9-Octadecenoic acid (Z)-, monoester with 1,2,3-propanetriol ester with boric acid (H3BO3)

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 November 1981 to 27 December 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

9-Octadecenoic acid (Z)-, monoester with 1,2,3-propanetriol ester with boric acid (H3BO3)

EC Number:

264-092-6

EC Name:

9-Octadecenoic acid (Z)-, monoester with 1,2,3-propanetriol ester with boric acid (H3BO3)

Cas Number:

63310-16-7

Molecular formula:

C21H39O5B (based on the representative structure below)

IUPAC Name:

2-hydroxy-3-{[hydroxy({2-hydroxy-3-[(9Z)-octadec-9-enoyloxy]propoxy})boranyl]oxy}propyl (9Z)-octadec-9-enoate; 3-{[bis({2-hydroxy-3-[(9Z)-octadec-9-enoyloxy]propoxy})boranyl]oxy}-2-hydroxypropyl (9Z)-octadec-9-enoate; {2-hydroxy-3-[(9Z)-octadec-9-enoyloxy]propoxy}boronic acid

Test material form:

liquid: viscous

Details on test material:

- EC no.: 264-092-6
- Physical state: liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts, USA
- Age at study initiation: 67 days (males); 78 days (females)
- Weight at study initiation: 281 - 353 g (males); 202 - 283 g (females)
- Housing: individually in wire-bottom cages
- Diet: Purina Certified Rodent Chow #5002 ad libitum
- Water: ad libitum
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 21 ºC
- Humidity (%): 61 - 78 %
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From 29 November 1981 to 27 December 1981

Administration / exposure

Type of coverage:

not specified

Vehicle:

other: mineral oil

Details on exposure:

TEST SITE
- The back of each rat was clipped free of hair prior to dosing on the first day of each week and as needed thereafter.

REMOVAL OF TEST SUBSTANCE
- The test material was removed 6 hours after application by wiping the animals backs using gauze squares moistened with diethyl ether.

TEST MATERIAL
- 1.0 mL/kg test material at the following concentrations was applied to each dose group: unchanged (high-dose), 50% (w/w) in mineral oil (mid-dose), 20% (w/w) in mineral oil (low-dose), or 1.0 mL/kg mineral oil (control group). The test material was applied to an unabraded site which were alternated between the scapula and the sacrum to reduce skin irritation.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
- To prevent oral ingestion of the test material each animal was fitted with a Queen Ann collar prior to dosing each day and removed at the end of the 6 hour exposure period.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

Animals were treated for 6 hours per day for an average of five days per week (six days during the first week, five days during the second and third weeks, and four days during the fourth week) for four consecutive weeks.

Doses / concentrations

Remarks:

Doses / Concentrations:
100%, 50%, 20% and 0% w/w
Basis:

No. of animals per sex per dose:

15 males and 15 females

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION: Yes
- Time schedule for examinations: at the beginning and end of each treatment period. Animals scored according to Draize et al (1944)

BODY WEIGHT: Yes
- Time schedule for examinations: at the beginning and end of each weekly treatment period and prior to sacrifice

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes (overnight prior to sacrifice)
- How many animals: 10 animals of each sex per dose group
- Parameters checked included: white blood cell count, red blood cell count, hemaglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemaglobin concentration. A differential count was also performed.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes (overnight prior to sacrifice)
- How many animals: 10 animals of each sex per dose group
- Parameters checked included: glucose, iron, calcium, phosphorus, uric acid, blood urea nitrogen, creatinine, sodium, potassium, chloride, lactate dehydrogenase, serum glutamic oxalacetic transminase, serum glutamic pyruvic transaminase, alkaline phosphatase, total bilirubin, direct bilirubin, indirect bilirubin, cholesterol, triglycerides, total protein, albumin and globulin.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

All of the animals were fasted overnight prior to sacrifice. Terminal sacrifice was performed by exsanguination. Necropsy commenced four days following the last treatment and continued over the next three days until all of the animals had been sacrificed.

GROSS PATHOLOGY: Yes
- The following organs were examined for gross pathological changes and weighed: brain, heart, liver, gonads, kidneys and adrenals.
- The following organs and tissues were also examined for gross pathological changes: skin, spleen, pancreas, stomach, small and large intestine, uterus or seminal vesicles, bladder, heart, thymus, salivary glands, lungs, trachea, esophagus, thyroid and fat.

HISTOPATHOLOGY: Yes
- Samples of the tissues taken at necropsy, and any gross lesions observed, were preserved in 10% (v/v) neutral formalin and submitted for histopathological examination.

Statistics:

Body weights, weekly body weight changes, organ weights, organ/body weight ratios, hematology and serum chemistry data were statistically analysed by one-way analysis of variance among the four test groups. Monocyte, eosinophil, basophil, and banded neutrophil differential counts were not statistically analysed since this data were very sparse. Incidences of histopathological changes were analysed using a Chi-Square Test with Yates' correction for continuity, or the Fisher's Exact Test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for further information

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:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for further information

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths occurred and no signs of toxicity were observed during the study.

DERMAL IRRITATION
At the end of the first week of treatment, very slight to well defined erythema appeared in male animals and very slight to moderate erythema appeared in female animals in an apparent dose-related manner. Very slight edema was also present in males in the high dose group and in females in the high and mid-dose groups. there was also a high incidence of dry, flaky and/or cracked skin in male and female animals. Skin irritation had subsided by the end of the second week of treatment and appeared sporadically in a few treated animals through the end of the study.

BODY WEIGHT AND WEIGHT GAIN
There were no significant differences in mean bodyweight, or bodyweight gain, between control and treated males or females during the study.

HAEMATOLOGY
THere were no significant differences between any hematological parameter between control and treated animals that could be attributed to treatment with the test material. There was a significant decrease in the mean percentage of neutrophils between control and treated males but since the decrease was not dose-related it was concluded not to be related to treatment with the test material.

CLINICAL CHEMISTRY
Mean serum calcium and phosphorus levels were significantly greater than control in male and female animals of all treatment groups. Mean blood urea nitrogen levels and mean blood urea nitrogen/creatinine ratios were significantly lower than control in female animals of all treatment groups. Mean serum alkaline phosphatase activity was significantly lower than the control in the mid- and low-dose groups but not in the high-dose group. None of the changes in serum chemistry were dose-related, and no other changes in serum chemistry were observed that could be attributed to treatment with the test material.

ORGAN WEIGHTS
There were no significant differences in mean organ weights or mean organ/body weight ratios between control and treated males. The mean left ovary weight was significantly lower than control in the female high and mid dose groups and the changes were apparently dose-related. There were no differences in the mean right ovary weight . Mean brain weight in females in the high dose group was significantly greater than control but there was no difference in the mean brain/body weight ratio between females in these two groups.

GROSS PATHOLOGY
At necropsy, no gross pathological changes were observed that could be attributed to treatment with the test material.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of tissues from the control, low and high-dose groups did not reveal any histopathological changes or any significantly increased incidences of any pathological lesions that could be attributed to treatment with the test material.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No mortality or signs of toxicity were noted at this treatment level.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Incidence of Skin Irritation from 6 Days of Repeated Dermal Application of 1 mg/kg Test Material

Treatment group	Males			Females
	Erythema	Edema	Dry, flaky and/or cracked skin	Erythema	Edema	Dry, flaky and/or cracked skin
Mineral oil (control)	3/15	0/15	7/15	0/15	0/15	0/15
20% (w/w) test material	6/15	0/15	8/15	2/15	0/15	4/15
50% (w/w) test material	5/15	0/15	5/15	9/15	2/15	10/15
100% test material	11/15	5/15	12/15	10/15	6/15	12/15

Table 2: Organ Weights

Treatment group	Males				Females
	Mineral oil control	20% (w/w) test material	50% (w/w) test material	100%test material	Mineral oil control	20% (w/w) test material	50% (w/w) test material	100% test material
Organ
Brain (g)	2.0	2.0	2.0	2.0	1.9	1.9	1.9	2.0†
Liver (g)	10.3	10.5	10.5	10.4	7.0	7.2	7.1	7.3
Heart (g)	1.2	1.2	1.2	1.2	0.9	0.8	0.8	0.9
Left kidney (g)	1.3	1.3	1.3	1.4	0.9	0.9	0.9	0.9
Right kidney (g)	1.3	1.3	1.4	1.3	0.9	0.9	0.9	0.9
Left testicle (g)/ovary (mg)	1.5	1.6	1.6	1.6	73.4	71.7	62.9†	59.5†
Right testicle (g)/ovary (mg)	1.5	1.6	1.7	1.6	64.0	69.9	66.2	63.4
Left adrenal (mg)	32.6	35.2	32.8	31.7	41.4	40.8	41.9	40.1
Right adrenal (mg)	31.1	31.9	29.4	31.8	35.0	38.6	37.8	37.1

† Significantly different from control (p ≤ 0.01)

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, subacute dermal treatment of male and female rats with test material did not cause any mortality or any signs of toxicity. Mild skin irritation appeared in both sexes during the first week of the study, but subsided thereafter. The statistically significant changes in mean serum calcium levels, serum phosphorus levels, blood urea nitrogen, blood urea nitrogen/creatinine rations, left ovary weight, left ovary/body weight ratio and brain weight were not considered biologically significant. No other significant changes occurred that could be attributed to treatment with the test material. The No Observed Adverse Effect Level was subsequently determined to be 1000 mg/kg bw/day. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Executive summary:

The dermal repeat dose toxicity of the test material was determined following a methodology equivalent to standardised guideline OECD 410. Groups of fifteen male and fifteen female rats were dermally administered 20% w/w, 50% w/w or 100% test material in mineral oil for a period of 4 weeks. Animals were treated for 6 hours per day, six times a week during week 1, fives time per week during weeks 2 and 3 and four times a week during week 4. An additional group of fifteen males and fifteen females were treated with mineral oil alone and served as controls. Clinical observations, bodyweights and skin irritation parameters were measured throughout the study. At the end of the study, the animals were killed and subjected to an examination post mortem. Cardiac blood samples were taken for clinical pathology, selected organs were weighed and specified tissues were taken for subsequent histopathology examination. Under the conditions of the study, subacute dermal treatment of male and female rats with test material did not cause any mortality or any signs of toxicity. Mild skin irritation appeared in both sexes during the first week of the study, but subsided thereafter. The statistically significant changes in mean serum calcium levels, serum phosphorus levels, blood urea nitrogen, blood urea nitrogen/creatinine ratios, left ovary weight, left ovary/body weight ratio and brain weight were not considered biologically significant. No other significant changes occurred that could be attributed to treatment with the test material. The No Observed Adverse Effect Level was subsequently determined to be 1000 mg/kg bw/day.