Octanal, 2-(phenylmethylene)-, (2E)-

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1979.12.13-1980.7.20

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

other: to meet the requirements of the Food and Drug Administration

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

open coverage, no details on test site

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Hexyl Cinnamic Aldehyde
- Molecular formula (if other than submission substance): Not reported
- Molecular weight (if other than submission substance): Not reported
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: Not reported
- Physical state: liquid
- Analytical purity: Not reported
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: Not reported
- Isomers composition: Not reported
- Purity test date: Not reported
- Lot/batch No.: 262
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Not reported
- Storage condition of test material: Room temperature
- Other:

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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): volumn
- Concentration (if solution): not diluted
- Constant volume or concentration used: administered by volumn using specific gravity (1.0 at 20 degrees)
- For solids, paste formed: yes/no: Not applicable


VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable
- Amount(s) applied (volume or weight with unit): Not applicable
- Concentration (if solution): Not applicable
- Lot/batch no. (if required): Not applicable
- Purity: Not applicable


USE OF RESTRAINERS FOR PREVENTING INGESTION: no (Not reported)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, seven days a week

Doses / concentrations

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

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.


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


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
- Time schedule for examinations: Not applicable


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
- Parameters checked in table [No.?] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 6 and week 13
- Animals fasted: Yes
- How many animals: five animals/sex/group
- Parameters checked in table [No.?] were examined.


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
- Parameters checked in table [No.?] were examined.


NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: Not applicable
- Dose groups that were examined: Not applicable
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Not applicable


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes
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:

Not reported in detail

Results and discussion

Results of examinations

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).

BODY WEIGHT AND WEIGHT GAIN:
Treatment with Hexyl cinnamic aldehyde at 0.50 and 1.00 g/kg 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 suggested a treatment-related effect, the absence of similar findings among the corresponding males and the intermediate dose female (0.25 g/kg) 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 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 groups, it is evident that the conversion of food consumed into body weight gain was negatively affected by treatment at these dose levels.

FOOD EFFICIENCY:
Not reported.

WATER CONSUMPTION:
Not reported.

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.

NEUROBEHAVIOUR:
Not tested.

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.

GROSS PATHOLOGY:
Not reported.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Percutaneous administration of Hexyl cinnamic aldehyde at a dose level of 1.00 g/kg 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.

HISTOPATHOLOGY: NEOPLASTIC (if applicable):
Not applicable.

HISTORICAL CONTROL DATA (if applicable):
Not applicable.

OTHER FINDINGS

Effect levels

1

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: Skin irritation

2

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

125 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: Based on increased liver and kidney weights at 250 mg/kg bw/d taking into account that both dermal and oral exposures occurred.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. 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

Table 2. 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

Table 3. 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**
x10^3	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**
x10^3	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

Table 4. 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

Table 5. 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

Table 6. 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

Table 7. 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

Table 8. 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

Table 9. 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

Applicant's summary and conclusion

Conclusions:

LOAEL local effects = 125 mg/kg bw/d
NOAEL systemic effects = 125 mg/kg bw/d

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/day 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 level. Absolute food consumption (g/rat/day) was increased in females at 250, 500 and 1000 mg/kg bw. 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 (hematology and clinical biochemistry) demonstrated inconsistent changes in hemoglobin, hematocrit, erythrocyte cout, 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. Histopathological examination revealed morphological alteration at the 1000 mg/kg bw 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 %, see § 7.1.2) supports this hypothesis.

Based on the observed effets, 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 taking into account that both dermal and oral exposures occurred.