elevated white cell counts in the higher dose levels. Necropsy revealed gastrointestinal mucosal irritation and dermal irritation.  The observation of gastrointestinal effects suggested ingestion via grooming may have contributed more to systemic effects than any material absorbed via the skin (dermal absorption is very low for HCA = 0.183%) .   In the second subchronic investigation the highest dose tested, 25 mg/kg bw/d elicited no notable systemic effects following dermal application. Local dermal effects were limited to instances of skin dryness, cracking and sloughing and local erythema at the site of application.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1973
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published study which contains sufficient detail, including in results, to judge it reliable for hazard assessment purposes. Non GLP but close to guideline study and well documented.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no opthalmological examination; No sensory reactivity determination; some serum determinations not conducted i.e. creatinine, cholesterol, sodium and potassium. Some organ histology not conducted .
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: CFE
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding colony (not clear whether sourced at BIBRA)
- Age at study initiation: no data
- Weight at study initiation: males 100-125g; females 90-120g
- Fasting period before study:
- Housing: 5 per cage
- Diet (e.g. ad libitum): Ad libitum. Spillers' Laboratory Small Diet.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-1C
- Humidity (%): 50-60%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: From: To: no data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The loss of amyl cinnamic aldehyde from the diet was determined prior to the study. This was done by the analysis (gas chromatography) of a diet which had been prepared with amyl cinnamic aldehyde at 10,000ppm and had been exposed in the animal room for 48, and compared to the same mixture in an air tight container. Only 1% of amyl cinnamic aldehyde was lost from the diet. Since this was not considered significant amyl cinnamic aldehyde was administered in food prepared every 4-5 days at the appropriate concentrations.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
Daily (continuous) dietary exposure
Remarks:
Doses / Concentrations:
0, 80, 400 or 4000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
15 for 14 weeks treatment (0, 80, 400, or 4000ppm)
Additional 5 for 2 weeks treatment (0, 400, or 4000ppm)
Additional 5 for 6 weeks treatment (0, 400, or 4000ppm)
Control animals:
yes, plain diet
Details on study design:
No information provided in publication
Positive control:
None
Observations and examinations performed and frequency:
Body weights: At the beginning of the study and then weekly up to week 14.
Food and water consumption were measured over the 24hour period preceding each weighing.
Sacrifice and pathology:
At completion of the study the animals were necropsied. Any macroscopic abnormalities were noted.
The following organs were weighed: brain, pituitary, thyroid, heart, liver, spleen, kidneys, adrenals, gonads, stomach, small intestine and caecum.
Samples of the organs weighed were histologically examined. Also, tissues and samples from the following were histologically examined: salivary gland, trachea, aorta, thymus, lymph nodes, urinary bladder, colon, rectum, pancreas, uterus, skeletal muscle, and any other tissue that appeared to be abnormal.
Other examinations:
Blood samples were obtained before necropsy for haematological and serum chemistry investigations. In the final week of treatment, urine produced in a 6 hour period of water deprivation. At week 6 and 13, urine over a 2hr period was collected following a 25ml/kg of waterload and between 16 and 20hr after the water load.
Statistics:
Student's t-test. Significance p<0.05 or p<001
Clinical signs:
no effects observed
Mortality:
no mortality observed
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):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver and kidney
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
One female in the 400ppm group died but histological and necropsy findings showed to be due to infection and therefore, unrelated to treatment.
Mammary adenomas were noted in two females (1 at 80ppm and 1 at 400ppm) during the last 3 week of the study. These were considered spontaneous and non-treatment related. Significant lower stomach weights in males given 400ppm and lower small-intestine weights in females given 4000ppm were observed after treatment for 6 weeks. The latter was not significant when expressed per body weight. Also, there were significant increases in relative liver weights in both sexes after 14week treatment and in males after 6 week treatement. Relative kidney weights were also increased after 14weeks at 4000ppm. There were some macroscopic and histological findings but none associated with substance intake. These were red, patchy lungs in three male rats (two at 80ppm, one in control), and pale kidneys in some male rats in all groups including controls. Also, vacuolation of some liver cells, protein casts in the kidney tubules and signs of chronic lung infection, with low and similar incidence in both treated and controls.
Dose descriptor:
NOAEL
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Liver and kidney enlargement observed at 4000 ppm
Critical effects observed:
not specified

The increase in relative liver and kidney weights was not associated with any histological abnormality.

Mean intakes of amyl cinnamic aldehyde were 6.1, 29.9 and 287.3 mg/kg bw/d for males and 6.7, 34.9, and 320.3 mg/kg bw/d for females. The 400 ppm NOAEL corresponded to approximately 23 mg/kg bw/d in males and 36 mg/kg bw/d in females.

Conclusions:
An oral NOAEL of approximately 30 mg/kg bw/d can be derived under the conditions of this study.
Executive summary:

Male and female rats were fed a diet containing amyl cinnamic aldehyde at 0, 80, 400 or 4000 ppm for 14 weeks. No differences were observed in body weight gain, haematology, urinary analysis, or clinical chemistry between control and treatment groups. Macroscopic and histological examination did not find any treatment-related changes. There were small mammary adenomas in two females from the two lowest doses which were considered spontaneous. At the end of the study there were a significant relative kidney weight increase in females and relative liver weight increase in male and females at the 4000 ppm dose and consequently a NOAEL of 400 ppm (approximately) 30 mg/kg bw/d can be established.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1965
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This is a published study containing sufficient details to regard it as reliable for use in hazard assessment. Only basic experimental details were provided in the publication.
Qualifier:
no guideline followed
Principles of method if other than guideline:
One feeding level was administered continuously for 90 days. Dose was selected by multiplying by a factor of 100 the daily intake of the test substance in food.
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
other: FDRL
Sex:
male/female
Details on test animals and environmental conditions:
No further information
Route of administration:
oral: feed
Vehicle:
cotton seed oil
Remarks:
: prior to incorporation in the diet
Details on oral exposure:
No further details provided
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily (continuous) exposure via dietary admixture
Remarks:
Doses / Concentrations:
6.1-6.6 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
15
Control animals:
yes, plain diet
Details on study design:
No details provided
Positive control:
No information
Observations and examinations performed and frequency:
No information
Sacrifice and pathology:
No information
Other examinations:
No information
Statistics:
No information
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Details on results:
No treatment related effects observed at the single dose level investigated
Dose descriptor:
NOAEL
Effect level:
6.1 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no treatment related effects observed at the only dose tested
Dose descriptor:
NOAEL
Effect level:
6.6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no treatment related effects observed at the only dose tested
Critical effects observed:
not specified
Conclusions:
No effects of treatment were observed at the single dose level used in this study, equivalent to 6.1 and 6.6 mg/kg bw/d in male and female rats, respsectively.
Executive summary:

Male and female rats received dietary dose levels equivalent to 6.1 and 6.6 mg/kg bw/d alpha-amylcinnamaldehyde respectively, for 90 days. Body weights, food consumption, haematology and blood chemistry, were determined at 6 and 12 weeks. At necropsy, the kidney and liver weights were measured and histological examination of 20 organs and tissues was conducted. There were no treatment-related adverse effects at the only dose level investigated in this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two published studies are available for this endpoint; studies report comparable results but one is limited by the use of a single low dose level.

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

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 December 1979 to 20 July 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Organ weight measurements and histopathological examinations were performed with limited organs, but the study was conducted under GLP and in accordance with FDA guideline.
Qualifier:
according to
Guideline:
other: US FDA
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Camm Research Institute Inc.
- Age at study initiation: 40-50 days
- Weight at study initiation: 225-290 g (males), 170-230 g (females)
- Fasting period before study: Not applicable
- Housing: individually in metalic cages of conventional design
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): Not reported


IN-LIFE DATES: From: 1979-12-13 To: 1980-3-20
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: Not reported
- Type of wrap if used: Not applicable
- Time intervals for shavings or clipplings: every three days or as needed during the treatment period

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): volume
- Concentration (if solution): not diluted
- Constant volume or concentration used: administered by volume using specific gravity (1.0 at 20 degrees)

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily, 7 days per week
Remarks:
Doses / Concentrations:
0, 125, 250, 500, 1000 mg/kg bw/d
Basis:
nominal per unit body weight
No. of animals per sex per dose:
Group 1 (cage control) consisted of 30 males and 30 females. Each of test substance groups (2-5) consisted of 15 males and 15 females.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: based on the result of a range finding (28 day) percutaneous toxicity study
- Rationale for animal assignment (if not random): randomly assigned
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not reported
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a more detailed physical examination was conducted when animals found to be "abnormal" during the daily general examinations.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: No details reported

BODY WEIGHT: Yes
- Time schedule for examinations: A weekly mean variance per group will be calculated and statistically analyzed using Student's t test.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once in the pretreatment period and again after three months of substance administration.
- Dose groups that were examined: all amimals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 6 and week 13
- Anaesthetic used for blood collection: Yes (ether anesthesia)
- Animals fasted: Yes
- How many animals: five animals/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 6 and week 13
- Animals fasted: Yes
- How many animals: five animals/sex/group

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (approximately 16 hours) collection from five rats/sex/group during week 6 and week 13.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
Sacrifice and pathology:
Microscopic examination was performed the following tissues; skin (treated and untreated), adrenals, brain, heart, kidneys, liver, stomach, lung, bronchi, mesentric lymph node, pituitary, sternum (bone marrow), spinal cord, testes with epididymis, ovaries, spleen, urinary bladder and nerve with muscle. Some organs were indicators of neurotoxicity (spinal cord, nerve with muscle) and immunotoxicity (spleen, bone marrow, lymph nodes)
Statistics:
No information
Clinical signs:
effects observed, treatment-related
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
Percutaneous administration of Hexyl cinnamic aldehyde caused irritation of the treated skin at all dose levels investigated. The irritation was characterized by erythema, cracking, drying and sloughing of the skin and the severity was related to increasing dose. The behavioural changes were most probably a result of conditioning produced by the irritant effect of the test article and the animals' attempts to avoid treatment. Treatment-related deaths occurred only at the high dose level (1.00 g/kg bw/d).

BODY WEIGHT AND WEIGHT GAIN:
Treatment with Hexyl cinnamic aldehyde at 0.50 and 1.00 g/kg bw/d produced statistically significant reductions in the body weights of both sexes in relation to corresponding values in the control group. Although decreases for females treated at 0.125 g/kg bw/d suggested a treatment-related effect, the absence of similar findings among the corresponding males and the intermediate dose female (0.25 g/kg bw/d) made the biological significance of this finding doubtful.

FOOD CONSUMPTION:
A negative effect of treatment with Hexyl cinnamic aldehyde on this parameter could not be demonstrated in the males and some intervals cited indicated an increase in the absolute amount of food consumed in comparison with the corresponding controls. Females treated at 0.25, 0.50 and 1.00 g/kg bw/d revealed increased absolute food consumption during the treatment period. Since the relative food consumption values were significantly increased in both sexes of the treated animals and corresponding body weights were significantly decreased in the 0.50 and 1.00 g/kg bw/d groups, it is evident that the conversion of food consumed into body weight gain was negatively affected by treatment at these dose levels.

OPHTHALMOSCOPIC EXAMINATION:
After three months of Hexyl cinnamic aldehyde administration ophthalmological examinations did not reveal any effect due to treatment. The findings at the preterminal examination were consistent with those that would be expected during an infection with sialodacryoadenitis.

HAEMATOLOGY:
The administration Hexyl cinnamic aldehyde resulted in consistent differences (alteration at both 6 and 13 weeks of treatment) with respect to the following hematological parameters: White blood cell and segmented neutrophil counts - significant elevation in Group 4 and 5 males, respectively. The high dose males revealed significantly reduced lymphocyte counts on both assessment occasions. The Group 2, 3 and 4 females displayed elevated white blood cell counts on both occations while the Group 4 females had consistent segmented neutrophil elevations. Only the Group 3 females displayed significantly reduced lymphocyte counts at both 6 and 13 weeks of administration.

CLINICAL CHEMISTRY:
The administration of Hexyl cinnamic aldehyde resulted in reduced serum glucose values and increased BUN and SAP in both sexed after 13 weeks of treatment.

URINALYSIS:
Examinaton of the urinalysis data did not reveal differences between treated and control rats.

ORGAN WEIGHTS:
Male organ weights were not affected by treatment; the increased relative values resulted from significantly decreased body weights. Among the females, the increase in kidney and liver weights in comparison with the controls, was supported by both absolute and relative data. Therefore treatment with Hexyl cinnamic aldehyde for 90 days resulted in significantly increased liver and kidney weights for the female rats dosed at 0.25, 0.50 and 1.00 g/kg bw/d.

GROSS PATHOLOGY:
Not reported.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Percutaneous administration of Hexyl cinnamic aldehyde at a dose level of 1.00 g/kg bw/d for 90 days produced hepatic hydropic vacuolization and single cell degeneration, splenic lymphoid depletion and fibrosis, focal gastric ulceration and chronic nectotizing dermatitis with acanthosis, hyperkeratosis and sebaceous gland hyperplasia. In addition dose-related incrases in the myeloid-erythroid and decreases in the cell-fat ratios of the bone marrow occurred.
Dose descriptor:
LOAEL
Remarks:
(local effects)
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Local effects (skin irritation) observed at all dose levels
Dose descriptor:
NOAEL
Remarks:
(systemic effects)
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on increased liver and kidney weights at 250 mg/kg bw/d
Critical effects observed:
not specified

Body weight change (group mean)

Sex Male Female
Group Number 1 2 3 4 5 1 2 3 4 5
Test Article Control HCA HCA HCA HCA Control HCA HCA HCA HCA
Dosage Level(mg/kg/day) - 125 250 500 1000 - 125 250 500 1000
Number of Animals 30 15 15 15 15 30 15 15 15 15
Body weight (g)                    
Week -1 208,5 207,4 211,3 203,7 205,1 184,8 180,6 182,8 181,5 180,5
Week 0 255,6 252,9 258,8 249,6 251,4 199,9 196,3 199,4 195,8 197,9
Week 1 282,1 279,5 278,8 244.1** 227.9** 215,7 207.7** 209.1* 199.4** 191.5**
Week 2 310,3 311,3 309 276.4** 245.8** 224,1 219,4 224,7 212.3** 199.9**
Week 3 329 328,8 326,6 288.9** 252.1** 232,9 224.5* 232,1 219.1** 205.7**
Week 4 346,2 345,8 346,1 302.5** 267.7** 239,8 232,9 242,1 228.6* 220.4**
Week 5 360,1 355,5 356,2 309.8** 377.1** 246,8 237.9* 247,7 235.7* 221.2**
Week 6 368,8 362,7 358,2 312.5** 274.4** 248,1 237.9* 251,3 235.6** 228**
Week 7 380,2 378,1 371,6 320.6** 279.5** 255,2 245.1* 258,5 239.6** 238.5**
Week 8 389,4 385,8 373,9 324.7** 275.5** 260 248.1* 263,3 246.7* 248.8*
Week 9 398,8 395,4 380 337.1** 297.9** 262,7 252.5* 268,2 255 256,2
Week 10 406,2 402,3 388,3 337.7** 296.8** 265,8 252.7** 271,9 256 257,6
Week 11 412 406,8 393,8 342.5** 301.4** 267,3 256* 273,2 257.9* 257.8*
Week 12 416,2 404,5 391* 341.2** 298.7** 271,8 259.2* 273,2 257.9* 257.8*
Week 13 406,5 396,5 379.1* 332.9** 282** 262,3 250.1* 268,1 255,3 254,3

*P<0.05, **P<0.01

Food consumption

Sex Male Female
Group Number 1 2 3 4 5 1 2 3 4 5
Test Article Control HCA HCA HCA HCA Control HCA HCA HCA HCA
Dosage Level(mg/kg/day) - 125 250 500 1000 - 125 250 500 1000
Number of Animals 30 15 15 15 15 30 15 15 15 15
Food consumption                     
(g/rat/day)     Week -1 23,8 23,3 23,9 23,2 23,5 Data are missing (page 29)
Week 0 24,3 24 23.1* 19.2** 17.8**
Week 1 24,4 25,3 26* 25,4 24,7
Week 2 25 26 27.3** 25,8 25,5
Week 3 25,1 26,3 26,3 25,8 26,3
Week 4 25,2 26 26,3 25,5 24,2
Week 5 24,3 25,5 25,4 24,6 24,2
Week 6 24,2 26.2** 26.8** 25,5 24,7 18,5 19,7 21** 23** 26.1**
Week 7 24,6 26.5** 25,9 25,5 24 18,7 19,4 21.2** 22.6** 24.9**
Week 8 24 26.4** 25,9 25,3 24,4 18,5 19,2 20.8** 21.9** 23.1**
Week 9 24,6 27** 28.3** 26.4* 25,7 19,6 19,9 22.2* 22.7** 26.9**
Week 10 23,3 26.3** 26.4** 26.2** 26.2** 18,4 19,3 21.9** 22.2** 25.5**
Week 11 23,3 25.6** 25.8** 25.4** 26.4** 18,9 19,9 21.3* 22.8** 26.6**
Week 12 21,2 21,6 22,3 24.2* 22 15,7 16,3 18.9** 22.8** 23.7**
Food consumption                    
(g/kg/day)     Week -1 102,4 101,2 101,6 102,4 103 96,6 95,9 97,8 97,6 99,4
Week 0 90,5 90,3 85.7** 77.8** 74** 91,5 92,4 96,5 86.2* 102.2**
Week 1 82,6 85,6 88.5** 97.4** 104.7** 83,1 91.4** 95.2** 101** 122.1**
Week 2 78,2 81.3* 85.6** 91.7** 103** 86,3 94.7** 94.7** 100.1** 158.9**
Week 3 74,4 78* 78.2* 87.4** 102.2** 82 89.4* 90.9** 96.1** 122.3**
Week 4 71,3 74,3 74.9* 83.4** 88.4** 82,9 85,5 89.6* 92.4** 115.9**
Week 5 66,7 71.2* 71.1** 79.1** 88.9** 74,5 80,3 80,8 92.2** 108.3**
Week 6 64,6 71** 73.4** 80.6** 91.1** 73,7 81.5** 82.7** 97.1** 115.4**
Week 7 63,9 69.6** 69.4** 79.3** 86.7** 72,7 78.8* 81.6** 93.1** 103**
Week 8 61 67.9** 68.6** 76.7** 85.2** 70,9 76.7* 78.5* 87.3** 91.8**
Week 9 61,1 67.7** 74.2** 78.3** 86.6** 74,1 78,7 82.3* 89.1** 104.7**
Week 10 57 65** 67.5** 77.1** 87.9** 69 76** 80.2** 86.5** 99.3**
Week 11 56,1 63.3** 65.8** 74.3** 88.4** 70,3 77.3* 77.8* 88.4** 103.6**
Week 12 51,4 54,1 58* 71.8** 75.7** 58,6 64,2 70** 89** 92.9**

*P<0.05, **P<0.01

Hematology

Sex Male Female
Group Number 1 2 3 4 5 1 2 3 4 5
Test Article Control HCA HCA HCA HCA Control HCA HCA HCA HCA
Dosage Level(mg/kg/day) - 125 250 500 1000 - 125 250 500 1000
Number of Animals 30 15 15 15 15 30 15 15 15 15
Hematology                    
Leucocyte(WBC)  6week 8,2 10.1** 11* 14.9** 10,2 6,3 8.5** 9.6** 9** 10.9**
x103 13week 10 13 7,8 11,6 15.3* 6,9 10.5** 11.7** 13** 7,4
Erythrocyte(RBC)   6week 8,06 8.54* 8.47* 8 8.7* 7,71 7,78 7,45 7,84 8.24**
x103 13week 8,69 8,7 8,66 8,76 8,87 7,52 7,56 7.24* 7,84 7,96
Hemoglobin  6week 15,5 16 15,9 15,1 16.4* 15 15 14,8 14,8 15,5
 (g/100ml) 13week 16,2 16,1 16 16,1 16,4 14,7 14,7 13,7 14,6 14,9
Hematocrit  6week 44,2 46,2 45,8 44 48** 42,6 43,6 42,4 43,6 45.8**
(%) 13week 47 47 46,8 47,8 48,6 41,8 41,8 40.6* 42,4 43,4
Neutrophil  6week 14,6 29.6** 28.4** 35.6** 45.8** 13,4 30.8** 21.8** 38.8** 54.4**
 (% of total) 13week 33 45.6* 42 43.6* 52.2** 38,6 44,4 43,4 56.4** 40,6
Lymphocyte 6week 83 66.8** 69.2** 61.6** 52.4** 85 65.2** 75** 58.8** 44.2**
( % of total) 13week 63 51 54,2 52,6 44** 58,6 53 51 40.2** 55,4
Platelet (10) 13week 490,8 392,4 911,8 719** 744,4 629 622,8 436 368 972.8*

*P<0.05, **P<0.01

MCV NT NT NT NT NT NT NT NT NT NT
MCH NT NT NT NT NT NT NT NT NT NT
MCHC NT NT NT NT NT NT NT NT NT NT
Monocyte  (% of total) NT NT NT NT NT NT NT NT NT NT
Eosinophil (% of total) NT NT NT NT NT NT NT NT NT NT
Basophil   (% of total) NT NT NT NT NT NT NT NT NT NT
Nucleated RBC/100WBC NT NT NT NT NT NT NT NT NT NT

NT: not tested, *P<0.05, **P<0.01

Serum Chemistry

Sex Male Female
Group Number 1 2 3 4 5 1 2 3 4 5
Test Article Control HCA HCA HCA HCA Control HCA HCA HCA HCA
Dosage Level(mg/kg/day) - 125 250 500 1000 - 125 250 500 1000
Number of Animals 30 15 15 15 15 30 15 15 15 15
Serum Chemistry                    
Total Bilirubin 6week 0,4 0,4 0.3** 0.3** 0.2** 0,4 0,4 0.3** 0.3** 0.3*
(mg/dl) 13week 0,3 0,3 0,2 0,3 0,3 0,3 0,3 0,3 0.2* 0,3
BUN 6week 16,4 20** 18.4* 24** 32** 17,4 19 19,8 23.2* 25**
(mg/dl) 13week 15,8 16,4 19,6 26** 24.6** 14,6 17.4** 16.4* 17 26.8**
GPT 6week 39,4 42,8 43 50.2** 72.4* 27,4 33** 44.6** 42.8** 48.8**
(IU/L) 13week 46 49,2 54,6 47,4 50,6 34,6 42 40.6** 48.6** 40.6*
GOT 6week 104,2 97,2 96 127 170,6 91,2 93,3 137** 140.4** 129.8**
(IU/L) 13week 162,4 177,6 143 170 177,2 177,8 215,2 233,6 214 149,4
ALP 6week 104,8 144.8** 139.4* 152.2* 211.2** 83,4 108.5** 124.6** 122** 164.6**
(IU/L) 13week 78,6 109** 114,6 146.4** 224.6** 70 105.2** 110.6** 113.8** 164**
Glucose 6week 103,4 94 89.4* 80.2** 77.4** 111,2 91.6** 87.8** 86** 88.4**
(mg/dl) 13week 103,4 101 85.4* 97,4 78** 98,8 96 88.6** 84.4** 84*
LDH (IU/L) 13week 1135,2 984,6 457.8** 1296 909,6 1300,2 969 823,4 1276,8 975,6
CPK(IU/L) 13week 543,4 645,6 159.6* 534,2 389,4 562,6 448,8 667,2 511,6 507,4

*P<0.05, **P<0.01

 Albumin        (g%) NT NT NT NT NT NT NT NT NT NT
 Total Protein   (g%) NT NT NT NT NT NT NT NT NT NT
 Creatinin     NT NT NT NT NT NT NT NT NT NT
 gamma-GTP   NT NT NT NT NT NT NT NT NT NT
 Cholesterol    (mg%) NT NT NT NT NT NT NT NT NT NT
 Calcium       (mg%) NT NT NT NT NT NT NT NT NT NT
 Chloride       NT NT NT NT NT NT NT NT NT NT
 Phosphorus   (mg%) NT NT NT NT NT NT NT NT NT NT
 Potassium      NT NT NT NT NT NT NT NT NT NT
 Sodium  NT NT NT NT NT NT NT NT NT NT

NT: not tested, *P<0.05, **P<0.01

Urinalysis

Sex Male Female
Group Number 1 2 3 4 5 1 2 3 4 5
Test Article Control HCA HCA HCA HCA Control HCA HCA HCA HCA
Dosage Level(mg/kg/day) - 125 250 500 1000 - 125 250 500 1000
Number of Animals 30 15 15 15 15 30 15 15 15 15
Urinalysis - - - - - - - - - -

-: no effect

Ophthalmology

Sex Male Female
Group Number 1 2 3 4 5 1 2 3 4 5
Test Article Control HCA HCA HCA HCA Control HCA HCA HCA HCA
Dosage Level(mg/kg/day) - 125 250 500 1000 - 125 250 500 1000
Number of Animals 30 15 15 15 15 30 15 15 15 15
Ophthalmology - - - - - - - - - -

-: no effect

Organ weight (Absolute)

Sex Male Female
Group Number 1 2 3 4 5 1 2 3 4 5
Test Article Control HCA HCA HCA HCA Control HCA HCA HCA HCA
Dosage Level(mg/kg/day) - 125 250 500 1000 - 125 250 500 1000
Number of Animals 30 15 15 15 15 30 15 15 15 15
Organ weight (Absolute)  
 Brain 1,892 1,877 1,847 1.839* 1.814* 1,795 1,765 1,766 1,788 1,796
 Left Gonad 2,927 2,928 2,951 2.652** 2.394** 0,059 0,054 0,065 0,057 0,059
 Right Gonad 3,01 3,013 2.863* 2.815** 2.465** 0,057 0,051 0,06 0,062 0,054
 Left Kidney 1,551 1,599 1,613 1,562 1.402** 0,943 0,953 1.026** 1.035** 1.08**
 Right Kidney 1,555 1,66 1,593 1,591 1.394** 0,972 0,973 1.072** 1.049* 1.094**
 Liver 12,598 13,431 13,182 12,903 11.004* 7,617 8.19* 9.257** 9.357** 10.475**

*P<0.05, **P<0.01

Organ weight (Relative)

Sex Male Female
Group Number 1 2 3 4 5 1 2 3 4 5
Test Article Control HCA HCA HCA HCA Control HCA HCA HCA HCA
Dosage Level(mg/kg/day) - 125 250 500 1000 - 125 250 500 1000
Number of Animals 30 15 15 15 15 30 15 15 15 15
Organ weight (Relative)  
 Body weight (g) 388,8 380,2 365.8* 319.2** 262.8** 251,9 242.3* 258,7 244,1 237.8*
 Brain 0,488 0,495 0.508* 0.578** 0.696** 0,714 0,732 0,689 0,734 0.758**
 Left Gonad 0,755 0,772 0,809 0.834* 0.917** 0,023 0,023 0,025 0,023 0,025
 Right Gonad 0,776 0,794 0,79 0.883** 0.949** 0,023 0,021 0,023 0,026 0,023
 Left Kidney 0,4 0,419 0.443** 0.489** 0.535** 0,375 0,393 0.397* 0.424** 0.456**
 Right Kidney 0,4 0.435* 0.436** 0.498** 0.534** 0,387 0,402 0.415** 0.43** 0.461**
 Liver 3,234 3.516* 3.609** 4.035** 4.176** 3,018 3.377** 3.577** 3.83** 4.4**

*P<0.05, **P<0.01

Histopathological findings

group Liver Alterations 1) Spleen Alterations 2) Skin Alterations 3) Gastric Ulceration
control 0/60 0/60 0/60 0/60
HCA 1.00 kg/kg 14(male 10, female 4)/30 10(male 6, female 4)/30 30(male 15, female 15)/30 4(male 2, female 2)/30

1) accentuated regeneration; single cell degeneration and hydropic vacuolization

2) lymphoid depletion, fibrosis and hemosiderosis

3) chronic dermatitis with acanthosis, hyperkeratosis and sebaceous hyperplasia

Conclusions:
Based on the effects observed in this study, a LOAEL of 125 mg/kg bw/d can be set for local effects based on skin irritation and a NOAEL of 125 mg/kg bw/d is proposed for systemic effects; the NOAEL for systemic effects is likely to be a conservative derivation, taking into account that both dermal and oral exposures are likely to have occurred.
Executive summary:

In a sub-chronic toxicity study performed similarly to OECD guideline No. 411, Hexyl cinnamic aldehyde was administered percutaneously to Albino rats (15/sex/doses) at 125, 250, 500 and 1000 mg/kg bw/d for 90 consecutive days. For control purposes, a fifth group containing 30 animals/sex was maintained without treatment.

Percutaneous administration of the test article resulted in dose-dependent dermal irritation characterized by erythema, cracking, dryness and sloughing. Five male and three female rats did not survive 90 days of treatment at 1000 mg/kg bw/d. Body weights (both sexes) were significantly decreased during treatment at the 500 and 1000 mg/kg bw/d dose level. Absolute food consumption (g/rat/day) was increased in females at 250, 500 and 1000 mg/kg bw/d. Although absolute food intake in males was unaffected, both sexes had increased relative values at all dose level. Ophthalmological examination after approximately 13 weeks did not reveal any alteration due to treatment.

Laboratory studies (haematology and clinical biochemistry) demonstrated inconsistent changes in haemoglobin, haematocrit, erythrocyte count, SGOT and SGPT at the six and 13 week sampling occasions. Consistent significant elevations occurred in the white blood cell and the segmented neutrophil counts of the treated Group 4 and 5 males, respectively. However only the high dose males revealed significantly reduced lymphocyte counts on both assessment occasions indicating a differential shift to the left. With respect to the females, Group 2, 3 and 4 displayed elevated white blood cell counts on both occasions, but only the Group 3 females had significantly reduced lymphocyte counts at both 6 and 13 weeks of administration. Clinical biochemistry indicated reduced serum glucose values and increased BUN and SAP in all treated animals after 13 weeks of test article administration. Urinalysis data obtained during the conduct of the study were unremarkable.

Gross examination at necropsy revealed dose-related irritation of the gastrointestinal tract mucosa and the treated skin. The liver and kidney weights of treated females were significantly increased at 250, 500 and 1000 mg/kg bw/d. Histopathological examination revealed morphological alteration at the 1000 mg/kg bw/d dose level in the form of hepatic hydropic vacuolization and single cell degeneration, splenic lymphoid depletion and fibrosis, focal gastric ulceration and chronic necrotizing dermatitis. Bone marrow examination revealed dose-related increase in the myeloid-erythroid and decrease of the cell-fat ratios.

Application of HCA induced cracking and dryness of the skin in all the treated animals. Study authors suggested that the gastrointestinal irritation observed at the highest dose tested was a consequence of HCA intake during grooming and ingestion of necrotic skin. Indeed neither coverage nor restrainers were used to prevent ingestion. Systemic effects observed at this dose are more likely the consequence of HCA ingestion rather than the results of HCA skin application itself. The skin absorption potential of HCA which is estimated to be very low (0.183%) supports this hypothesis.

Based on the observed effects, a LOAEL of 125 mg/kg bw/d can be set for local effects based on skin irritation and a NOAEL of 125 mg/kg bw/d is proposed for systemic effects; the NOAEL for systemic effects is likely to be a conservative derivation, taking into account that both dermal and oral exposures are likely to have occurred.

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12 August 1979 to 19 February 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Only male animals (2 males/each dose) were used in this study. However, the study was conducted under GLP.
Qualifier:
according to
Guideline:
other: US FDA
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Camm Research Institute Inc.
- Age at study initiation: 50-60 days
- Weight at study initiation: 150-175 g (males) upon arrival, 211-248 g at treatment initiation
- Fasting period before study: Not applicable
- Housing: individually in stainless steel wire-mesh cages of conventional design
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): Not reported


IN-LIFE DATES: From: 1979-10-29 To: 1979-11-26
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: Not reported
- % coverage: Not reported
- Type of wrap if used: Not applicable
- Time intervals for shavings or clipplings: every three days or as needed during the treatment period

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not applicable
- Time after start of exposure: Not applicalbe

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): volume
- Concentration (if solution): not diluted
- Constant volume or concentration used: administered by volume, using specific gravity (1.0 at 20 degrees)
- For solids, paste formed: yes/no: Not applicable

USE OF RESTRAINERS FOR PREVENTING INGESTION: Not reported
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
150, 375, 750, 1500, 3000 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
Only male animals used. Each of the test substance groups (1-5) consisted of 2 males.
Control animals:
no
Details on study design:
- Dose selection rationale: Not reported
- Rationale for animal assignment (if not random): randomly assigned
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not reported
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- During the course of the experiment all animals were observed daily for evidence of possible treatment-related changes in behaviour and/or general appearance. Particular attention was paid to the condition of the dorsum of each animal in the area of Hexyl Cinnamic Aldehyde application.


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: No details reported


BODY WEIGHT: Yes
- Time schedule for examinations: Individual rat body weights were determined twice a week and the dosage levels for all experimental animals were adjusted twice weekly in accordance with changes in body weight.


FOOD CONSUMPTION:
- Food consumption for each animal determined once a week, and expressed as g food/rat/day and g food/kg body weight/day: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 4
- Anaesthetic used for blood collection: Not reported
- Animals fasted: Yes
- How many animals: two males/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 4
- Animals fasted: Yes
- How many animals: two males/group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Clinical signs:
effects observed, treatment-related
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
All treated animals had erythema and eschar formation with cracking and dryness of the skin where Hexyl Cinnamic Aldehyde was administered. The severity of these findings was dose-related, the higher dose levels (1.5 and 3.0 g/kg bw/d) animals being most severely affected as evidenced by erythema and sloughing of necrotic skin.
Signs of hyperirritability were noted at 0.75, 1.5 and 3.0 g/kg bw/d with frequency of occurrence and severity being dose-dependent. There animals were easily startled and occasionally overtly aggressive when handled. In addition, abnormally arched backs occurred in animals at 1.5 and 3.0 g/kg bw/d. The gonads of males no. 401 and 402 (1.5 g/kg bw/d) appeared atrophied and were contained in a taut scrotum that gave them the appearance of being improperly descended. Animals at the 1.5 and 3.0 g/kg bw/d dose levels tended to insert their heads into food jars for extended periods of time. This behaviour was not exhibited in other treated groups.
Other noteworthy observations were related to the deaths of rat no. 501 and 502 (3.0 g/kg bw/d) after 7 and 11 days of treatment respectively. Both animals became emaciated early during the treatment period then developed ataxia prior to dying spontaneously. In addition, rat no. 502 developed breathing difficulties three days before succumbing to treatment.

BODY WEIGHT AND WEIGHT GAIN:
No significant differences in body weight gain were noted among animals receiving 0.15, 0.375 and 0.75 g/kg bw/d. However, animals at 1.5 g/kg bw/d suffered a substantial reduction in body weight gain while at 3.0 g/kg bw/d, the rats lost weight rapidly until time of death.

FOOD CONSUMPTION:
The absolute and relative amount of food consumed were not adversely affected in treated groups with the exception of one group 5 (3.0 g/kg bw/d) animal for which the amount of food consumed was substantially reduced.

FOOD EFFICIENCY:
Not reported.

WATER CONSUMPTION:
Not reported.

OPHTHALMOSCOPIC EXAMINATION:
Not examined.

HAEMATOLOGY:
Examination of the parameters investigated by means of intergroup comparison revealed a dose-related negative effect on clotting time and white blood cell count manifested by a significant decrease in platelets and white blood cells. There was a progressive prolongation of the clotting time in association with increasing dose levels. The differential counts for group 1 (0.15 g/kg bw/d) and 2 (0.375 g/kg bw/d) were considered normal. However, the 1.5 and 3.0 g/kg bw/d levels demonstrated a marked shift in the proportion of segmented neutrophils to lymphocytes.

CLINICAL CHEMISTRY:
Intergroup comparison of the various blood biochemical parameters suggested an adverse dose-related effect revealed by a progressive increase in BUN, SAP and SGPT values. The SGOP and Glucose values indicated a dose-related trend (decrease in Glucose and increase in SGOT) especially in group 3 (0.75 g/kg bw/d) and 4 (1.5 g/kg bw/d). With respect to Total Bilirubin, no differences among groups were found.

URINALYSIS:
Not examined.

NEUROBEHAVIOUR:
Not tested.

ORGAN WEIGHTS:
Both absolute and relative organ weights indicated decrease in the weights of the thymus and spleen at 1.5 g/kg bw/d Hexyl Cinnamic Aldehyde in comparison with other treated groups . Organ weights were not obtained at the 3.0 g/kg bw/d dose level due to mortality suffered during the study conduct. The relative organ weight data for the kidneys (right and left) revealed slightly heavier weights for the 1.5 g/kg bw/d group than other treated groups. However, this was not as evident in the absolute organ weight data. Similarly, the relative organ weight data for the gonads (right and left) displayed slightly heavier weights at the 1.5 g/kg bw/d dose level.

GROSS PATHOLOGY:
Gross pathological examination at necropsy failed to reveal and effect of treatment at 0.15 g/kg. However, at 0.375 and 0.75 g/kg bw/d dose levels, thickening of the skin at the application sites was noted. Both animals in group 4 (Hexyl Cinnamic Aldehyde, 1.5 g/kg bw/d) displayed thickened skin at the site of application with erythema of the dermis and epidermis along the lateral aspects of the application site. Both members of the high dose group were emaciated, had congested lungs and displayed severe gastrointestinal irritation with the presence of free blood. It was felt that the gastrointestinal irritation in part was a consequence of Hexyl Cinnamic Aldehyde intake during grooming and ingestion of necrotic skin. Male no. 502 had congested adrenals and dry and cracked skin at the site of test article application. The gross pathological findings for male no. 501 did not include a description of skin irritation at the site of application probably as a result of early mortality during the study conduct. Male no. 501 also exhibited a pale spleen.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Animals at all dose levels exhibited pulmonary alterations consisting of mild and diffuse interstitial thickening that was considered to be a background lesion possibly due to a mild infection with murine mycoplasmosis. Dermatitis with mild to severe hyperkeratosis was observed at the application sites at all dose levels except 0.15 g/kg bw/d. Dermatitis was characterized by focal infiltration of a mixed of a mixed thickening of the stratum corneum. At 0.75 and 1.5 g/kg bw/d, kidney alterations consisting of focal dilatation of the tubules were noted. At 3.0 g/kg bw/d, vascular congestion of the gastrointestinal tract with sub-acute to chronic necrotizing and hemorrhagic enteritis of the small intestine was observed. Lymphoid depletion and necrosis with reticulo-endothelial hyperplasia of the spleen were observed in male 501 at 3.0 g/kg bw/d.
Dose descriptor:
NOAEL
Remarks:
(local effects)
Effect level:
<= 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Local effects (erythema and eschar formation) were seen at all tested doses
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Critical effects observed:
not specified

Individual body weights (g)

Group No. RAT No. 0,1 0,4 1,1 1,4 2,1 2,4 3,1 3,4 4,1
1                    
HCA 101 211 226 249 272 282 295 308 316 298
0.15 g/kg 102 226 235 252 261 273 274 284 293 280
2                  
HCA 201 216 218 242 253 245 247 262 273 255
0.375 g/kg 202 233 245 269 288 298 308 322 333 311
3                    
HCA 301 230 243 259 270 277 276 281 294 267
0.75 g/kg 302 248 244 249 270 277 287 298 312 293
4                    
HCA 401 214 213 197 201 219 229 237 247 211
1.50 g/kg 402 220 210 213 225 220 217 229 249 224
5                    
HCA 501 220 200 - - - - - - -
3.00 g/kg 502 223 203 173 135 - - - - -

-: aminal died

Absolute food consumption (g/rat/day)

Group No. RAT No. 0.1-1.1 1.1-2.1 2.1-3.1 3.1-3.7
1          
HCA 101 23,1 23,0 27,1 26,8
0.15 g/kg 102 22,4 22,4 24,0 24,2
2          
HCA 201 19,8 18,8 23,6 23,7
0.375 g/kg 202 24,4 25,0 26,1 27,0
3          
HCA 301 23,6 23,4 25,7 25,3
0.75 g/kg 302 21,1 24,1 29,0 28,7
4          
HCA 401 17,4 21,0 27,8 22,5
1.50 g/kg 402 19,6 20,1 35,3 30,5
5          
HCA 501 - - - -
3.00 g/kg 502 14,8 14,5 - -

-: aminal died

Relative food consumption (g/kg/day)

Group No. RAT No. 0.1-1.1 1.1-2.1 2.1-3.1 3.1-3.7
1          
HCA 101 100,6 86,6 92,0 88,6
0.15 g/kg 102 93,8 85,4 86,2 85,7
2          
HCA 201 86,7 77,4 93,0 91,6
0.375 g/kg 202 97,3 88,2 84,3 85,3
3          
HCA 301 96,4 87,4 92,2 92,4
0.75 g/kg 302 85,1 91,8 100,9 97,0
4          
HCA 401 84,8 101,0 122,2 100,4
1.50 g/kg 402 90,4 93,0 157,2 134,6
5          
HCA 501 - - - -
3.00 g/kg 502 75,0 - - -

-: aminal died

Hemograms

WBC differencial %
Group No. RAT No. RBC (x10E6) HGB (g/100 ml) HCT (%) MCV (x10E3) MCH (UUG) MCHC (%) Platelet (x10E3) Clotting time (min) WBC (x10E3)  Neut. SEG. Neut. NON-SEG. Lympho. Mono Eosin Baso
1                                
HCA 101 7,52 15,0 45 60 20 33 872 3,50 9,9 26 0 72 2 0 0
0.15 g/kg 102 8,13 15,9 47 58 20 34 1053 3,50 5,1 21 0 79 0 0 0
2                                
HCA 201 7,82 15,5 45 58 20 34 1014 4,50 7,8 22 0 78 0 0 0
0.375 g/kg 202 7,67 14,7 45 58 19 33 1195 4,50 6,9 22 0 77 0 1 0
3                                
HCA 301 7,83 15,3 48 61 20 32 940 4,50 8,4 53 0 43 2 2 0
0.75 g/kg 302 7,56 14,3 43 57 19 33 944 5,00 5,8 38 0 62 0 0 0
4                                
HCA 401 7,64 14,1 44 57 18 32 821 5,00 4,4 65 0 35 0 0 0
1.50 g/kg 402 7,98 14,9 45 57 19 33 799 6,00 5,6 66 0 33 1 0 0
5                                
HCA 501 - - - - - -   - - - - - - - -
3.00 g/kg 502 - - - - - -   - - - - - - - -

-: aminal died

Blood Biochemical Analyses

Group No. RAT No. Glucose (mg/dl) BUN (mg/dl) Total Bilirubin (mg/dl) ALP (IU/L) SGOT (IU/L) SGPT (IU/L)
1              
HCA 101 141 15 0,3 123 139 23
0.15 g/kg 102 117 17 0,2 160 108 31
2              
HCA 201 92 23 0,2 144 118 33
0.375 g/kg 202 114 18 0,2 136 127 25
3              
HCA 301 107 22 0,2 265 136 48
0.75 g/kg 302 97 19 0,2 154 102 35
4              
HCA 401 90 33 0,2 422 157 64
1.50 g/kg 402 80 23 0,2 266 137 65
5              
HCA 501 - - - - - -
3.00 g/kg 502 - - - - - -

-: aminal died

Gross pathological findings

Rat No   Observations
Group 1 0.15 g/kg  
101   No abnormal findings
102   No abnormal findings
Group 2 0.375 g/kg  
201   The skin on the dorsum was thickened at the site of administration.
202   The skin on the dorsum was thickened at the site of administration.
Group 3 0.75 g/kg  
301   The skin on the dorsum was thickened at the site of administration.
302   The skin on the dorsum was thickened at the site of administration.
Group 4 1.50 g/kg  
401   The skin on the dorsum was thickened at the site of administration.
Erythema of the dermis and epidermis along both lateral aspects of the application site was noted.
402   The skin on the dorsum was thickened at the site of administration.
Erythema of the dermis and epidermis along both lateral aspects of the application site was noted.
Group 5 3.00 g/kg  
501   Externally the animal was emaciated. The lungs were moderately congested throughout. The adrenals were pale and surrounded by a clear fluid. The spleen was pale. Blood was present in the lumen of the small intestine with moderate to severe irritation of the mucos membrane. The mucos membrane of the stomach was mildly to moderately irritated.
502   Externally the animal was emaciated. The skin on the dorsum was dry and cracked at the site of administration. The lungs were moderately to severely congested throughout. The adrenals were congensted. Free blood was present in the lumen of the stomach and intestines.

Absolute organ weight (g)

Organ Group 1 Group 2 Group 3 Group 4 Group 5
HCA HCA HCA HCA HCA
0.15 g/kg 0.375 g/kg 0.75 g/kg 1.50 g/kg 3.00 g/kg
101 102 201 202 301 302 401 402 501 502
Body weight (g) 298 280 255 311 267 293 211 224 - -
Adrenal Lt. 0,027 0,021 0,021 0,018 0,031 0,021 0,024 0,026 - -
Adrenal Rt. 0,026 0,021 0,025 0,019 0,029 0,020 0,023 0,026 - -
Brain 1,826 1,833 1,838 1,895 1,849 1,847 1,784 1,710 - -
Gonad Lt. 1,775 1,795 1,651 1,757 1,740 1,794 1,647 1,624 - -
Gonad Rt. 1,799 1,789 1,682 1,783 1,783 1,766 1,677 1,659 - -
Heart 1,078 1,013 0,946 1,017 0,863 1,045 0,812 0,854 - -
Kidney Lt. 1,071 1,112 0,982 1,180 1,094 1,111 0,934 1,043 - -
Kidney Rt. 1,170 1,074 0,914 1,168 1,153 1,080 0,953 1,056 - -
Liver 9,416 7,703 7,242 10,375 8,849 9,516 6,757 8,233 - -
Lungs 1,571 1,341 1,409 1,720 1,502 1,441 1,083 1,280 - -
Prostate 0,697 0,579 0,530 0,623 0,588 0,730 0,408 0,502 - -
Spleen 0,628 0,503 0,589 0,641 0,475 0,679 0,266 0,371 - -
Thymus 0,332 0,295 0,465 0,583 0,369 0,432 0,118 0,197 - -
Thyroid Lt. 0,009 0,005 0,007 0,007 0,006 0,008 0,006 0,008 - -
Thyroid Rt.. 0,006 0,007 0,008 0,009 0,005 0,008 0,005 0,005 - -

-: aminal died

Relative organ weight (g %)

Organ Group 1 Group 2 Group 3 Group 4 Group 5
HCA HCA HCA HCA HCA
0.15 g/kg 0.375 g/kg 0.75 g/kg 1.50 g/kg 3.00 g/kg
101 102 201 202 301 302 401 402 501 502
Body weight (g) 298 280 255 311 267 293 211 224 - -
Adrenal Lt. 0,009 0,008 0,008 0,006 0,012 0,007 0,011 0,012 - -
Adrenal Rt. 0,009 0,008 0,010 0,006 0,011 0,007 0,011 0,012 - -
Brain 0,613 0,655 0,721 0,609 0,692 0,630 0,845 0,763 - -
Gonad Lt. 0,596 0,641 0,647 0,565 0,652 0,612 0,780 0,725 - -
Gonad Rt. 0,604 0,639 0,660 0,573 0,668 0,603 0,795 0,741 - -
Heart 0,362 0,362 0,371 0,327 0,323 0,357 0,385 0,381 - -
Kidney Lt. 0,359 0,397 0,385 0,379 0,410 0,379 0,443 0,466 - -
Kidney Rt. 0,393 0,384 0,358 0,408 0,432 0,369 0,452 0,471 - -
Liver 3,160 2,751 2,840 3,336 3,314 3,248 3,202 3,675 - -
Lungs 0,527 0,479 0,552 0,553 0,562 0,492 0,513 0,571 - -
Prostate 0,234 0,207 0,208 0,200 0,220 0,249 0,193 0,224 - -
Spleen 0,211 0,180 0,231 0,206 0,178 0,232 0,126 0,166 - -
Thymus 0,111 0,105 0,182 0,187 0,138 0,147 0,056 0,088 - -
Thyroid Lt. 0,003 0,002 0,003 0,002 0,002 0,003 0,003 0,004 - -
Thyroid Rt.. 0,002 0,002 0,003 0,003 0,002 0,003 0,002 0,002 - -

-: aminal died

Histopathological findings

Rat No   Organ  
Group 1 0.15 g/kg    
101   Lung Interstitial thickening, mild, diffuse.
102   Lung Interstitial thickening, mild, diffuse.
Group 2 0.375 g/kg    
201   Lung Interstitial thickening, mild, diffuse.
201   Skin Dermatitis, mild, focal with mild hyperkeratosis and necrosis of stratum corneum.
202   Lung Interstitial thickening, mild, diffuse.
202   Skin Nopathological alteration.
Group 3 0.75 g/kg    
301   Lung Interstitial thickening, mild, diffuse.
301   Skin Nopathological alteration.
302   Lung Interstitial thickening, mild, diffuse.
302   Skin Dermatitis, mild, focal.
302   Kidney Dilation, tubular, focal.
Group 4 1.50 g/kg    
401   Lung Interstitial thickening, mild, diffuse.
401   Skin Dermatitis, mild, focal with mild hyperkeratosis and acanthosis.
402   Lung Interstitial thickening, mild, diffuse.
402   Skin Nopathological alteration.
402   Kidney Dilation, tubular, focal.
Group 5 3.00 g/kg    
501     Note: This animal died during the course of the study. Tissues examined revealed some autolytic changes.
501   Lung Interstitial thickening, mild, diffuse. Congestion.
501   Kidney Congestion.
501   Liver Congestion.
501   Gastrointestinal tract Vascular congestion throughout with sub-acute to chronic necrotizing and hemorrhagic enteritis in the small intestine.
501   Spleen Lymphoid depletion and necrosis with reticulo-endothelial hyhperplasia.
501   Adrenal Nopathological alteration.
502     Note: This animal died during the course of the study. Tissues examined revealed widespread autolysis.
502   Skin Dermatitis, mild, with severe hyperkeratosis.
502   Lung Pulmonary congestion and edema (post-mortem). Interstitial thickening, mild, diffuse. 
502   Adrenal Nopathological alteration.
502   Gastrointestinal tract Not examined.

Incidence of dermatitis and hyperkeratosis

Group No. Dermatitis Hyperkeratosis
Mild Moderate Severe Mild Moderate Severe
1 - - - - - -
HCA
0.15 g/kg
2 201* - - 201 - -
HCA
0.375 g/kg
3 302 - - - - -
HCA
0.75 g/kg
4 401 - - 401 - -
HCA
1.50 g/kg
5 501 - - - - 501
HCA
3.00 g/kg

*: animal number

Conclusions:
Based on the results of this study, doses selected for the main 90-day percutaneous study were 0, 125, 250, 500 and 1000 mg/kg bw/d.
Executive summary:

Hexyl cinnamic aldehyde was applied percutaneously to the shaved dorsa of 10 male Sprague-Dawley rats at dose levels of 150, 375, 750, 1500 and 3000 mg/kg bw/day for 28 consecutive days.

Erythema and eschar formation with cracking and dryness of the skin at the test article application site were noted at all dose levels. Hyperirritability was noted at 750, 1500 and 3000 mg/kg bw/d and abnormally arched backs at 1500 and 3000 mg/kg bw/d. The two high dose (3000 mg/kg bw/d) rats developed ataxia and survived only 7 and 11 days of treatment. Body weights and food consumption differed little at the 150, 375 and 750 mg/kg bw/d dose levels. At 1500 and 3000 mg/kg bw/d, body weights were reduced substantially but food intake was depressed only at 3000 mg/kg bw/d.

Intergroup comparison with respect to blood biochemical parameters revealed a dose-related negative effect on clotting time and white blood cell count. The 1500 and 3000 mg/kg bw/d levels demonstrated a marked shift in the proportion of segmented neutrophils to lymphocytes. A review of the various blood biochemical parameters investigated, suggested an adverse dose-related effect revealed by a progressive increase in BUN, SAP and SGPT values. The SGOT and Glucose values indicated a dose-related trend (decrease in Glucose and increase in SGOT) especially in group 3 (750 mg/kg bw/d) and 4 (1500 mg/kg bw/d).

Gross pathological examination revealed thickening of the skin at the application sites for the dose levels 375 and 750 mg/kg bw/d. At 1500 mg/kg bw/d the test substance caused thickening of the skin and erythema of both the dermis and epidermis along the lateral aspects of the application site. Application of Hexyl Cinnamic Aldehyde at 3000 mg/kg bw/d produced dry and cracked skin at the administration site, body emaciation, congested lungs and gastrointestinal irritation. Both absolute and relative organ weight data displayed decreases in the weights of the thymus and spleen at 1500 mg/kg bw/d and other treated groups. The relative organ weight data for the gonads (right and left) displayed slightly heavier weights at the 1500 mg/kg bw/d dose level.

Microscopic examination of the skin at the application dose levels except 150 mg/kg bw/d. At 750 and 1500 mg/kg bw/d, kidney alterations consisting of focal dilation of the tubules were noted. Congestion of the gastrointestinal tract with sub-acute to chronic necrotizing and hemorrhagic enteritis of the small intestine was observed at 3000 mg/kg bw/d. Lymphoid depletion and necrosis with reticulo-endothelial hyperplasia were observed in one animal at 3000 mg/kg bw/d.

Application of HCA induced cracking and dryness of the skin in all the treated animals. Study authors suggested that the gastrointestinal irritation observed at the highest dose tested was a consequence of HCA intake during grooming and ingestion of necrotic skin. Indeed neither coverage nor restrainers were used to prevent ingestion. Systemic effects observed at this dose are more likely the consequence of HCA ingestion rather than the results of HCA skin application itself. The skin absorption potential of HCA which is estimated to be very low (0.183 %) supports this hypothesis.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed before guideline implementation but meets basic scientific principles. However only one dose was tested which was not a limit dose, only 5 animals/sex were used, test sites were open, ophthalmoscopic examinations were not done, organ weight measurements were performed on limited organs.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Assessment of repeated dose dermal toxicity (single dose level) over 90 days
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ace Animals, Inc., P.O.Box 122, Boyertown, PA 19512
- Age at study initiation: Not reported.
- Weight at study initiation: 75-100 g
- Fasting period before study: Not applicable
- Housing: individually
- Diet: Purina Rat Mash (#5001) ad libitum
- Water: ad libitum
- Acclimation period: 21 days for control animals ; 7 days for treated animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled
- Humidity (%): controlled
- Air changes (per hr): Not reported
- Photoperiod: 12 hrs dark/12 hrs light

IN-LIFE DATES:
- control : From: 1981-02-24 To: 1981-05-24
- treated: From: 1981-03-10 To: 1981-06-07
Type of coverage:
open
Vehicle:
other:
Details on exposure:
TEST SITE
- Area of exposure: the back were divided into a seven (7) area grid and a separate area was treated daily to minimize local irritation
- Time intervals for shavings or clipplings: once a week or as needed

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not done

TEST MATERIAL
- Amount(s) applied: 0.5 mL/kg
- Concentration (if solution): 5%
- Constant volume or concentration used: Dosage volumes were based on weekly body weights.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Amount(s) applied (volume or weight with unit): 1.0 mL/kg
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days/week
Remarks:
Doses / Concentrations:
25 mg/kg bw/d
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not reported
- Rationale for animal assignment (if not random): randomly assigned
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not reported
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- The rats were observed daily for signs of toxicity, pharmacological effects and mortality. Dermal observation were recorded daily.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: recorded weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Not reported
- Animals fasted: Yes
- How many animals: two animals/group
- Parameters checked in table 7.5.2/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: two animals/group
- Parameters checked in table 7.5.2/1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 13 weeks
- Metabolism cages used for collection of urine: Not reported
- Animals fasted: Not reported
- Parameters checked in table 7.5.2/1 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.2/2)
HISTOPATHOLOGY: Yes (see table 7.5.2/2)
Other examinations:
No other examinations were performed
Statistics:
The details were not reported.
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
All but one of the aminals survived the test in generally good health. A male rat (2912), Group 2 (HCA), died on Day 14 after losing, weight and refusing to drink water. Emaciation, starting Day 7, lethargy, and ptosis were also noted. For dermal reactions, all sites were normal at all times.

BODY WEIGHT AND WEIGHT GAIN:
Body weights were generally comparable to control. In Group 2, the rate of gain was very slightly less for females and slightly less for males. However, none of the differences was statistically significant.

HAEMATOLOGY:
There were no remarkable differences from control.

CLINICAL CHEMISTRY:
Most parameters were comparable to control. There was a slight increase in alkaline phosphatase values in males of both treated groups. This was not noted in females. A relationship of treatment is considered to be questionable.

URINALYSIS:
There was no remarkable difference from control.

ORGAN WEIGHTS:
There was no remarkable difference from control.

GROSS PATHOLOGY:
Necropsy findings in the rats which died, 2912M, Group 2, HCA, were indication of a lung infection. Other findings at sacrifice on Day 91, appeared to be spontaneous in nature and unrelated to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC:
There were no significant lesions on treated or untreated skin, bone marrow of spinal cord.
Findings in the liver and kidney were similar to lesions found in normal, non-experimental animals and were therefore considered to be unrelated to treatment.
Dose descriptor:
NOAEL
Effect level:
>= 25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects (local or systemic) were observed at the only dose level used in this study
Critical effects observed:
not specified

Daily observations

Group

Dose

Animal Number & Sex

Sign

Days

1 (control)

 

1.0 mL/kg

 

2882-M

Diarrhea

50-69

2883-M

Urine Stains

79-86

Emaciation

84-85

Chromorhinorrhea

84

2884-M

Diarrhea

52-56

2 (HCA)

 

25 mg/kg

 

2911-M

Diarrhea

3, 7, 16-24

2912-M

Emaciation

 7-13

Adipsia

 7-13

Lethargy

 9-13

Ptosis

 10-13

Death

14

2913-M

Diarrhea

2, 3, 22-52, 71, 72

Urine Stains

78-79

Emaciation

78-79

Chromorhinorrhea

78

2914-M

Diarrhea

17-52

2915-M

Diarrhea

2, 3, 17-52

Mean body weight change (g)

Time

Male

Female

Vehicle

HCA

Vehicle

HCA

Pre 3

99 ± 7.8

no data

91 ± 9.0

no data

Pre 2

145 ± 20.8

no data

135 ± 16.4

no data

Pre 1

201 ± 20.1

108 ± 5.4

172 ± 19.7

99 ± 5.9

Week 1

225 ± 25.3

132 ± 14.1

193 ± 22.7

122 ± 7.7

Week 2

263 ± 20.8

156 ± 30.7

202 ± 22.2

165 ± 9.3

Week 3

309 ± 16.7

229 ± 25.6

228 ± 26.4

194 ± 13.9

Week 4

351 ± 15.8

272 ± 28.6

240 ± 31.1

212 ± 16.8

Week 5

374 ± 20.8

319 ± 33.5

256 ± 31.7

227 ± 16.9

Week 6

403 ± 14.3

357 ± 38.0

260 ± 22.6

241 ± 17.7

Week 7

427 ± 34.6

381 ± 46.0

271 ± 28.0

244 ± 12.2

Week 8

457 ± 37.6

399 ± 58.3

283 ± 32.4

260 ± 15.4

Week 9

477 ± 29.3

426 ± 53.5

282 ± 32.8

268 ± 17.3

Week 10

495 ± 35.8

460 ± 58.0

296 ± 30.3

277 ± 16.9

Week 11

514 ± 34.5

480 ± 57.0

299 ± 30.4

275 ± 17.5

Week 12

526 ± 34.9

456 ± 94.1

307 ± 31.7

286 ± 14.6

Week 13

525 ± 50.7

486 ± 61.6

307 ± 31.9

296 ± 14.4

Mean Haematology

Parameters

Male

Female

Vehicle

HCA

Vehicle

HCA

Leucocyte(WBC) (103)

9.2 ± 2.2

8.6 ± 2.3

9.6 ± 1.8

12.8 ± 3.2

Erythrocyte(RBC) (106)

8.31 ± 0.8

8.13 ± 0.3

8.35 ± 0.7

8.05 ± 0.3

Hemoglobin       (g)

15.4 ± 1.3

15.2 ± 0.4

15.7 ± 1.6

15.6 ± 0.4

Hematocrit       (%)

48.3 ± 4.1

47.1 ± 1.2

49.2 ± 5.0

49.2 ± 1.9

Platelet

NT

NT

NT

NT

MCV

NT

NT

NT

NT

MCH

NT

NT

NT

NT

MCHC

NT

NT

NT

NT

Neutrophil (% of total)

15 ± 5.4

34 ± 8.7

15 ± 4.7

23 ± 7.9

Lymphocyte ( % of total)

85 ± 5.4

63 ± 12.7

85 ± 5.1

76 ± 8.1

Monocyte  (% of total)

NT

NT

NT

NT

Eosinophil (% of total)

0.0 ± 0.0

1.0 ± 0.8

0.2 ± 0.5

0.4 ± 0.6

Basophil   (% of total)

NT

NT

NT

NT

Nucleated RBC/100WBC

no data

no data

no data

0.2 ± 0.5

NT = Not Tested

 

Mean Serum Chemistry

Parameters

Male

Female

Vehicle

HCA

Vehicle

HCA

 Albumin        (g%)

3.6 ± 0.2

3.8 ± 0.2

3.9 ± 0.4

3.8 ± 0.1

 Total Protein    (g%)

6.0 ± 0.6

5.9 ± 0.1

6.1 ± 0.4

5.7 ± 0.4

 Total Bilirubin  (mg%)

0.07 ± 0.11

0.09 ± 0.12

0.07 ± 0.11

0.07 ± 0.11

 BUN        (mg%)

14.7 ± 1.1

13.0 ± 1.0

14.5 ± 1.1

19.7 ± 4.4

 Creatinin    

NT

NT

NT

NT

 GPT          (Units)

49.3 ± 11.7

47.5 ± 8.6

37.0 ± 6.4

69.3 ± 45.6

 GOT          (Units)

115 ± 8.2

112 ± 16.8

92 ± 18.5

106 ± 17.8

 ALP          (IU/L)

23.0 ± 1.5

36.9 ± 8.6*

24.8 ± 3.2

25.7 ± 2.7

 LDH          (IU/L)

1320 ± 73.5

1320 ± 54.8

1128 ± 190.5

1143 ± 72.3

 gamma-GTP    

NT

NT

NT

NT

 URIC (mg%)  

1.4 ± 0.1

1.5 ± 0.2

1.4 ± 0.1

1.5 ± 0.2

 Cholesterol    (mg%)

78.9 ± 14.5

81.9 ± 10.5

65.5 ± 9.7

71.4 ± 8.5

 Glucose     (mg%)

126 ± 28.7

114 ± 13.7

115 ± 8.1

110 ± 9.5

 Calcium       (mg%)

9.6 ± 0.9

9.6 ± 0.4

10.1 ± 0.5

9.7 ± 0.7

 Chloride      

NT

NT

NT

NT

 Phosphorus   (mg%)

5.9 ± 0.3

5.6 ± 0.6

6.0 ± 0.9

6.0 ± 1.0

 Potassium     

NT

NT

NT

NT

 Sodium       

NT

NT

NT

NT

*: P<0.05, NT: not tested

Urinalysis (Males) 

Animal Number & Sex

Appearance

S.GR

pH

PROT

GLUC

KETONE

BILI

BLOOD

EPI

RBC

WBC

CEYSTALS

Group: 1 (1.0 ml/kg control)

2881-M

Clear

no data

6,5

+++

N

N

N

N

Few

N

1-2

Oxalate

2882-M

Clear

1,050

6,0

++

N

N

N

N

Few

1-2

1-2

Oxalate

2883-M

Clear

1,045

6,0

TR

N

N

N

N

Few

N

N

Oxalate

2884-M

Clear

1,040

6,0

++

N

N

N

N

Few

1-2

2-3

Oxalate

2885-M

Cloudy Amber

1,040

6,5

++++

N

N

N

N

Few

1-2

2-3

Oxalate

Group: 2 (25 mg/kg HCA)

2911-M

Clear

1,045

6,5

+

N

N

N

N

Few

N

1-2

Oxalate

2912-M

death

 

 

 

 

 

 

 

 

 

 

 

2913-M

Clear

1,010

6,0

++

N

N

N

N

Few

N

N

Oxalate

2914-M

Clear

1,040

6,0

N

N

N

N

N

Few

N

2-3

Oxalate

2915-M

Amber

1,050

6,5

++

N

N

N

N

Few

1-2

2-3

Oxalate

S.GR. = specific gravity, PROT = protein mg%, GLUC = glucose g%, BILI = bilirubin, EPI = epitherial cells, RBC = red blood cell, WBC = white blood cell

N = normal or negative, + = small/slight/few, ++ = moderate, +++ = severe/large, TR = 2/2

Urinalysis (Females)

Animal Number & Sex

Appearance

S.GR

pH

PROT

GLUC

KETONE

BILI

BLOOD

EPI

RBC

WBC

CRYSTALS

Group: 1 (1.0 ml/kg control)

2886-F

Clear

1,005

6,0

N

N

N

N

N

Few

N

1-2

Oxalate

2887-F

Clear Amber

1,040

6,0

+

N

N

N

N

Few

N

2-3

Oxalate

2888-F

Clear

1,055

6,0

+

N

N

N

N

Few

N

1-2

Oxalate

2889-F

Clear

1,048

6,0

+

N

N

N

N

Few

N

N

Oxalate

2890-F

Clear

1,040

6,0

+

N

N

N

N

Few

1-2

1-2

Oxalate

Group: 2 (25 mg/kg HCA)

2916-F

Turbid

1,040

6,0

N

N

N

N

N

Few

N

2-3

Phosphate

2917-F

Amber

1,050

6,0

N

N

N

N

N

Few

1-2

2-3

Oxalate

2918-F

Turbid

1,045

6,5

+

N

N

N

N

Few

2-3

2-3

Oxalate

2919-F

Clear

1,045

7,0

N

N

N

N

N

Few

1-2

2-3

Oxalate

2920-F

Clear

1,040

6,0

N

N

N

N

N

Few

N

1-2

Oxalate

S.GR. = specific gravity, PROT = protein mg%, GLUC = glucose g%, BILI = bilirubin, EPI = epitherial cells, RBC = red blood cell, WBC = white blood cell

N = normal or negative, + = small/slight/few, ++ = moderate, +++ = severe/large, TR = 2/20

  

Necropsy observations

Group

Dose

Animal Number & Sex

Tissue

Observations

1 (control)

1.0 ml/kg

2885-M

Heart

Appears enlarged.

2 (HCA)

25 mg/kg

2911-M

Urinary Bladder

Slightly distended.

2912-M

Lungs

All lobes covered with white/yellowish nodules. Urine stains (day 14 spontaneous death)

2914-M

Testes

Both appear smaller than normal

2915-M

Kidney

Right one slightly hydronephrotic

  

Conclusions:
Under the conditions of the study there was no evidence of toxicity induced by treatment with HCA. The NOAEL is therefore 25 mg/kg bw/d, i.e. the highest dose tested.
Executive summary:

In a repeated dose sub-chronic dermal toxicity study, Hexyl Cinnamic Aldehyde (HCA, 5% in phenyl ethyl alcohol) was applied to the shaved skin of Sprague-Dawley Albino rat (5/sex/dose) at a single dose level of 25 mg/kg bw/d (0.5 mL/kg bw), daily, 7 days/week during a 90-day period. A control group received phenyl ethyl alcohol alone (1.0 mL/kg bw). All rats were observed daily and skin reactions were recorded. Body weights were recorded weekly. At termination, selected haematology, clinical chemistry and urinalysis parameters were evaluated. All animals were examined grossly and liver and kidneys were weighed. Microscopic examination of the skin, liver, kidney, sterna bone marrow and spinal cord was conducted. One male rat treated with HCA died on Day 14. At necropsy, there was evidence of a lung infection. The death was not considered to be related to treatment.  There were no dermal reactions observed on any rats in any group. Under the conditions of the study there was no evidence of toxicity induced by treatment with HCA. The NOAEL is therefore 25 mg/kg bw/d, i.e. the only dose level tested.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two 90-day studies are supported by a 28-day range-finding study.

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed before guideline implementation but meets basic scientific principles. However only one dose was tested which was not a limit dose, only 5 animals/sex were used, test sites were open, ophthalmoscopic examinations were not done, organ weight measurements were performed on limited organs.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Assessment of repeated dose dermal toxicity (single dose level) over 90 days
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ace Animals, Inc., P.O.Box 122, Boyertown, PA 19512
- Age at study initiation: Not reported.
- Weight at study initiation: 75-100 g
- Fasting period before study: Not applicable
- Housing: individually
- Diet: Purina Rat Mash (#5001) ad libitum
- Water: ad libitum
- Acclimation period: 21 days for control animals ; 7 days for treated animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled
- Humidity (%): controlled
- Air changes (per hr): Not reported
- Photoperiod: 12 hrs dark/12 hrs light

IN-LIFE DATES:
- control : From: 1981-02-24 To: 1981-05-24
- treated: From: 1981-03-10 To: 1981-06-07
Type of coverage:
open
Vehicle:
other:
Details on exposure:
TEST SITE
- Area of exposure: the back were divided into a seven (7) area grid and a separate area was treated daily to minimize local irritation
- Time intervals for shavings or clipplings: once a week or as needed

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not done

TEST MATERIAL
- Amount(s) applied: 0.5 mL/kg
- Concentration (if solution): 5%
- Constant volume or concentration used: Dosage volumes were based on weekly body weights.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Amount(s) applied (volume or weight with unit): 1.0 mL/kg
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days/week
Remarks:
Doses / Concentrations:
25 mg/kg bw/d
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not reported
- Rationale for animal assignment (if not random): randomly assigned
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not reported
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- The rats were observed daily for signs of toxicity, pharmacological effects and mortality. Dermal observation were recorded daily.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: recorded weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Not reported
- Animals fasted: Yes
- How many animals: two animals/group
- Parameters checked in table 7.5.2/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: two animals/group
- Parameters checked in table 7.5.2/1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 13 weeks
- Metabolism cages used for collection of urine: Not reported
- Animals fasted: Not reported
- Parameters checked in table 7.5.2/1 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.2/2)
HISTOPATHOLOGY: Yes (see table 7.5.2/2)
Other examinations:
No other examinations were performed
Statistics:
The details were not reported.
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
All but one of the aminals survived the test in generally good health. A male rat (2912), Group 2 (HCA), died on Day 14 after losing, weight and refusing to drink water. Emaciation, starting Day 7, lethargy, and ptosis were also noted. For dermal reactions, all sites were normal at all times.

BODY WEIGHT AND WEIGHT GAIN:
Body weights were generally comparable to control. In Group 2, the rate of gain was very slightly less for females and slightly less for males. However, none of the differences was statistically significant.

HAEMATOLOGY:
There were no remarkable differences from control.

CLINICAL CHEMISTRY:
Most parameters were comparable to control. There was a slight increase in alkaline phosphatase values in males of both treated groups. This was not noted in females. A relationship of treatment is considered to be questionable.

URINALYSIS:
There was no remarkable difference from control.

ORGAN WEIGHTS:
There was no remarkable difference from control.

GROSS PATHOLOGY:
Necropsy findings in the rats which died, 2912M, Group 2, HCA, were indication of a lung infection. Other findings at sacrifice on Day 91, appeared to be spontaneous in nature and unrelated to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC:
There were no significant lesions on treated or untreated skin, bone marrow of spinal cord.
Findings in the liver and kidney were similar to lesions found in normal, non-experimental animals and were therefore considered to be unrelated to treatment.
Dose descriptor:
NOAEL
Effect level:
>= 25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects (local or systemic) were observed at the only dose level used in this study
Critical effects observed:
not specified

Daily observations

Group

Dose

Animal Number & Sex

Sign

Days

1 (control)

 

1.0 mL/kg

 

2882-M

Diarrhea

50-69

2883-M

Urine Stains

79-86

Emaciation

84-85

Chromorhinorrhea

84

2884-M

Diarrhea

52-56

2 (HCA)

 

25 mg/kg

 

2911-M

Diarrhea

3, 7, 16-24

2912-M

Emaciation

 7-13

Adipsia

 7-13

Lethargy

 9-13

Ptosis

 10-13

Death

14

2913-M

Diarrhea

2, 3, 22-52, 71, 72

Urine Stains

78-79

Emaciation

78-79

Chromorhinorrhea

78

2914-M

Diarrhea

17-52

2915-M

Diarrhea

2, 3, 17-52

Mean body weight change (g)

Time

Male

Female

Vehicle

HCA

Vehicle

HCA

Pre 3

99 ± 7.8

no data

91 ± 9.0

no data

Pre 2

145 ± 20.8

no data

135 ± 16.4

no data

Pre 1

201 ± 20.1

108 ± 5.4

172 ± 19.7

99 ± 5.9

Week 1

225 ± 25.3

132 ± 14.1

193 ± 22.7

122 ± 7.7

Week 2

263 ± 20.8

156 ± 30.7

202 ± 22.2

165 ± 9.3

Week 3

309 ± 16.7

229 ± 25.6

228 ± 26.4

194 ± 13.9

Week 4

351 ± 15.8

272 ± 28.6

240 ± 31.1

212 ± 16.8

Week 5

374 ± 20.8

319 ± 33.5

256 ± 31.7

227 ± 16.9

Week 6

403 ± 14.3

357 ± 38.0

260 ± 22.6

241 ± 17.7

Week 7

427 ± 34.6

381 ± 46.0

271 ± 28.0

244 ± 12.2

Week 8

457 ± 37.6

399 ± 58.3

283 ± 32.4

260 ± 15.4

Week 9

477 ± 29.3

426 ± 53.5

282 ± 32.8

268 ± 17.3

Week 10

495 ± 35.8

460 ± 58.0

296 ± 30.3

277 ± 16.9

Week 11

514 ± 34.5

480 ± 57.0

299 ± 30.4

275 ± 17.5

Week 12

526 ± 34.9

456 ± 94.1

307 ± 31.7

286 ± 14.6

Week 13

525 ± 50.7

486 ± 61.6

307 ± 31.9

296 ± 14.4

Mean Haematology

Parameters

Male

Female

Vehicle

HCA

Vehicle

HCA

Leucocyte(WBC) (103)

9.2 ± 2.2

8.6 ± 2.3

9.6 ± 1.8

12.8 ± 3.2

Erythrocyte(RBC) (106)

8.31 ± 0.8

8.13 ± 0.3

8.35 ± 0.7

8.05 ± 0.3

Hemoglobin       (g)

15.4 ± 1.3

15.2 ± 0.4

15.7 ± 1.6

15.6 ± 0.4

Hematocrit       (%)

48.3 ± 4.1

47.1 ± 1.2

49.2 ± 5.0

49.2 ± 1.9

Platelet

NT

NT

NT

NT

MCV

NT

NT

NT

NT

MCH

NT

NT

NT

NT

MCHC

NT

NT

NT

NT

Neutrophil (% of total)

15 ± 5.4

34 ± 8.7

15 ± 4.7

23 ± 7.9

Lymphocyte ( % of total)

85 ± 5.4

63 ± 12.7

85 ± 5.1

76 ± 8.1

Monocyte  (% of total)

NT

NT

NT

NT

Eosinophil (% of total)

0.0 ± 0.0

1.0 ± 0.8

0.2 ± 0.5

0.4 ± 0.6

Basophil   (% of total)

NT

NT

NT

NT

Nucleated RBC/100WBC

no data

no data

no data

0.2 ± 0.5

NT = Not Tested

 

Mean Serum Chemistry

Parameters

Male

Female

Vehicle

HCA

Vehicle

HCA

 Albumin        (g%)

3.6 ± 0.2

3.8 ± 0.2

3.9 ± 0.4

3.8 ± 0.1

 Total Protein    (g%)

6.0 ± 0.6

5.9 ± 0.1

6.1 ± 0.4

5.7 ± 0.4

 Total Bilirubin  (mg%)

0.07 ± 0.11

0.09 ± 0.12

0.07 ± 0.11

0.07 ± 0.11

 BUN        (mg%)

14.7 ± 1.1

13.0 ± 1.0

14.5 ± 1.1

19.7 ± 4.4

 Creatinin    

NT

NT

NT

NT

 GPT          (Units)

49.3 ± 11.7

47.5 ± 8.6

37.0 ± 6.4

69.3 ± 45.6

 GOT          (Units)

115 ± 8.2

112 ± 16.8

92 ± 18.5

106 ± 17.8

 ALP          (IU/L)

23.0 ± 1.5

36.9 ± 8.6*

24.8 ± 3.2

25.7 ± 2.7

 LDH          (IU/L)

1320 ± 73.5

1320 ± 54.8

1128 ± 190.5

1143 ± 72.3

 gamma-GTP    

NT

NT

NT

NT

 URIC (mg%)  

1.4 ± 0.1

1.5 ± 0.2

1.4 ± 0.1

1.5 ± 0.2

 Cholesterol    (mg%)

78.9 ± 14.5

81.9 ± 10.5

65.5 ± 9.7

71.4 ± 8.5

 Glucose     (mg%)

126 ± 28.7

114 ± 13.7

115 ± 8.1

110 ± 9.5

 Calcium       (mg%)

9.6 ± 0.9

9.6 ± 0.4

10.1 ± 0.5

9.7 ± 0.7

 Chloride      

NT

NT

NT

NT

 Phosphorus   (mg%)

5.9 ± 0.3

5.6 ± 0.6

6.0 ± 0.9

6.0 ± 1.0

 Potassium     

NT

NT

NT

NT

 Sodium       

NT

NT

NT

NT

*: P<0.05, NT: not tested

Urinalysis (Males) 

Animal Number & Sex

Appearance

S.GR

pH

PROT

GLUC

KETONE

BILI

BLOOD

EPI

RBC

WBC

CEYSTALS

Group: 1 (1.0 ml/kg control)

2881-M

Clear

no data

6,5

+++

N

N

N

N

Few

N

1-2

Oxalate

2882-M

Clear

1,050

6,0

++

N

N

N

N

Few

1-2

1-2

Oxalate

2883-M

Clear

1,045

6,0

TR

N

N

N

N

Few

N

N

Oxalate

2884-M

Clear

1,040

6,0

++

N

N

N

N

Few

1-2

2-3

Oxalate

2885-M

Cloudy Amber

1,040

6,5

++++

N

N

N

N

Few

1-2

2-3

Oxalate

Group: 2 (25 mg/kg HCA)

2911-M

Clear

1,045

6,5

+

N

N

N

N

Few

N

1-2

Oxalate

2912-M

death

 

 

 

 

 

 

 

 

 

 

 

2913-M

Clear

1,010

6,0

++

N

N

N

N

Few

N

N

Oxalate

2914-M

Clear

1,040

6,0

N

N

N

N

N

Few

N

2-3

Oxalate

2915-M

Amber

1,050

6,5

++

N

N

N

N

Few

1-2

2-3

Oxalate

S.GR. = specific gravity, PROT = protein mg%, GLUC = glucose g%, BILI = bilirubin, EPI = epitherial cells, RBC = red blood cell, WBC = white blood cell

N = normal or negative, + = small/slight/few, ++ = moderate, +++ = severe/large, TR = 2/2

Urinalysis (Females)

Animal Number & Sex

Appearance

S.GR

pH

PROT

GLUC

KETONE

BILI

BLOOD

EPI

RBC

WBC

CRYSTALS

Group: 1 (1.0 ml/kg control)

2886-F

Clear

1,005

6,0

N

N

N

N

N

Few

N

1-2

Oxalate

2887-F

Clear Amber

1,040

6,0

+

N

N

N

N

Few

N

2-3

Oxalate

2888-F

Clear

1,055

6,0

+

N

N

N

N

Few

N

1-2

Oxalate

2889-F

Clear

1,048

6,0

+

N

N

N

N

Few

N

N

Oxalate

2890-F

Clear

1,040

6,0

+

N

N

N

N

Few

1-2

1-2

Oxalate

Group: 2 (25 mg/kg HCA)

2916-F

Turbid

1,040

6,0

N

N

N

N

N

Few

N

2-3

Phosphate

2917-F

Amber

1,050

6,0

N

N

N

N

N

Few

1-2

2-3

Oxalate

2918-F

Turbid

1,045

6,5

+

N

N

N

N

Few

2-3

2-3

Oxalate

2919-F

Clear

1,045

7,0

N

N

N

N

N

Few

1-2

2-3

Oxalate

2920-F

Clear

1,040

6,0

N

N

N

N

N

Few

N

1-2

Oxalate

S.GR. = specific gravity, PROT = protein mg%, GLUC = glucose g%, BILI = bilirubin, EPI = epitherial cells, RBC = red blood cell, WBC = white blood cell

N = normal or negative, + = small/slight/few, ++ = moderate, +++ = severe/large, TR = 2/20

  

Necropsy observations

Group

Dose

Animal Number & Sex

Tissue

Observations

1 (control)

1.0 ml/kg

2885-M

Heart

Appears enlarged.

2 (HCA)

25 mg/kg

2911-M

Urinary Bladder

Slightly distended.

2912-M

Lungs

All lobes covered with white/yellowish nodules. Urine stains (day 14 spontaneous death)

2914-M

Testes

Both appear smaller than normal

2915-M

Kidney

Right one slightly hydronephrotic

  

Conclusions:
Under the conditions of the study there was no evidence of toxicity induced by treatment with HCA. The NOAEL is therefore 25 mg/kg bw/d, i.e. the highest dose tested.
Executive summary:

In a repeated dose sub-chronic dermal toxicity study, Hexyl Cinnamic Aldehyde (HCA, 5% in phenyl ethyl alcohol) was applied to the shaved skin of Sprague-Dawley Albino rat (5/sex/dose) at a single dose level of 25 mg/kg bw/d (0.5 mL/kg bw), daily, 7 days/week during a 90-day period. A control group received phenyl ethyl alcohol alone (1.0 mL/kg bw). All rats were observed daily and skin reactions were recorded. Body weights were recorded weekly. At termination, selected haematology, clinical chemistry and urinalysis parameters were evaluated. All animals were examined grossly and liver and kidneys were weighed. Microscopic examination of the skin, liver, kidney, sterna bone marrow and spinal cord was conducted. One male rat treated with HCA died on Day 14. At necropsy, there was evidence of a lung infection. The death was not considered to be related to treatment.  There were no dermal reactions observed on any rats in any group. Under the conditions of the study there was no evidence of toxicity induced by treatment with HCA. The NOAEL is therefore 25 mg/kg bw/d, i.e. the only dose level tested.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/cm²
Study duration:
subchronic
Species:
rat
Quality of whole database:
Three studies are available, which consistently identify local dermal effects at higher dose levels.

Additional information

Sub-chronic oral toxicity

A 14-week oral toxicity study performed in the rat with amyl cinnamic aldehyde identifies a NOAEL of 400 ppm (equivalent to approximately 30 mg/kg bw/d) based on increased liver and kidney weights. Findings did not have any histopathological correlates. A second 9-day study performed in the rat using amyl cinnamic aldehyde did not identify any effects of treatment at the single dose level of 6.1-6.6 mg/kg bw/d.

Sub-chronic dermal toxicity

A 28-day rat dermal toxicity study performed with the read-across substance hexyl cinnamic aldehyde (HCA) identified local effects at the lowest dose level tested of 150 mg/kg bw/d. A 90-day dermal study performed with HCA identified a NOAEL for systemic effects of 125 mg/kg bw/d; however this is a conservative derivation as oral exposure in this study cannot be excluded and the dermal absorption of HCA is likely to be very low. A second 90-day study performed with HCA identified a NOAEL for local and systemic effects of 25 mg/kg bw/d; this was the only dose level tested in this study.

Repeated inhalation exposure investigations

Study requirement waived - Sub-chronic studies are available for both oral and dermal routes of exposure, based on read-across to the structural analogue hexyl cinnamaldehyde. Since repeated exposure data (sub-acute and subchronic duration) are available for the primary routes of potential exposure, studies via the inhalation route of exposure are not required. A waiver for inhalation testing is based on data being available by read across for other routes and the absence of effects observed in the acute inhalation toxicity study with hexyl cinnamaldehyde.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Although this study identifies a higher NOAEL, adverse effects were identified.  A second study was less comprehensive and used only a single (and relatively low) dose level which did not identify any effects.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

This study uses the longest exposure duration; a further 90-day study is available but is of limited value as it used only one low dose level.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

This study identifies a NOAEL for dermal effects; other studies performed at higher dose levels show local dermal effects at all dose levels.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Repeated dose toxicity: dermal - systemic effects (target organ) digestive: other

Justification for classification or non-classification

The NOAEL values obtained for oral and dermal long-term exposure do not warrant classification for repeated exposure toxicity (STOT-RE) according to the CLP Regulation.