Benzyltriphenylphosphonium chloride

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

(14-day study in male rats)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19-SEP-1988 to 27-JUN-1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1989

Materials and methods

Principles of method if other than guideline:

- Principle of test: standard protocol similar to sighting assays for repeated dose toxicity studies.
- Short description of test conditions: groups of 10 male rats were exposed by inhalation, nose-only, 6hr/day, 5 days/week for 14 days to 3 test concentrations and to concurrent air control. Five animals per group were sacrificed at the end of the exposure period (on day 12, after the 10th exposure), and the remaining 5 animals per group were examined at the end of a 14-day recovery period (day 26 of the study).
- Parameters analysed / observed: clinical signs, body weights, organ weights, haematology, clinical chemistry, urinalysis, gross and microscopic pathology.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzyltriphenylphosphonium chloride

EC Number:

214-154-3

EC Name:

Benzyltriphenylphosphonium chloride

Cas Number:

1100-88-5

Molecular formula:

C25H22P.Cl

IUPAC Name:

benzyltriphenylphosphonium chloride

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD BR

Details on species / strain selection:

standard strain well characterised in the test laboratory

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC, USA
- Age at study initiation: approx. 8 weeks old at the start of exposures
- Weight at study initiation: 218 - 267 g
- Fasting period before study: No
- Housing: suspended stainless steel, wire-mesh cages (except during exposure periods).
- Diet : ad libitum
- Water: ad libitum
- Acclimation period: a minimum of 6 days prior to testing

DETAILS OF FOOD AND WATER QUALITY:
Feed was periodically analysed for selected food contaminants: heavy metals, pesticides and nitrosamines.
Water was periodically monitored for selected contaminants: selected microorganisms, pesticides, heavy metals and halogens.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target, 23 +/- 2 °C
- Humidity (%): target: 50 +/- 10% (40-60%)
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs/dark cycle

IN-LIFE DATES: From: 19-SEP-1988 To: 14-OCT-1988

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 1.3 - <= 2.2 µm

Remarks on MMAD:

See details in Table 1
Volume median diameter : 24 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The previously milled test material was metered to the dust generator with a K-TRON Model T-20 twin screw Volumetric feeder using a K-Tron digital speed controller. Nitrogen passed continuously through the bin feeder reservoir to minimize abosption of atmospheric moisture by the test material.
- Method of holding animals in test chamber: individually restrained in a perforated, stainless steel cylinder with conical, non-perforated nose piece. Each restrainer is inserted into a face plate attached to a stainless steel and polymethylmethacrylate exposure chamber such that only the nose of each rat protrudes into the chamber (150-L volume).
- Source and rate of air: conditioned filtered air at 39-56 L/min in the dust generators
- Method of conditioning air: filtered
- System of generating particulates/aerosols: the milled BTPPC was suspended in a high pressure air stream. For the 5 and 60 mg/3 atmospheres the test material was metered into a Model 00 Jet-0-Mizer particle microniser. The jet mill disrupted the larger particles with high pressure air jets and discharged the dust into either a 1-L or a 4-L glass cyclone elutriator. For the 15 mg/m3 concentration, BTPPC was metered diretly into a high pressure air jet wich discharged the test material into a 1-L glass cyclonic elutriator. The smaller particles then passed into the 150-L exposure chamber.
- Temperature in air chambers: 18 - 26 °C ; control group chamber: 18 - 25%
- Humidity in air chambers: 46 - 60% ; control group chamber: 49 - 62%
- oxygen concentration in air chambers: 21%
- Air flow rate: Control rats were exposed to conditioned, filtered houseline air at approx 25 L/min.
- Air change rate: at least 10 ACH
- Method of particle size determination: particle size (MMAD and % < 3 or 10 µm) was determined using a Sierra Series 210 cyclone preseparator/cascade impactor and Sierra 110 Constant Flow Air Sampler.
- Treatment of exhaust air: high capacity dust filters and MSA activated charcoal/HEPA cartridge filters prior to discharge into fume hoods

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric analysis at approx every 30 minutes through a 25 mm filter cassette containing a preweighed Gelman Type A/E glass fiber. The filters were weighed on a a Cahn Model 26 Automatic Electrobalance. The difference in the pre- and post-sampling filter weights were
used to calculate the atmospheric concentration of test material.
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle: for control group, the conditioned filtered houseline air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

See Table 2
In addition to the gravimetric analysis reported, a colorimetric method was also used during some of the earlier exposures to verify the presence of chlorine in the chamber filter samples. Chlorine analysis conducted on 10 filters from the high and low-concentration chambers showed results consistent with the amount of chlorine in BTPPC.

Duration of treatment / exposure:

6 hour/day, for 2 weeks, followed by a 14-day recovery period
The starting time of each exposure is defined as the time when the generation system is turned on, and ending time when the generator is turned off.

Frequency of treatment:

5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air (analytical)

Remarks:

(air control)

Doses / concentrations 2

Dose / conc.:

5.1 mg/m³ air (analytical)

Doses / concentrations 3

Dose / conc.:

15 mg/m³ air (analytical)

Doses / concentrations 4

Dose / conc.:

60 mg/m³ air (analytical)

No. of animals per sex per dose:

10 males/group
5 rat/group euthanised after completion of the 10th exposure (Day 12 of study), and 5 rat/group euthanised on the 14th day of recovery (Day 26 of study).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: a range finding test was performed prior to the study with 2 groups of 6 male rats exposed 6 hr/day for 3-4 days at 50 or 60 mg/m3. Severe body weight loss was observed in both groups after the 1st exposure and subsequent days. Clinical signs (ruffled fur, lung noise) were mainly observed at 50 mg/m3. Based on this preliminary assay, 60 mg/m3 was selected as the highest concentration for the 14-day study.
- Animal assignment by computer randomisation to obtain similar pretest mean body weights
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 14-day (5 rats / group) (up to day 26 of study)
- Section schedule rationale (if not random): no data

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs observed daily prior to exposures, during exposure, and after the exposure period before they are returned to their cages

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: daily prior to exposure, daily during the 14-day recovery period (except week ends and holidays).

FOOD CONSUMPTION : No
WATER CONSUMPTION : No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from 10 rats/group after the 10th exposure, and from 5 rat/group on the 14th day of recovery.
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Not specified
- How many animals: from 10 rats/group after the 10th exposure, and from 5 rat/group on the 14th day of recovery
- List of Parameters examined:
Erythrocyte count (RBC)
Haemoglobin concentration (Hb)
Haematocrit (Ht)
Platelet count (PLAT)
Leukocytes count (WBC)
Neutrophils (relative number) (Neut)
Band neutrophils (relative number) (Band)
Lymphocytes (Lymph)
Atypical lymphocytes (Alym)
Eosinophils (Eosin)
Monocytes (Mono)
Basophils (Baso)
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from 10 rats/group after the 10th exposure, and from 5 rat/group on the 14th day of recovery
- Animals fasted: Not specified
- How many animals: from 10 rats/group after the 10th exposure, and from 5 rat/group on the 14th day of recovery
- List of Parameters examined:
Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Alkaline Phosphatase (ALP)
Urea nitrogen (BUN)
Creatinine (CREAT)
Total protein (TPROT)
Cholesterol (CHOL)

URINALYSIS: Yes
- Time schedule for collection of urine: 10 rats / group after the 9th exposure, and from 5 rats /group on the 13th day of recovery
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- List of Parameters examined:
Volume (VOL)
Osmolality (OSMOL)
Urobilinogen (UROBL)
pH
presence of hemoglobin or occult blood
glucose
protein
bilirubin
ketone

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see Table 3)

Statistics:

ANOVA test and Bartlett's test used to asses parameters for each sampling time.
When the F-test from ANOVA was significant, the Dunnett test was use to compare means from the control group and each of the groups exposed to BTPPC.
When the results of the Bartlett test were significant (p < 0.005), the Kruskal-Wallis test was used and the Mann-Whitney U test was used to compare means from the control group and each of the groups exposed to BTPPC.
Significance was judged at the 5% probability level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Effects were observed in some animals exposed to the high concentration group and consisted in lung noise in 1 or 2 animals (Result Table 2)
No relevant effects were observed in the mid-concentration group (ocular effects on 1 animal were attributed to blood sampling on day 12 after the last exposure).
Clinical signs in one animal that was killed in extremis on day 3 of recovery consisted in lung noise, lethargy, weakness, pallor, a black ocular discharge and a swollen paw.

Mortality:

no mortality observed

Description (incidence):

However, one rat from the 60 mg/m3 was killed in extremis 3 days after the last exposure (study day 15, during the recovery period).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Details in Result Table 1.
At the highest concentration (60 mg/m3), the body weight were significantly lower than the control group starting from the second exposure day and throughout the treatment period.
At the mid-concentration (15 mg/m3), the difference was statistically significant only on the 10th exposure (Day 12). Although not reaching statistical significance, body weights were slightly lower also in the low and mid-concentration groups throughout the exposure period, compared to controls.
During the recovery period, the mean body weights remained generally slightly lower at 15 and 60 mg/m3 but no longer statistically significant. In the 15 mg/m3, the mean body weight was similar to controls at the end of the recovery period.

The mean body weight gain was significantly lower in the 15 and 60 mg/m3 groups over the treatment period (day 1 to 12). The mean weight gain in the 3 treated groups was then higher than the control group during the recovery period, but without statistical significance.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significant effects were observed in animals of the mid- and high concentrations examined at the end if the exposure period (details in Result Table 3): Increased mean circulating erythrocytes (RBC, Hb, Ht) and serum total protein concentration suggested dehydration, but this was not confirmed by renal findings in the histopathology examination.
No remarkable effects were observed at the low concentration.
No significant differences were seen in treated groups compared to control group after the 14-day recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Details in Result Table 4:
An increase in liver enzymes (ALT and AST) was observed at the end of the 2-week exposure period in the mid and high concentration groups. No significant differences were observed at the low concentration..
No effects were seen at the end of the recovery period, except for total proteins in serum that were found lower than in the control group.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased urine volume in groups treated at 15 and 60 mg/m3 after the 10th exposure, and a lower pH in the high concentration animals. No significant effects were seen at the end of the recovery period.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The statistically significant increase in mean relative lung to body weight at the end of the exposure period, in groups exposed to 15 and 60 mg/m3 was considered treatment-related (Result Table 5).
No effects were seen at the end of the recovery period.
The decreased mean absolute liver and testes weights at 5.1 and 60 mg/m3 occured in the absence of histopathology findings, were of low magnitude and not concentration-related. The decreased absolute and relative spleen weight especially at 60 mg/m3 may be secondary to inanition or weight loss and stress.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Result Table 6. The main finding considered treatment-related was periocular chromodacryorrhea in the high concentration animals with a higher incidence (2 to 3-times) than usual.
treatment period (incidence in groups 0, 5.1, 15, 60 mg/m3 respectively): 1 / 0 / 0 / 3
Recovery period (incidence in groups 0, 5.1, 15, 60 mg/m3 respectively): 0 / 0 / 0 / 1

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Summary of microscopic findings in the lung and nasal cavities (Result Table 7). The incidence and severity of lesions increased with the test concentrations.
- At the end of the exposure period:
Lung: minimal to moderate hypertrophy and hyperplasia of the pulmonary bronchial and bronchiolar epithelium.
Nasal cavities: minimal to moderate inflammation and necrosis of respiratory and olfactory epithelium and minimal to moderate squamous metaplasia of respiratory epithelium. Inflammation and necrosis were more severe throughout the dorsal and medial aspects of the nasal cavities.
Squamous metaplasia was observed along the dorsal-lateral aspects of the anterior nasal cavity. The severity of nasal lesions were concentration-related.

- At the end of the recovery period:
Effects were observed only in the nasal cavities of the high concentration animals.
Lung: no lesions observed
Nasal cavities: minimal to mild necrosis and inflammation of the respiratory and olfactory epithelium, and minimal to mild squamous metaplasia of respiratory epithelium.
Minimal to mild Goblet cell hyperplasia was observed along the nasal septum, and focal respiratory epithelial hyperplasia on nasoturbinates. These lesions were considered reparative in nature.
Minimal to mild atrophy of the olfactory epithelium along the dorsal meatus and dorsal medial septum, and was considered as a stage of incomplete repair.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

ca. 5.1 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

15 mg/m³ air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

lungs

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

Result Table 1a: Mean body weights with Standard Deviation (SD)

Concentration:	0 mg/m3	5.1 mg/m3	15 mg/m3	60 mg/m3
	Mean (SD)	Mean (SD)	Mean (SD)	Mean (SD)
Days on test
Exposure period
1	244.1 (9.4)	242.2 (10.6)	245.1 (8.9)	249.9 (11.6)
2	248.1 (8.7)	241.4 (12.4)	243.2 (8.0)	231.4 (11.1)*
3	250.7 (10.5)	247.1 (12.9)	249.6 (8.1)	233.1 (6.3)*
4	256.3 (10.0)	250.7 (14.9)	252.5 (6.3)	233.2 (5.7)*
5	263.5 (12.4)	256.8 (13.3)	258.3 (5.7)	236.4 (7.0)*
8	288.5 (13.6)	279.8 (17.0)	285.1 (7.9)	265.6 (10.3)*
9	293.0 (16.3)	286.9 (18.0)	290.7 (11.3)	262.5 (7.2)*
10	298.0 (16.0)	289.6 (18.9)	290.9 (12.7)	262.0 (6.9)*
11	304.0 (14.1)	295.5 (17.4)	294.3 (10.4)	266.4 (11.2)*
12 (10^thexposure)	306.8 (12.1)	294.0 (21.3)	287.9 (12.3)*	255.4 (19.5)*
Recovery period
15	315.5 (22.0)	313.2 (20.7)	306.2 (15.3)	258.4 (43.2)*
16	327.4 (21.7)	328.6 (22.6)	320.4 (16.7)	294.4 (5.6)
17	328.3 (21.5)	332.0 (24.1)	325.5 (17.4)	302.5 (5.5)
18	335.7 (22.4)	338.4 (24.2)	328.1 (20.8)	309.4 (4.5)
19	339.1 (21.2)	337.5 (21.9)	330.5 (22.5)	310.5 (7.1)
22	362.3 (31.2)	363.9 (27.0)	355.6 (27.6	337.2 (8.8)
23	364.7 (31.1)	369.8 (28.8)	357.0 (27.9)	344.4 (11.4)
24	367.4 (26.3)	372.9 (30.1)	361.6 (32.1)	347.5 (12.3)
25	372.7 (28.3)	381.9 (31.6)	367.4 (35.7)	357.1 (15.8)
26	374.6 (28.3)	375.3 (25.1)	375.2 (32.0)	362.2 (19.2)

 *Dunnett's test (p<0.05) (comparison to control group)

(based on 10 animals during exposure period, 5 animals during recovery period)

Result Table 1b: Mean body weight gains with standard deviations (SD)

days 1-12 (exposure)	62.7 (13.0)	51.8 (14.8)	42.8 (13.5)*	5.5 (19.0)*
days 15-26 (recovery)	59.1 (7.8)	62.1 (8.1)	69.0 (21.0)	84.4 (18.5)

Significant different from controls by Least Significant Differences (LSD) and Dunnett's tests (p<0.05)

Result Table 2: Summary of clinical observations (number of rats with signs and median day of 1st observation)

Concentration:	0 mg/m3	5.1 mg/m3	15 mg/m3	60 mg/m3
Observations	Number of rats with signs (median day of 1^stobservation)
Alopecia	0	0	1 (2)	0
Colored discharge, eye	0	0	1 (25) (°)	1 (15) (x)
Enophthalmos	0	0	1 (25) (°)	0
Lethargy	0	0	0	1 (15) (x)
Lung noise	0	0	0	3 (12)
Pallor	0	0	0	1 (15) (x)
Scab	0	0	1 (15) (°)	0
Swollen paw, right rear	0	0	0	1 (15) (x)
weak	0	0	0	1 (15) (x)
Total group incidence	0	0	4	8

(°) Ocular effects attributed to injury during blood sampling in one rat on day 12

(x) animal killed in extremis

Result Table 3: Results of Haematology parameters for the exposure period (samples from day 12)

Concentration:	0 mg/m3	5.1 mg/m3	15 mg/m3	60 mg/m3
	Mean (SD)	Mean (SD)	Mean (SD)	Mean (SD)
Exposure period
RBC (10E6/µl)	6.89 (0.30)	7.07 (0.34)	7.34 (0.12)*	7.49 (0.33)*
Hb (g/dl)	14.3 (0.6)	14.6 (0.7)	15.5 (0.5)*	15.8 (0.8)*
Ht (%)	46.0 (2.0)	47.0 (1.0)	49.0 (1.0)*	50.0 (2.0)*
MCV (fl)	67.0 (2.0)	66.0 (2.0)	67.0 (1.0)	66.0 (2.0)
MCH (pg)	21.0 (1.0)	21.0 (1.0)	21.0 (0.0)	21.0 (1.0)
MCHC (g/dl)	31.0 (0.0)	31.0 (1.0)	32.0 (0.0)	32.0 (1.0)*
Platelet (10E3/µl)	1271 (323)	1216 (390)	1049 (175)	1285 (383)
WBC (10E3/µl)	14.6 (2.3)a	12.8 (2.9)	11.3 (2.6)*	9.3 (3.8)*
Neut (WBCx%	3191 (1234)	3462 (882)	3119 (1618)	3004 (1638)
Band (WBCx%)	0	0	0	0
Lymph (WBCx%)	10950 (2588)	8976 (2212)	7781 (2013)*	6061 (2500)*
Alymph (WBCx%)	45 (73)	48 (89)	90 (185)	5 (17)
Mono (WBCx%)	378 (208)	311 (251)	330 (166)	231 (114)
Eosin (WBCx%)	47 (76)	14 (43)	0 (0)	29 (62)
Baso (WBCx%)	0	0	0	0

* Significant difference between treated and control groups at 5% by Dunnett's test

All parameters were not found significantly different at the end of the recovery period (day 26)

Based on analysis from 10 animals per group.

Result Table 4: Results of clinical chemistry (serum and urine) for the exposure period (samples from day 12)

Concentration:	0 mg/m3	5.1 mg/m3	15 mg/m3	60 mg/m3
	Mean (SD)	Mean (SD)	Mean (SD)	Mean (SD)
Exposure period (d12)
Serum
ALP (U/l)	400 (100)	341 (108)	367 (116)	366 (131)
ALT (U/l)	44 (8)	47 (13)	49 (8)	57 (11)*
AST (U/l)	71 (10)	78 (12)	90 (10)#	148 (64)#
BUN (mg/dl)	17 (3)	16 (2)	16 (2)	15 (2)
Creatinine (mg/dl)	0.6 (0.1)	0.6 (0.1)	0.6 (0.1)	0.6 (0.1)
Total Prot (g/dl)	5.7 (0.3)	5.7 (0.4)	6.1 (0.3)*	6.1 (0.3)*
Cholesterol (mg/dl)	63 (16)	56 (7)	52 (11)	62 (12)
Urine
Volume (ml)	10.3 (1.8)	9.0 (2.1)	7.0 (2.2)*	6.2 (2.8)*
Osmolality (mOs)	1615 (394)	1755 (314)	1955 (378)	1788 (534)
pH	7.6 (0.6)	7.3 (0.3)	7.2 (0.5)	6.8 (0.5)*
Blood in urine (mg/dl)	0.1 (0.0)	0.1 (0.0)	0.1 (0.0)	0.1 (0.0)
Recovery period (d26)
Serum
Total Prot (g/dl)	6.5 (0.2)	6.2 (0.2)	6.1 (0.2)*	5.9 (0.4)*

*Significant difference between treated and control groups at 5% by Dunnett's test

# Significant difference between treated and control groups at 5% by Mann-Whitney U test

Except total serum protein, all parameters were not found significantly different at the end of the recovery period (day 26)

Based on analysis from 10 animals per group.

Result Table 5 : Organ weights

Concentration	Final body weight	Liver	Kidneys	Lungs	Spleen	Testes
Exposure day 12	Mean (SD)	Mean (SD)	Mean (SD)	Mean (SD)	Mean (SD)	Mean (SD)
Absolute weight
0	310.1 (8.7)	10.724 (0.695)	2.499 (0.116)	1.455 (0.086)	0.735 (0.152)	3.377 (0.230)
5.1 mg/m3	289.6 (15.2)+	8.994 (1.607)#	1.920 (0.676)	1.344 (0.087)	0.627 (0.119)	3.023 (0.135)+
15 mg/m3	288.5 (14.6)+	9.863 (0.773)	2.349 (0.152)	1.557 (0.113)	0.590 (0.090)	3.371 (0.231)
60 mg/m3	264.4 (15.8)#	9.047 (0.521)#	2.169 (0.072)	1.505 (0.100)	0.428 (0.065)#	3.011 (0.254)#
Relative weight
0 mg/m3		3.4578 (0.1882)	0.8061 (0.0348)	0.4691 (0.0230)	0.2365 (0.0444)	1.0890 (0.0645)
5.1 mg/m3		3.0905 (0.4202)	0.6572 (0.2043)	0.4645 (0.0282)	0.2131 (0.0452)	1.0445 (0.0381)
15 mg/m3		3.4173 (0.1782)	0.8154 (0.0614)	0.5404 (0.0440)#	0.2040 (0.0228)	1.1734 (0.1326)
60 mg/m3		3.4240 (0.1466)	0.8227 (0.0561)	0.5701 (0.0377)#	0.1623 (0.0243)#	1.1384 (0.0596)
Recovery day 26
Absolute weight
0	374.6 (28.3)	12.840 (1.713)	3.053 (0.203)	1.716 (0.053)	0.756 (0.141)	3.295 ( 0.501)
5.1 mg/m3	375.3 (25.1)	13.282 (1.836)	3.007 (0.401)	1.706 (0.251)	1.030 (0.585)	3.241 (0.238)
15 mg/m3	375.2 (32.0)	13.012 (2.387)	2.691 (0.315)	1.745 (0.233)	0.861 (0.066)	3.075 (0.968)
60 mg/m3	362.1 (19.2)	13.369 (1.213)	2.917 (0.218)	1.653 (0.038)	1.653 (0.038)	3.418 (0.327)
Relative weight
0 mg/m3		3.4185 (0.2350)	0.8173 (0.0623)	0.4607 (0.0443	0.2008 (0.0276)	0.8763 (0.0842)
5.1 mg/m3		3.5300 (0.3444)	0.7998 (0.0780)	0.4541 (0.0548)	0.2716 (0.1442)	0.8647 (0.0526)
15 mg/m3		3.4525 (0.3961)	0.7167 (0.0435)	0.4653 (0.0498)	0.2306 (0.0234)	0.8302 (0.2715)
60 mg/m3		3.6906 (0.2467)	0.8087 (0.0943)	0.4574 (0.0273)	0.2109 (0.0326)	0.9481 (0.1259)

+ significantly different (p<0.05) from control group by Least Significant Differences (LSD) test

# significantly different (p<0.05) from control group by LSD and Dunnett’s test

Based on examination from 5 animals per group.

Result Table 6: gross observations

Concentration:	0 mg/m3	5.1 mg/m3	15 mg/m3	60 mg/m3
	Mean (SD)	Mean (SD)	Mean (SD)	Mean (SD)
Exposure period
Skin stain	0	0	1	3
Periocular chromodachryorrhea	1	0	0	3
Kidneys deformity	0	1	0	0
Renal pelvis dilatation	0	0	1	0
Epididymides nodule	0	0	0	1
Adrenals deformity	0	1	0	0
Recovery period
Whole body, thin	0	0	0	1
Periocular chromodachryorrhea	0	0	0	1
Kidneys, large	1	0	0	0
Renal pelvis, dilatation	1	0	0	0
Testes, small	0	0	1	0
Epididymides, discoloration	0	0	0	1
Eyes, enophthalmus	0	0	1	0

Based on analysis on 5 animals per group.

Result Table 7: Summary of microscopic observations in Lungs and Nasal cavities

	Concentration:	5.1 mg/m3	15 mg/m3	60 mg/m3	5.1 mg/m3	15 mg/m3	60 mg/m3
		Exposure	Exposure	Exposure	Recovery	Recovery	Recovery
Lungs	Hypertrophy/hyperplasia, respiratory epithelium (#) Grade 1 (minimal) Grade 2 (mild) Grade 3 (moderate) Grade 4 (severe)	(4) 4 - - -	(5) 2 3 - -	(5) 1 3 1 -	-	-	-
Nose, Levels I & II	Atrophy, olfactory epithelium (#) Grade 1 (minimal) Grade 2 (mild) Grade 3 (moderate) Grade 4 (severe)	-	-	-	-	-	(4) 2 2 - -
Nose, Levels I & II	Goblet cell hyperplasia, respiratory mucosa (#) Grade 1 (minimal) Grade 2 (mild) Grade 3 (moderate) Grade 4 (severe)	-	-	-	-	(2) 2 - -	(2) 1 1 -
Nose, Levels I & II	Hyperplasia, focal, respiratory epithelium (#) Grade 1 (minimal) Grade 2 (mild) Grade 3 (moderate) Grade 4 (severe)	-	-	-	-	-	(2) - 1 1 -
Nose, Levels I & II	Inflammation/necrosis, acute/subacute, mucosa (#) Grade 1 (minimal) Grade 2 (mild) Grade 3 (moderate) Grade 4 (severe)	(3) 3 - - -	(5) 2 3 - -	(5) - 2 3 -	-	-	(3) 2 1 - -
Nose, Levels I & II	Squamous metaplasia, respiratory mucosa (#) Grade 1 (minimal) Grade 2 (mild) Grade 3 (moderate) Grade 4 (severe)	-	(3) 3 - - -	(5) 1 3 1 -	-	-	(3) 2 1 - -
Nose, Levels III & IV	Atrophy, olfactory epithelium (#) Grade 1 (minimal) Grade 2 (mild) Grade 3 (moderate) Grade 4 (severe)	-	-	-	-	-	(4) 2 2 - -
Nose, Levels III & IV	Inflammation/necrosis, acute/subacute, mucosa (#) Grade 1 (minimal) Grade 2 (mild) Grade 3 (moderate) Grade 4 (severe)	(3) 3 - - -	(5) 1 4 - -	(5) - 1 4 -	-	-	(2) 1 1 - -

5 animals examined for each group.

#: total number affected in each group; incidence for each grade.

Applicant's summary and conclusion

Conclusions:

Exposure to BTPPC during 14 days up to 60 mg/m3 caused concentration-related reductions in body weight and lower body weight gains. Lesions in the lung and nasal cavities were observed after the 14-day exposure. No lesions were observed in the lung following the recovery period, but some nasal lesions persisted in some animals of the mid- and high concentration groups with a lower incidence and severity. Considering the minimal effects at the lowest concentration, the NOAEC is considered at 5.1 mg/m3. The Lowest Adverse Effect Concentration is considered to be 15 mg/m3.

Executive summary:

A 14-day repeated inhalation exposure to BTPPC particles of respirable size was conducted in male Crl:CD BR rats in compliance with GLP principles. Groups of 10 animals were exposed nose-only to concentrations of 0 (air control), 5.1, 15 or 60 mg/m3 BTPPC (MMAD ca. 1.3-2.2 µm) for 6 hours/day, 5 days/week, for a total of 10 exposures. At the end of the exposure period, blood and urine samples were collected from all the animals for clinial analysis and 5 rats per group were euthanised for pathologic examination. The remaining rats were observed during a 14-day post-exposure recovery period and necropsied on day 26 of the study for the same examinations.

Clinical signs were observed in the high concentration group and included mainly lung noise following exposure and chromodacryorrhea.

Lower mean body weights and mean body weight gains were observed in treated rats compared to controls during the exposure period. The mean body weight were significantly lower than controls starting from the second exposure day and throughout the exposure period at the high concentration, and the difference was statistically significant only on day 12 at the mid-concentration. At the low concentration, the mean body weights were slightly lower than controls throughout the treatment period, without statistical significance. At the end of the recovery period, mean body weight were similar to controls at 5.1 and 15 mg/m3, and only slightly lower at 60 mg/m3.

The test substance caused significant increases in red blood cells count, haematocrit, hemoglobin and serum total proteins, and decreased urine volume in rats exposed to 15 or 60 mg/m3. These changes were thought to be consistent with dehydration but there was no supporting microscopic findings.

Reduction of total lymphocytes and a slight increase in ALAT, and a larger increase in ASAT were also observed in the same treatment groups.

These parameters had returned to control values following the 14-day recovery period, except mean serum total protein values that were found lower in the 2 higher concentration groups.

The only substance-related change in organ weight was a significant increase in mean relative lung to body weights observed in rats exposed to 15 and 60 mg/m3 at the end of the exposure period, and no longer seen after the recovery period.

Substance-related microscopic findings consisted in lesions in the lungs and nasal cavities of rats from all treatment groups following the exposure period, and in the nasal cavities of rats from the 15 and 60 mg/m3 groups after the 14-day recovery period. The severity and incidence of lesions were concentration-related. At the end of the exposure period, they consisted of minimal to moderate hypertrophy and hyperplasia of bronchial and bronchioalveolar epithelium, necrosis and inflammation of nasal olfactory epithelium, and necrosis, inflammation and squamous metaplasia of nasal respiratory epithelium. At the end of the recovery period, the incidence and severity of nasal lesions observed only at 15 and 60 mg/m3 were lowered and consisted of necrosis and inflammation of nasal respiratory and olfactory epithelium, squamous metaplasia, goblet cell hyperplasia and focal hyperplasia of the respiratory epithelium, and atrophy of olfatcory epithelium.

Under the conditions of this study, based on the minimal effects observed at the low concentration, 5.1 mg/m3 is considered a No-observed Adverse Effect Concentration (NOAEC) and the Lowest Adverse Effect Concentration (LOAEC) is set at 15 mg/m3.