3-iodo-2-propynyl butylcarbamate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

1981

Deviations:

yes

Remarks:

adrenals were not weighed

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-iodo-2-propynyl butylcarbamate

EC Number:

259-627-5

EC Name:

3-iodo-2-propynyl butylcarbamate

Cas Number:

55406-53-6

Molecular formula:

C8H12INO2

IUPAC Name:

3-iodoprop-2-yn-1-yl butylcarbamate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2DR-293-TSI

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 (UK) Ltd
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: Group mean weight first delivery: males: 268 - 273 g; females: 188 - 193 g; second delivery: males: 312 g; females: 219 g
- Housing: individually
- Diet: ad libitum
- Water:ad libitum

DETAILS OF FOOD AND WATER QUALITY: There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test substance could reasonably be expected to be present in the diet or in the drinking water.


ENVIRONMENTAL CONDITIONS
- Temperature: 15.5 - 23.5°C
- Humidity: 25 - 54 %
- Photoperiod: 12 / 12 hrs dark / hrs light

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

> 1.55 - < 3.5 µm

Remarks on MMAD:

Examination of the data indicates that the test aerosol did not contain a normal distribution of particle sizes. The test aerosols consisted of at least 2 populations of particles, one with a median aerodynamic diameter of between approximately 1.55 and 3.5 µm and one with median aerodynamic diameter less than 0.5 µm. Size distributions of this type cannot be described by a single mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD).
67.5 to 83.1 % of the population of particles is considered to be less than 6 µm in aerodynamic diameter in each dose group, respectively. These particles are considered to be respirable to the rat.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chambers were of stainless steel and glass construction, approx. 0.75 m^3 internal volume
- System of generating particulates/aerosols: Wright dust feed mechanism (WDFM)

TEST ATMOSPHERE
- Brief description of analytical method used: Samples of each test atmosphere were withdrawn through 37 mm diameter glass microfibre filters (Type GF/A, Whatman International Ltd, Maidstone, Kent, England) supported in an open-face holder, at a rate of 10 litres per minute. The total volume sampled was measured using an in-line wet-type gas meter (Model DM3D Alexander Wright and Co (Westminster) Ltd). The test substance deposited on the filters was extracted into acetonitrile and the resultant solutions were analysed by an HPLC method.

VEHICLE: air

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

6 h

Frequency of treatment:

5 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air

Remarks:

control

Doses / concentrations 2

Dose / conc.:

0.25 mg/m³ air

Remarks:

analytical: 0.30 mg/m^3

Doses / concentrations 3

Dose / conc.:

1.25 mg/m³ air

Remarks:

analytical: 1.16 mg/m^3

Doses / concentrations 4

Dose / conc.:

6.25 mg/m³ air

Remarks:

analytical: 6.70 mg/m^3

Doses / concentrations 5

Dose / conc.:

0.25 mg/m³ air

Remarks:

analytical: 0.23 mg/m^3 (repeat low dose)

No. of animals per sex per dose:

Main groups: 10
Satellite groups: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: random
- Post-exposure recovery period in satellite groups: no

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during each exposure period and at least twice daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: for allocation to groups and weekly, starting one week before exposures commenced until the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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 AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during week 13 of exposure
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all animals of the main groups
- Parameters examined: packed cell volume (PCV), haemoglobin concentration, erythrocyte count, total and differential white cell count, platelet count, mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), reticulocyte count, cell morphology, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 13
- Animals fasted: Not specified
- How many animals: all animals of the main groups
- Parameters examined: sodium, potassium, calcium, inorganic phosphorus, chloride, glucose, cholesterol, urea nitrogen, bilirubin, creatinine, total protein, albumin, globulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), gamma glutamyl transpeptidase (gGT)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
ORGAN WEIGHTS: Yes, all animals: liver, kidneys, testes with epididymides main groups: brain

HISTOPATHOLOGY: Yes
- tissues examined:
control and high dosed groups: adrenals, aorta, caecum, colon, duodenum, eyes, gross abnormalities, heart, ileum, jejunum, lymph nodes, mammary gland, nasal passages, oesophagus, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, sternum with marrow, testes with epididymides, thymus, thyroids with parathyroids, trachea, urinary bladder, uterus
all rats: kidneys, liver, lungs (all lobes and bronchi)
control, low, repeat low, and high dosed groups: larynx
main groups only: brain (medullary cerebellar and cortical sections)

Other examinations:

Plasma and erythrocyte cholinesterase activity was determined in the animals of the satellite groups after 2 and 13 weeks. Brain cholinesterase activity was determined in the satellite groups after terminal sacrifice. For determination of cholinesterase activity the method of Ellman was used.

Statistics:

Bartlett’s test: heterogeneity of variance between treatment when the relative frequency of data does not exceed 75%. One-way analysis of variance: for data not showing statistically significant heterogeneity using Bartlett’s test. Kruskal-Wallis analysis for data with significant heterogeneity of variance. Student’s t-test and Williams test for a dose-related response. Analysis of covariance it appropriate. Fischer’s Exact Test for histopathological incidences.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

During the first three exposures, closed/half closed eyes were seen in the high dosed rats. Red ears were noted following exposures 3, 4, and 5. These effects were considered to be attributable to the irritating potential of the test substance and not of toxicological relevance. There were no other treatment-related signs noted. Clinical signs attributable to cholinesterase inhibition were also not noted.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two male and two female rats of the low dose groups were sacrificed during week 10 on study following the period of overdosing. There were no deaths attributable to treatment with the test substance.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean group body weight and body weight gain were comparable between control and treated dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean group food consumption was comparable between control and treated dose groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopy was comparable between controls and treated groups.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant differences from concurrent control values were observed:
In the high dosed females a greater erythrocyte count, higher PCV, lower MCHC and lower reticulocyte count when compared to controls were observed. In all treated male groups MCV was lower when compared to concurrent controls.
All the above differences were either of low magnitude, not dose-related or inconsistent between sexes. Thus, these changes are not considered to be of toxicological relevance.
Values for haematological parameters of the repeat low dose group were also similar to the controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes in cholinesterase activity determination were noted:
During week 2 a statistically significant lower plasma cholinesterase activity (-21.7%) in high dosed males when compared to concurrent controls and a statistically significant lower erythrocyte cholinesterase activity (-38.9%) in high dosed females when compared to concurrent controls was observed. During week 13 plasma cholinesterase activity in high dosed males was statistically significant lower (-18.2%) when compared to concurrent controls. Brain cholinesterase activity in the mid dosed females (-24.6%) and in males and females of the high dose group was statistically significantly reduced (-16.8% and –35.3%, respectively) when compared to concurrent controls.

Plasma cholinesterase activity is considered to be only a marker of exposure and should not be considered to be of toxicological relevance.

Erythrocyte cholinesterase activity was statistically significant reduced in the high dosed females after two weeks. After 13 weeks of treatment, however, there was no statistically significant difference to concurrent control in erythrocyte cholinesterase activity noted. In the absence of time consistency, reduced erythrocyte cholinesterase activity in the high dose females was not considered to be of toxicological relevance.

At termination, brain cholinesterase activity was statistically significantly reduced in the mid dosed females (-25% lower than concurrent control) as well as in the high dosed males and females (-17% and 36% lower than concurrent control, respectively). However, the trigger of 20% reduction is not reached when females brain cholinesterase activities are compared to the historical control mean. At 1.16 mg/m3, the measured mean brain cholinesterase activity is 10.5% lower than historical control mean, at 6.7 mg/m3 12.3%. A clear dose-response relationship is further not observable.

However, when control brain cholinesterase activities are compared to the historical control data given in the report, one notes that activities show a great variability. Males concurrent control brain cholinesterase activity is 9% lower when compared to historical control mean. In females the concurrent mean control values exceeds the historical mean (+19%).

By inspection of individual data, one can see that the lowest concurrent control value (5.03 µmol/g/min for males and 4.74 µmol/g/min in females) is always comparable to the lowest figure of the treated animals (4.35 µmol/g/min for males and 4.2 µmol/g/min for females).
It is therefore concluded, that brain cholinesterase activity was not adversely affected during the course of the study up to and including the high dose animals and is within the normal biological variation.
The following statistically significant changes in further clinical chemistry parameters were noted: During week 13, higher albumin levels in the high dosed females when compared to controls were observed. ALT activity in the low dosed females and in males and females of the mid and high dosed groups was elevated when compared to concurrent controls.
Changes in albumin and glucose parameters were not considered to be related to treatment because they were inconsistent between sexes, of low magnitude, and/or without a dose response relationship. The increases in ALT activity were within the expected range for rats of this age and strain. In the absence of any histopathological findings in liver, the increase in ALT was not considered to be of toxicological importance.

Clinical chemistry parameters of the repeat low dose group were well within the expected range for animals of this age and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute and adjusted organ weights were comparable to control values.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no changes in macroscopic pathology noted in which could be attributed to test item administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related histopathological changes were noted larynx and lungs:
In lungs, macrophage aggregates were observed in a slightly higher incidence in the high dosed males when compared to control males. In all treated females and in the low and mid dosed males, the incidence in macrophage aggregates in lungs was comparable to the concurrent control.

Histopathological findings in the larynx were necrosis in the ventral cartilage, epithelial hyperplasia in ventral region, and squamous metaplasia in ventrolateral region in all animals of the high dose group.

In addition, epithelial hyperplasia over the arytenoid projections in all high dosed males and a proportion of the high dosed females was noted. Epithelial ulceration in the ventral region was observed in low incidence in the high dosed males. In some of the high dosed males and females, additionally, atrophy of submucosal glands was noted.
Similar findings (epithelial hyperplasia and/or necrosis of the ventral cartilage) were observed in low incidence in the initial low dosed animals at terminal sacrifice. This was not observed in the animals of this dose group sacrificed during the course of the study. Furthermore, no histopathological lesions were observed in the repeat low dose group.

In conclusion, histopathological lesions in the larynx observed in the initial low dosed animals are not considered to be related to test item treatment due to the lack of a dose-response relationship and thus are considered to be incidental.

The larynx is often damaged during inhalation studies especially in the high dosed animals due to its morphology. Especially the ventral cartilage – a small U-shaped cartilage - is affected. The ventral cartilage lies immediately beneath the thin surface squamous epithelium. The damage of the epithelium normally results in necrosis of the underlying cartilage. Subsequently the epithelium of the cartilage undergoes reparative hyperplasia but the necrotic appearance of the cartilage persists after the epithelium has repaired.
The test item is known to be an irritating substance. The histopathological changes of the larynx in the high dosed animals may thus be associated with the intrinsic irritating properties of the test item.

It should further be considered during evaluation that rats respire obligatory through their nose whereas human tend to respire also through the mouth. The anatomical differences in the nasal passages as well as in the larynx should also be taken into account. For these reasons, histopathological changes seen in larynx are not considered to be of relevance for human exposed to the test item via air.
There were no histopathological changes noted in the other organs.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

LOAEC

Effect level:

6.7 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEC

Effect level:

1.16 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.007 mg/L air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

larynx

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

Table 1 Cholinesterase activity determination

Parameter	unit	time [week]	Dose level [mg/m3]
			0		0.3	0.23	1.16	6.7
			controls	historical
Males
plasma ChE	µmol/mL/min	2	0.46	0.47	0.46	0.40	0.40	0.36** (-21.7%)1)
RBC  ChE	µmol/mL/min		1.41	2.03	1.37	2.83	1.52	1.59
plasma ChE	µmol/mL/min	13	0.44	0.47	0.42	0.42	0.44	0.36** (-18.2%)1)
RBC  ChE	µmol/mL/min		1.68	2.03	1.74	1.37	1.54	1.07
brain ChE	µmol/g/min	14	5.77	6.31	5.02 (-13.0%)1 (-20.4%)2	7.17 (+13.6%)	5.05 (-12.5%)1 (-20.0%)2	4.80* (-16.8%)1 (-23.9%)2)
Females
plasma ChE	µmol/mL/min	2	0.83	1.30	0.79	1.01	0.88	0.73
RBC  ChE	µmol/mL/min		1.62	1.64	1.46	1.67	1.46	0.99* (-38.9%)
plasma ChE	µmol/mL/min	13	1.27	1.30	1.46	1.36	1.28	1.29
RBC  ChE	µmol/mL/min		1.90	1.64	1.59	1.22	2.03	2.00
brain ChE	µmol/g/min	14	6.59	5.55	6.38 (-3.2%)1 (+15.0%)2	7.21 (+29.9%)2	4.97* (-24.6%)1 (-10.5%)2	4.87* (-35.3%)1 (-12.3%)2

*          p < 0.05 compared with control values (William’s test)

**         p < 0.01 compared with control values (William’s test)

1         Values in parenthesis indicate the percentage change from concurrent control

2         Values in parenthesis indicate the percentage change from historical control

 

Table 2 Results of 90-day inhalation toxicity study in rats

Parameter	Dose level [mg/m3]										dose-response +/-
	0		0.3		0.23		1.16		6.7
	m	f	m	f	m	f	m	f	m	f	m	f
number of animals examined	15	15	15	15	15	15	15	15	15	15	15	15
Mortality	0	0	21)	21)	0	0	0	0	0	0	-	-
clinical signs	0	0	0	0	0	0	0	0	0	0	-	-
body weight			ne	ne	ne	ne	ne	ne	ne	ne	-	-
food consumption			ne	ne	ne	ne	ne	ne	ne	ne	-	-
clinical chemistry			ne	ne	ne	ne	ne	ne	ne	ne	-	-
haematology			ne	ne	ne	ne	ne	ne	ne	ne	-	-
cholinesterase activities			ne	ne	ne	ne	ne	ne	ne	ne	-	-
Larynx
weight	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd
gross pathology	ne	ne	ne	ne	ne	ne	ne	ne	ne	ne	-	-
microscopic pathology	152)	15	13	13	15	15	15	15	15	15
necrosis of the ventral cartilage	0	0	2	0	0	0	nd	nd	15	15	+	+
epithelial hyperplasia in ventral region	0	0	4	7	0	0	nd	nd	15	15	+	+
epithelial hyperplasia over arytenoid	0	0	0	1	0	0	nd	nd	15	5	+	+
squamous metaplasia in ventrolateral region	0	0	0	0	0	0	nd	nd	15	15	+	+
epithelial ulceration in ventral region	0	0	0	0	0	0	nd	nd	4	1	+	-
atrophy of submucosal glands	0	0	0	0	0	0	nd	nd	3	6	+	+
Lungs
weight			ne	ne	ne	ne	ne	ne	ne	ne	-	-
gross pathology			ne	ne	ne	ne	ne	ne	ne	ne	-	-
microscopic pathology	152)	15	13	13	15	15	15	15	15	15
aggregates of macrophages	4	1	3	1	1	0	3	3	7	2	+	-

 

1)       sacrificed due to overdosing

2)        number of animals examined at terminal sacrifice

3)        Please note: the low dosing regime was repeated due to overdosing of the original low dose. However, no concurrent control was used

Abbreviations:

ne        no effects
nd       not determined
na        not applicable (no concurrent control)

 

Applicant's summary and conclusion

Conclusions:

Based on the results and under the conditions of this study a NOAEC is considered to be 1.16 mg/m^3 based on larynx effects.

Executive summary:

Treatment of Crl:CD BR rats over 90 days with the test item via the inhalation route at doses of 0, 0.23, 0.3, 1.16, and 6.7 mg/m3 did not influence the survival, the body weight development, or the food consumption of all treated animals. Ophthalmoscopical examination did not show any treatment-related effects on the eyes of the treated rats. There were no treatment-related effects on haematological and clinical chemistry parameters noted.

There were no changes in organ weights noted. Histopathological examination of the organs showed no changes in treated animals when compared to controls except for larynx and lungs of the high dosed animals. In lungs an increased incidence in the aggregation of macrophages was noted in the high dosed males (7 of 15 animals compared to 4 of 15 animals in the concurrent control).

RBC and brain cholinesterase activities were within the normal biological variation.

Histopathological findings in the larynx of the high dose group were necrosis in the ventral cartilage, epithelial hyperplasia in ventral region, and squamous metaplasia in ventrolateral region in all animals. In addition, epithelial hyperplasia over the arytenoid projections in all high dosed males and in 5 of the 15 high dosed females was noted. Epithelial ulceration in the ventral region was observed in low incidence in the high dosed males (4 of 15 animals). In some of the high dosed males and females, additionally, atrophy of submucosal glands was noted (3 and 6 animals of 15, respectively).