Dipropoxymethane

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 04 APRIL 2019 to 11 SEPTEMBER 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

Some observed chamber temperatures and humidity were below the recommended range (recorded values 14.5 - 16.6 °C). These deviations had no impact on study integrity.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diethoxymethane

EC Number:

207-330-6

EC Name:

Diethoxymethane

Cas Number:

462-95-3

Molecular formula:

C5H12O2

IUPAC Name:

Diethoxymethane

Test material form:

liquid

Remarks:

Clear liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lambiotte & Cie - 1808131400R and 1904111400R
- Expiration date of the lot/batch: 13 August 2020 and 11 April 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2 to 8°C), under nitrogen and protected from
light

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The RccHan™®:WIST strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited (UK).
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 8 weeks.
- Weight at study initiation: Males: 193 to 240 g; Females: 143 to 178 g
- Housing: Five of the same sex, unless reduced by mortality or isolation
- Diet: Teklad 2014C Diet ad libitum (removed overnight before blood sampling for hematology or blood chemistry)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (except during exposure)
- Acclimation period: At least 11 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24ºC
- Humidity (%): Monitored and maintained within the range of 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours dark : 12 hours light.

IN-LIFE DATES: From: 04 APRIL 2019 To: 15 to 17 JULY 2019

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

snout only

Vehicle:

clean air

Remarks on MMAD:

Due to the physical nature of the test material atmosphere it was not possible to capture the samples required to determine a particle size distribution

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through nose-only chamber; Aluminum alloy construction comprising a base unit, three animal exposure sections, a top section and a pre-chamber
- Method of holding animals in test chamber: Plastic nose-only restraint tube
- Source and rate of air: From in-house compressed air system – breathing quality
- System of generating particulates/aerosols: Glass sintered vaporizer; The test item was supplied to the generator, via a feed line, from a syringe driven at a constant rate by a syringe pump
- Temperature, humidity in air chamber: 19 – 25°C; 30 - 70%
- Air flow rate: Generator flow: 19L/minute; Extract flow: 20L/minute
- Treatment of exhaust air: Drawn by in-house vacuum system and filtered locally

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test sample (Solvent trap collected in trapping solvent): Transfer to volumetric flask using extraction solvent. Further dilute, if necessary, using extraction solvent to provide a solution containing Ethylal at a nominal concentration in the range 20 – 200 µg/mL. Inject onto the GC in duplicate.

Duration of treatment / exposure:

13 weeks 6 hours daily

Frequency of treatment:

exposure for 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (analytical)

Doses / concentrations 2

Dose / conc.:

505 ppm (analytical)

Doses / concentrations 3

Dose / conc.:

1 520 ppm (analytical)

Doses / concentrations 4

Dose / conc.:

3 860 ppm (analytical)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previous inhalation toxicity study, three groups of rats were exposed to ethylal for 2 weeks (6 hours/day, 5 days/week) at mean achieved aerosol concentrations of 490, 1520 and 3930 ppm respectively. A fourth group received a single 4-hour exposure at an achieved concentration of 4810 ppm.
Exposures at mean achieved concentrations of 490, 1520 or 3930 ppm resulted in clinical signs of unsteady gait/swaying, salivation and piloerection. These signs were transient after dosing and were not considered to be adverse for these groups. Pathological examination did not show any test item related changes at 490 or 1520 ppm. Minimal olfactory epithelial degeneration was evident in two males and one female exposed at 3930 ppm.
A single exposure to ethylal at an achieved concentration 4810 ppm was stopped after 4 hours, due to adverse clinical signs. Signs seen on return to home cage included: unresponsiveness to external stimuli, decreased activity, breathing abnormalities, reduced body temperature and abnormal posture. The condition of these animals did not improve by 90 minutes post exposure and they were euthanized on grounds of animal welfare. Pathological examination showed moderate olfactory epithelial degeneration in all animals.
For this study, the highest target concentration of 4000 ppm was expected to result in treatment related effects, but not cause deaths. The lower target concentrations of 500 and 1500 ppm were chosen to explore the dose relationship of any effects seen over 13 weeks.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed weekly physical examination was performed on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded twice weekly one week before treatment commenced to Week 4, weekly from Weeks 5 to 13 and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded for the week before treatment started and for each week throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows: pretreatment for all animals and at week 13 for all animals of groups 1 and 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: all animals.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at termination
- Dose groups that were examined: all groups
- Number of animals: all animals

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

All statistical analyses were carried out separately for males and females using the individual
animal as the basic experimental unit.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Unsteady gait was occasionally observed for rats receiving ethylal at all exposure levels. In addition, swaying, decreased activity, lacrimation and wet rales were occasionally observed for a proportion of rats exposed to 3860 ppm. These signs were observed on return to home cage and resolved by the end of the working day.

On Week.Days 7.1 to 7.3, clinical signs evident for rats receiving the highest exposure level of ethylal were more marked than those seen after other exposures. Signs seen after exposure, for a proportion of rats, included locomotor and responsiveness effects (decreased activity, unsteady gait, unresponsiveness, flattened posture) and breathing effects (slow and/or shallow breathing and wet rales). Animal 138 (a high exposure level female) was removed from exposure on Week.Day 7.2, after approximately 4.5 hours, due to welfare concerns regarding clinical signs. Clinical signs evident for this animal included abnormal gait (unsteady and elevated), hunched posture, shallow breathing, decreased activity and pale ears. This rat had recovered by the end of the working day.
Signs associated with the administration procedure included red staining of the head and eyes, and/or wet fur on occasion. These were seen for rats of all groups, including control, on return to the home cage and at the end of the working day. These are signs considered to be due to the method of restraint and are not test item related.
Table 9 includes individual observation following detailed weekly physical examination

Mortality:

mortality observed, treatment-related

Description (incidence):

There was one unscheduled death due to overexposure to ethylal. Animal 134, a female receiving the highest exposure level of ethylal was found dead at the end of exposure on Week.Day 7.1. Macroscopic observations included incomplete deflation of the lungs, depressions and pale areas in the glandular mucosa of the stomach (not confirmed microscopically), and gaseous distension of the stomach, duodenum and jejunum. Incomplete deflation of the lungs and gaseous distension of the GIT were considered to be post-mortem changes. Other microscopic changes consisted in unilateral pyogranulomatous inflammation within the glandular epithelium of the nose (with presence of the bacteria and Splendore-Hoeppli material) and minimal bilateral dilation if the renal pelvis.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Differences in mean bodyweight gain were small compared with intragroup variation and were inconsistent between exposure levels and/or sexes. Small weight losses in the final week, for all groups, are considered to be associated with blood sampling techniques.
Table 1

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Minor fluctuations in food consumption lacked dose relationship, were inconsistent between the sexes and are considered to be unrelated to treatment.
Table 8

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All differences from control, that attained statistical significance, were small compared with intragroup variation, lacked dose relationship and/or were inconsistent between sexes.
Table 2

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

All differences from control, that attained statistical significance, were small compared with intragroup variation, lacked dose relationship and/or were inconsistent between sexes.
Table 3

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Mean liver weights (absolute and adjusted for terminal bodyweight) were higher than control for males exposed to 1520 ppm and both sexes exposed to 3860 ppm (up to 1.23X control).
Mean kidney weights (absolute and adjusted for terminal bodyweight) were higher than control for females exposed to 1520 ppm and both sexes exposed to 3860 ppm (up to 1.21X control).
Mean adrenal weights (absolute and adjusted for terminal bodyweight) were higher than control for both sexes exposed to 1520 or 3860 ppm (up to 1.26X control).
All other differences from control were small, lacked dose relationship and/or were inconsistent between sexes.
Table 4

Gross pathological findings:

no effects observed

Description (incidence and severity):

Table 6

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to exposure to ethylal were seen in thyroid glands and liver.
In the thyroids, minimal hypertrophy of the follicular cells was observed for one control male and 5 out of 10 males exposed to 3860 ppm.
In the liver, diffuse minimal centrilobular hepatocellular hypertrophy was recorded for four males out of 10 exposed to 3860 ppm.
Table 7

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on BAL parameters.
All differences from control were small, lacked dose relationship and/or were inconsistent between sexes.
Table 5

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

3 860 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

	Air control	Ethylal
Dose group	1	2	3	4
Exposure level (ppm)	0	505	1520	3860

 

Table 1: Body weight – group mean values (g)

Group /Sex		Day P1	P4	Day 1	4	8	11	15	18	22	25	29	36	43
Statistics test
1M	Mean	187	203	217	219	234	238	253	256	271	272	285	299	310
	SD	5.0	6.2	7.5	11.1	13.1	16.2	17.2	20.7	21.9	24.0	24.1	26.2	27.0
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
2M	Mean	186	200	213	211	227	230	243	248	262	264	277	286	298
	SD	5.8	6.5	7.8	8.6	10.8	11.7	13.9	13.8	16.4	17.3	17.7	19.7	21.0
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
	% of 1M
3M	Mean	182	200	214	217	234	240	253	259	274	275	286	296	308
	SD	4.9	7.0	9.5	10.8	12.6	13.3	15.3	15.4	17.6	19.9	21.1	21.4	22.8
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
	% of 1M
4M	Mean	185	203	220	220	237	238	253	254	270	266	280	290	300
	SD	7.6	9.8	11.0	10.9	13.6	14.6	18.0	18.7	21.0	20.8	22.0	23.8	25.0
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
	% of 1M

 

 

Table 1 (cont): Body weight – group mean values (g)

Group /Sex		Day 50	57	64	71	78	85	92	Change 1-92
Statistics test									Wi
1M	Mean	318	326	330	337	343	350	342	124
	SD	29.3	31.7	33.2	36.0	36.4	37.9	40.3	34.5
	N	10	10	10	10	10	10	10	10
2M	Mean	301	311	320	324	328	335	330	117
	SD	20.5	21.3	21.4	22.3	23.1	20.9	21.7	18.3
	N	10	10	10	10	10	10	10	10
	% of 1M								94
3M	Mean	311	323	330	335	340	348	344	129
	SD	22.3	23.6	23.4	25.0	26.4	27.1	26.2	20.2
	N	10	10	10	10	10	10	10	10
	% of 1M								104
4M	Mean	302	311	318	324	331	337	333	113
	SD	25.1	26.8	29.7	32.2	32.1	34.2	34.3	27.3
	N	10	10	10	10	10	10	10	10
	% of 1M								91

 

Table 1 (cont): Body weight – group mean values (g)

Group /Sex		Day P1	P4	Day 1	4	8	11	15	18	22	25	29	36	43
Statistics test
1F	Mean	138	148	156	158	167	167	174	176	183	183	189	196	202
	SD	6.2	8.4	9.1	8.4	8.4	11.0	12.1	12.2	12.2	14.6	13.6	12.6	15.7
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
2F	Mean	140	149	156	160	167	165	174	174	180	182	187	194	197
	SD	5.3	6.8	8.3	7.8	8.0	8.6	9.4	9.2	9.8	11.2	10.5	10.8	13.5
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
	% of 1F
3F	Mean	144	155	164	166	175	175	182	185	193	193	200	206	211
	SD	5.1	5.8	7.7	6.6	7.6	8.2	9.2	11.1	11.6	11.6	12.5	11.6	9.5
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
	% of 1F
4F	Mean	140	148	157	159	167	164	169	172	179	178	180	189	195
	SD	7.2	6.8	7.9	7.9	8.4	8.4	7.9	8.7	9.8	10.4	9.0	11.5	8.1
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
	% of 1F

 

Table 1 (cont): Body weight – group mean values (g)

Group /Sex		Day 50	57	64	71	78	85	92	Change 1-92
Statistics test									Wi
1F	Mean	203	206	210	212	211	215	213	58
	SD	16.7	17.4	15.5	17.8	18.3	19.2	17.1	13.2
	N	10	10	10	10	10	10	10	10
2F	Mean	199	205	207	206	208	211	209	52
	SD	12.3	13.8	13.5	14.2	14.6	13.0	15.5	8.6
	N	10	10	10	10	10	10	10	10
	% of 1F								90
3F	Mean	213	217	220	220	221	225	221	57
	SD	12.5	13.9	12.5	10.9	13.9	14.0	9.7	7.9
	N	10	10	10	10	10	10	10	10
	% of 1F								99
4F	Mean	197	201	201	204	206	209	206	49
	SD	13.4	8.8	13.2	11.2	12.5	9.6	12.1	8.9
	N	9	9	9	9	9	9	9	9
	% of 1F								85

 

 

Table 2: Hematology – group mean values during Week 13 of treatment

Group /Sex		Hct L/L	Hb g/dL	RBC x 10^12/L	Retic x 10^12/L	MCH pg	MCHC g/dL	MCV fL	RDW %
Statistics test		Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
1M	Mean	0.499	15.7	8.90	0.118	17.7	31.5	56.1	11.3
	SD	0.0121	0.45	0.304	0.0212	0.56	0.55	1.42	0.47
	N	10	10	10	10	10	10	10	10
2M	Mean	0.497	15.7	8.94	0.115	17.5	31.5	55.7	11.6
	SD	0.0206	0.63	0.438	0.0141	0.70	0.70	1.46	0.44
	N	10	10	10	10	10	10	10	10
3M	Mean	0.494	15.6	8.79	0.129	17.7	31.5	56.2	11.6
	SD	0.0103	0.37	0.293	0.0193	0.64	0.54	1.83	0.58
	N	10	10	10	10	10	10	10	10
4M	Mean	0.490	15.5	8.71	0.134	17.9	31.7	56.3	11.9*
	SD	0.0134	0.41	0.358	0.0230	0.54	0.29	1.50	0.45
	N	10	10	10	10	10	10	10	10

 

Table 2 (cont): Hematology – group mean values during Week 13 of treatment

Group /Sex		WBC x 10^9/L	N x 10^9/L	L x 10^9/L	E x 10^9/L	B x 10^9/L	M x 10^9/L	LUC x 10^9/L
Statistics test		Wi	Wi	lWi	Wi	Wi	Wi	Wi
1M	Mean	4.95	0.94	3.83	0.05	0.01	0.09	0.03
	SD	1.320	0.474	0.859	0.025	0.006	0.045	0.012
	N	10	10	10	10	10	10	10
2M	Mean	5.21	1.15	3.87	0.05	0.01	0.11	0.02
	SD	1.035	0.207	0.920	0.018	0.007	0.048	0.016
	N	10	10	10	10	10	10	10
3M	Mean	5.84	1.28	4.32	0.07	0.01	0.12	0.03
	SD	1.619	0.708	1.231	0.039	0.011	0.054	0.021
	N	10	10	10	10	10	10	10
4M	Mean	5.34	0.98	4.17	0.06	0.01	0.11	0.03
	SD	0.856	0.253	0.904	0.014	0.003	0.037	0.012
	N	10	10	10	10	10	10	10

 

Table 2 (cont): Hematology – group mean values during Week 13 of treatment

Group /Sex		Plt x 10^9/L	PT sec	APTT sec
Statistics test
1M	Mean	823	21.3	14.3
	SD	125.7	0.74	0.67
	N	10	10	10
2M	Mean	857	20.7	14.8
	SD	104.7	0.54	1.23
	N	10	9	9
3M	Mean	824	20.6	14.5
	SD	133.9	1.28	1.03
	N	10	10	10
4M	Mean	824	20.3*	14.4
	SD	70.6	0.90	0.97
	N	10	10	10

 

Table 2 (cont): Hematology – group mean values during Week 13 of treatment

Group /Sex		Hct L/L	Hb g/dL	RBC x 10^12/L	Retic x 10^12/L	MCH pg	MCHC g/dL	MCV fL	RDW %
Statistics test		Wi	Wi	Wi	Wi	Wi	Sh	Wi	Wi
1F	Mean	0.465	14.7	8.00	0.125	18.3	31.5	58.2	10.2
	SD	0.0144	0.50	0.243	0.0185	0.74	1.03	0.97	0.40
	N	10	10	10	10	10	10	10	10
2F	Mean	0.465	14.7	8.00	0.125	18.3	31.5	58.2	10.2
	SD	0.0144	0.50	0.243	0.0185	0.74	1.03	0.97	0.40
	N	10	10	10	10	10	10	10	10
3F	Mean	0.465	14.7	8.04	0.122	18.3	31.6	57.9	10.2
	SD	0.0107	0.36	0.225	0.0197	0.37	0.45	0.95	0.25
	N	10	10	10	10	10	10	10	10
4F	Mean	0.459	14.6	7.96	0.118	18.3	31.8	57.7	10.7**
	SD	0.0148	0.46	0.433	0.0199	0.64	0.48	1.68	0.27
	N	9	9	9	9	9	9	9	9

 

Table 2 (cont): Hematology – group mean values during Week 13 of treatment

Group /Sex		WBC x 10^9/L	N x 10^9/L	L x 10^9/L	E x 10^9/L	B x 10^9/L	M x 10^9/L	LUC x 10^9/L
Statistics test		Wi	Wi	Wi	Wi	Wi	Wi	Wi
1F	Mean	3.50	0.57	2.78	0.06	0.00	0.07	0.02
	SD	0.927	0.183	0.857	0.033	0.005	0.024	0.007
	N	10	10	10	10	10	10	10
2F	Mean	4.48*	0.60	3.74	0.05	0.01	0.07	0.02
	SD	1.031	0.218	0.893	0.012	0.005	0.045	0.011
	N	10	10#	10#	10#	10#	10#	9
3F	Mean	4.24*	0.75	3.33	0.05	0.01	0.08	0.02
	SD	0.742	0.248	0.650	0.019	0.009	0.027	0.008
	N	10	10	10	10	10	10	10
4F	Mean	4.30*	0.75	3.39	0.04	0.00	0.09	0.02
	SD	0.614	0.192	0.479	0.018	0.005	0.029	0.010
	N	9	9	9	9	9	9	9

#             Manual counts included

Table 2 (cont): Hematology – group mean values during Week 13 of treatment

Group /Sex		Plt x 10^9/L	PT sec	APTT sec
Statistics test		Wi	Sh	Wi
1F	Mean	818	22.8	11.3
	SD	106.2	3.41	2.01
	N	10	10	10
2F	Mean	891	21.8	11.2
	SD	120.6	0.91	1.67
	N	9	10	10
3F	Mean	801	21.8	10.8
	SD	76.3	1.31	0.95
	N	10	10	10
4F	Mean	861	20.7*	10.8
	SD	149.7	1.14	1.52
	N	9	9	9

 

Table 3: Blood chemistry – group mean values during Week 13 of treatment

Group /Sex		ALP U/L	ALT U/L	AST U/L	Bili µmol/L	Urea mmol/L	BUN mmol/L	Creat µmol/L	Gluc mmol/L	Chol mmol/L	Trig mmol/L
Statistics test		lWi	Wi	Wi	Du	Sh	Sh	Wi	Wi	Wi	Wi
1M	Mean	74	48	83	2	5.89	5.89	28	6.59	2.37	0.85
	SD	17.6	10.8	26.3	0.4	0.792	0.792	1.9	0.791	0.213	0.180
	N	10	10	10	10	10	10	10	10	10	10
2M	Mean	76	44	79	2	6.62	6.62	28	6.97	2.30	0.80
	SD	7.3	6.6	14.7	0.4	0.673	0.673	1.3	1.169	0.316	0.376
	N	10	10	10	10	10	10	10	10	10	10
3M	Mean	62	49	80	1*	6.09	6.09	28	6.71	2.42	0.93
	SD	7.1	12.5	14.3	0.5	0.653	0.653	2.8	1.021	0.318	0.385
	N	10	10	10	10	10	10	10	10	10	10
4M	Mean	76	60*	96	2	6.60	6.60	29	6.50	2.22	0.80
	SD	18.3	13.2	37.3	0.4	1.919	1.919	2.2	1.032	0.182	0.292
	N	10	10	10	10	10	10	10	10	10	10

 

 

 

Table 3 (cont): Blood chemistry – group mean values during Week 13 of treatment

Group /Sex		Na mmol/L	K mmol/L	Cl mmol/L	Ca mmol/L	Phos mmol/L	Total Prot g/L	Alb g/L	A/G Ratio
Statistics test		Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
1M	Mean	144	3.86	103.6	2.64	1.83	68	39	1.34
	SD	1.5	0.163	1.51	0.080	0.166	2.5	0.7	0.114
	N	10	10	10	10	10	10	10	10
2M	Mean	143	4.00	101.9**	2.66	1.98	69	39	1.32
	SD	1.4	0.114	1.52	0.056	0.146	2.2	1.1	0.071
	N	10	10	10	10	10	10	10	10
3M	Mean	143	4.02*	101.5**	2.67	1.93	70	39	1.27
	SD	0.7	0.199	0.68	0.062	0.079	1.4	0.9	0.077
	N	10	10	10	10	10	10	10	10
4M	Mean	144	4.24**	102.5**	2.72*	2.06**	68	39	1.34
	SD	1.1	0.164	0.99	0.082	0.155	2.1	1.3	0.078
	N	10	10	10	10	10	10	10	10

 

Table 3 (cont): Blood chemistry – group mean values during Week 13 of treatment

Group /Sex		ALP U/L	ALT U/L	AST U/L	Bili µmol/L	Urea mmol/L	BUN mmol/L	Creat µmol/L	Gluc mmol/L	Chol mmol/L	Trig mmol/L
Statistics test		Wi	Wi	Wi	Fe	Wi	Wi	Wi	Wi	Wi	Wi
1F	Mean	40	45	81	2	7.77	7.77	31	6.04	2.16	0.76
	SD	12.3	10.1	16.6	0.4	0.985	0.985	3.8	0.914	0.383	0.371
	N	10	10	10	10	10	10	10	10	10	10
2F	Mean	35	51	82	2	8.39	8.39	32	5.66	2.29	0.91
	SD	10.2	10.9	20.7	0.3	1.064	1.064	2.6	0.410	0.421	0.253
	N	10	10	10	10	10	10	10	10	10	10
3F	Mean	34	48	75	2	7.45	7.45	27*	6.50	2.38	1.02
	SD	9.2	9.1	15.2	0.6	1.131	1.131	2.9	1.193	0.558	0.407
	N	10	10	10	10	10	10	10	10	10	10
4F	Mean	40	68**	75	2	7.32	7.32	27**	6.87	2.47	0.76
	SD	10.2	20.1	11.4	0.4	0.962	0.962	3.2	1.029	0.312	0.194
	N	9	9	9	9	9	9	9	9	9	9

 

Table 3 (cont): Blood chemistry – group mean values during Week 13 of treatment

Group /Sex		Na mmol/L	K mmol/L	Cl mmol/L	Ca mmol/L	Phos mmol/L	Total Prot g/L	Alb g/L	A/G Ratio
Statistics test		Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
1F	Mean	141	3.63	102.0	2.63	1.67	70	42	1.49
	SD	1.0	0.240	1.26	0.067	0.189	4.0	2.0	0.103
	N	10	10	10	10	10	10	10	10
2F	Mean	142	3.73	101.9	2.73**	1.80	73	43	1.42
	SD	1.1	0.139	1.11	0.050	0.185	2.0	1.4	0.064
	N	10	10	10	10	10	10	10	10
3F	Mean	143	3.64	101.7	2.74**	1.77	73	43	1.40
	SD	0.8	0.157	1.36	0.088	0.200	3.0	1.9	0.100
	N	10	10	10	10	10	10	10	10
4F	Mean	142	3.77	101.2	2.68**	1.98**	69	40*	1.40
	SD	1.2	0.175	1.65	0.052	0.129	2.7	1.2	0.088
	N	9	9	9	9	9	9	9	9

 

Table 4: Organ weights – group mean absolute and adjusted values (g) for animals killed after 13 weeks of treatment

Group /Sex		Terminal Body weight	Adrenals	Brain	Epididymides	Heart	Kidneys	Liver	Lungs and Bronchi	Spleen	Testes
Statistics test		Wi
1M	Mean	341.3	0.061	1.955	0.984	0.977	1.768	10.930	1.310	0.667	2.704
	SD	40.5	0.008	0.108	0.159	0.114	0.292	1.850	0.204	0.126	0.319
	N	10	10	10	10	10	10	10	10	10	10
2M	Mean	331.1	0.063	2.017	1.023	0.981	1.746	11.070	1.294	0.647	2.696
	SD	21.8	0.011	0.104	0.153	0.128	0.173	1.252	0.156	0.096	0.559
	N	10	10	10	10	10	10	10	10	10	10
3M	Mean	343.6	0.069	2.015	1.013	1.047	1.934	13.154	1.317	0.706	2.921
	SD	25.5	0.008	0.125	0.125	0.155	0.248	1.527	0.088	0.127	0.476
	N	10	10	10	10	10	10	10	10	10	10
4M	Mean	332.3	0.074	2.093	0.942	1.010	2.137	13.102	1.372	0.641	2.709
	SD	34.0	0.009	0.115	0.140	0.192	0.728	1.773	0.077	0.084	0.457
	N	10	10	10	10	10	10	10	9	10	10
Statistics test			Wi	Wi	Wi	Wi	lWi	Wi	Wi	Wi	Wi
1M	Adjusted Mean		0.061	1.948	0.976	0.969	1.727	10.767	1.298	0.661	2.680
2M	Adjusted Mean		0.063	2.026	1.034	0.992	1.770	11.300	1.311	0.656	2.730
3M	Adjusted Mean		0.069	2.005	1.001	1.035	1.875	12.903**	1.298	0.697	2.884
4M	Adjusted Mean		0.074**	2.101**	0.951	1.018	2.092**	13.285**	1.388	0.648	2.736

 

Table 4 (cont): Organ weights – group mean absolute and adjusted values (g) for animals killed after 13 weeks of treatment

Group /Sex		Terminal Body weight	Thymus and Thyroids	Parathyroids
Statistics test		Wi
1M	Mean	341.3	0.237	0.022
	SD	40.5	0.087	0.007
	N	10	10	10
2M	Mean	331.1	0.192	0.020
	SD	21.8	0.041	0.004
	N	10	10	10
3M	Mean	343.6	0.226	0.019
	SD	25.5	0.048	0.004
	N	10	10	10
4M	Mean	332.3	0.198	0.021
	SD	34.0	0.057	0.004
	N	10	10	10
Statistics test			Wi	Wi
1M	Adjusted Mean		0.234	0.022
2M	Adjusted Mean		0.197	0.020
3M	Adjusted Mean		0.221	0.019
4M	Adjusted Mean		0.201	0.021

 

Table 4 (cont): Organ weights – group mean absolute and adjusted values (g) for animals killed after 13 weeks of treatment

Group /Sex		Terminal Body weight	Adrenals	Brain	Heart	Kidneys	Liver	Lungs and Bronchi	Ovaries	Spleen	Thymus
Statistics test		Wi						St
1F	Mean	212.5	0.066	1.928	0.740	1.252	7.656	1.012	0.079	0.512	0.199
	SD	19.2	0.011	0.115	0.135	0.097	0.780	0.126	0.016	0.096	0.063
	N	10	10	10	10	10	10	10	10	10	10
2F	Mean	209.1	0.071	1.923	0.692	1.274	7.572	1.025	0.083	0.527	0.229
	SD	14.5	0.006	0.072	0.104	0.091	0.560	0.066	0.016	0.094	0.041
	N	10	10	10	10	10	10	10	10	10	10
3F	Mean	221.1	0.081	1.942	0.791	1.386	8.316	1.108	0.084	0.520	0.224
	SD	10.8	0.007	0.108	0.103	0.113	0.700	0.088	0.014	0.056	0.059
	N	10	10	10	10	10	10	10	10	10	10
4F	Mean	206.5	0.083	1.870	0.675	1.369	8.710	0.996	0.088	0.493	0.180
	SD	11.5	0.008	0.118	0.045	0.099	0.787	0.020	0.021	0.069	0.046
	N	9	9	9	9	9	9	9	9	9	9
Statistics test			Wi	Wi	Wi	Wi	Wi		Wi	Wi	Wi
1F	Adjusted Mean		0.066	1.928	0.740	1.251	7.653		0.079	0.512	0.199
2F	Adjusted Mean		0.071	1.932	0.706	1.286	7.686		0.085	0.538	0.233
3F	Adjusted Mean		0.081**	1.919	0.755	1.355*	8.025		0.079	0.491	0.212
4F	Adjusted Mean		0.083**	1.883	0.700	1.391**	8.910**		0.091	0.512	0.189

 

Table 4 (cont): Organ weights – group mean absolute and adjusted values (g) for animals killed after 13 weeks of treatment

Group /Sex		Terminal Body weight	Thymus and Thyroids	Parathyroids
Statistics test		Wi
1F	Mean	212.5	0.018	0.732
	SD	19.2	0.006	0.234
	N	10	10	10
2F	Mean	209.1	0.017	0.676
	SD	14.5	0.006	0.178
	N	10	10	10
3F	Mean	221.1	0.017	0.638
	SD	10.8	0.003	0.171
	N	10	10	10
4F	Mean	206.5	0.016	0.598
	SD	11.5	0.003	0.231
	N	9	9	9
Statistics test			Wi	Wi
1F	Adjusted Mean		0.018	0.732
2F	Adjusted Mean		0.018	0.680
3F	Adjusted Mean		0.015	0.626
4F	Adjusted Mean		0.017	0.606

 

Table 5: Bronchoalveolar lavage – group mean differential cell counts in the right lung

Group /Sex		Different cell counts calculated per animal (10^6/animal)					Different cell counts as a percentage of total white blood cells (%)
		Total Cells	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils
Statistics test		lDu	sWi	sDu	Du	Wi	Wi	Wi	Wi	Wi
1M	Mean	3.60	0.57	0.26	2.70	0.07	14.45	6.77	76.00	1.92
	SD	1.64	0.44	0.21	1.16	0.05	8.15	3.34	10.34	1.22
	N	10	10	10	10	10	10	10	10	10
2M	Mean	1.40***	0.13*	0.06**	1.20***	0.02	8.19	4.64	85.46	1.71
	SD	0.48	0.12	0.05	0.45	0.03	7.23	3.52	9.18	2.57
	N	10	10	10	10	10	10	10	10	10
3M	Mean	1.57**	0.17*	0.13	1.23***	0.04	9.06	7.31	81.07	2.56
	SD	0.84	0.24	0.18	0.64	0.02	10.10	6.82	14.63	2.37
	N	10	10	10	10	10	10	10	10	10
4M	Mean	2.89	0.40*	0.21	2.23	0.05	10.89	7.10	79.56	1.49
	SD	1.46	0.56	0.12	0.90	0.04	8.37	2.35	8.99	1.21
	N	10	10	10	10	10	10	10	10	10

 

Table 5 (cont): Bronchoalveolar lavage – group mean differential cell counts in the right lung

Group /Sex		Different cell counts calculated per animal (10^6/animal)					Different cell counts as a percentage of total white blood cells (%)
		Total Cells	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils
Statistics test		Wi	Wi	sWi	Wi	Wi	Wi	Wi	Wi	Wi
1F	Mean	1.85	0.26	0.13	1.43	0.03	11.84	4.88	81.57	1.33
	SD	1.08	0.27	0.20	0.70	0.03	6.41	5.42	10.53	1.75
	N	10	10	10	10	10	10	10	10	10
2F	Mean	1.99	0.35	0.14	1.48	0.03	13.09	6.71	78.97	0.69
	SD	1.36	0.43	0.11	0.83	0.04	9.92	2.92	12.68	1.14
	N	10	10	10	10	10	10	10	10	10
3F	Mean	1.89	0.16	0.12	1.59	0.02	7.12	5.62	85.93	0.90
	SD	0.78	0.16	0.08	0.56	0.04	6.89	3.02	8.87	1.43
	N	9	9	9	9	9	9	9	9	9
4F	Mean	2.18	0.33	0.14	1.67	0.05	13.51	6.81	77.14	1.80
	SD	1.02	0.25	0.06	0.74	0.05	8.66	2.91	11.87	1.79
	N	9	9	9	9	9	9	9	9	9

 

 

Table 5 (cont): Bronchoalveolar lavage – group mean values at termination

Group /Sex		LDH U/L	Prot g/L
Statistics test		Wi	lWi
1M	Mean	457	0.65
	SD	341.3	0.659
	N	10	10
2M	Mean	228	0.17
	SD	132.1	0.084
	N	10	10
3M	Mean	263	0.30
	SD	184.2	0.466
	N	10	10
4M	Mean	297	0.25
	SD	164.5	0.146
	N	10	10

 

Table 5 (cont): Bronchoalveolar lavage – group mean values at termination

Group /Sex		LDH U/L	Prot g/L
Statistics test		Wi	Wi
1F	Mean	274	0.23
	SD	147.7	0.188
	N	10	10
2F	Mean	367	0.50
	SD	248.0	0.603
	N	10	10
3F	Mean	186	0.27
	SD	150.8	0.445
	N	10	10
4F	Mean	385	0.47
	SD	276.6	0.446
	N	9	9

 

Table 6: Macropathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Number of animals within normal limits		9	7	6	8	9	8	6	7
Epididymides Small		1	0	0	0	-	-	-	-
Kidneys Cyst(s) Dilated Pelvis Enlarged		0 0 0	0 0 0	0 0 0	0 1 1	0 1 0	0 0 0	1 0 0	1 0 0
Lungs and Bronchi Pale area(s)		0	1	1	0	1	2	3	1
Lymph Node, Mediastinal Abnormal color Enlarged		0 0	0 0	0 0	0 0	0 0	0 0	0 0	1 1
Prostate Abnormal color		0	1	0	0	-	-	-	-
Skin and subcutis Scab(s)		0	0	1	0	0	0	0	0

 

Table 6 (cont): Macropathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Testes Abnormal color Pale area(s) Small Soft		0 0 1 1	1 1 1 1	1 0 2 3	0 0 1 0	- - - -	- - - -	- - - -	- - - -
Ureters Distended		0	0	0	1	0	0	0	0

 

Table 7: Histopathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Adrenals	No. examined	10	0	0	10	10	0	0	9
Aorta Thoracic	No. examined	10	0	0	10	10	0	0	9
Bone, Femur incl Marrow	No. examined	10	0	0	10	10	0	0	9
Bone, Sternum Including Marrow	No. examined	10	0	0	10	10	0	0	9
Brain	No. examined	10	0	0	10	10	0	0	9
Brain, Olfactory Bulbs	No. examined	10	0	0	7	10	0	0	9
Cecum	No. examined	10	0	0	10	10	0	0	9
Colon	No. examined	10	0	0	10	10	0	0	9
Duodenum	No. examined	10	0	0	10	10	0	0	9

 

Table 7 (cont): Histopathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Epididymides Sperm, Reduced, Luminal         Edema	No. examined Minimal Slight Moderate Total   Slight Total	10 4 0 1 5   0 0	0 0 0 0 0   0 0	0 0 0 0 0   0 0	10 3 2 0 5   1 1	- - - - -   - -	- - - - -   - -	- - - - -   - -	- - - - -   - -
Epiglottis	No. examined	10	0	0	10	10	0	0	9
Esophagus	No. examined	10	0	0	10	10	0	0	9
Eyes Rosettes/Folds, Retina	No. examined Minimal Total	10 0 0	0 0 0	0 0 0	10 0 0	10 0 0	0 0 0	0 0 0	9 1 1

 

Table 7 (cont): Histopathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Harderian Glands	No. examined	10	0	0	10	10	0	0	9
Heart Cardiomyopathy     Infiltrate, Inflammatory Cell	No. examined Minimal Total   Minimal Total	10 1 1   1 1	0 0 0   0 0	0 0 0   0 0	10 0 0   0 0	10 0 0   0 0	0 0 0   0 0	0 0 0   0 0	9 0 0   0 0
Ileum	No. examined	10	0	0	10	10	0	0	9
Jejunum	No. examined	10	0	0	10	10	0	0	9
Kidneys Dilatation, Pelvic	No. examined Minimal Slight Moderate Marked Total	10 1 0 0 0 1	0 0 0 0 0 0	0 0 0 0 0 0	10 1 1 0 1 3	10 0 0 1 0 1	0 0 0 0 0 0	0 0 0 0 0 0	9 0 0 0 0 0

 

Table 7 (cont): Histopathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Lacrimal Glands	No. examined	10	0	0	10	10	0	0	9
Larynx Granuloma	No. examined Slight Total	10 1 1	10 0 0	10 0 0	10 0 0	10 0 0	10 0 0	10 0 0	9 0 0
Liver Pigment, Hepatocellular     Hypertrophy, Hepatocellular, Centrilobular	No. examined Slight Total   Minimal Total	10 1 1   0 0	0 0 0   0 0	0 0 0   0 0	10 1 1   5 5	10 0 0   0 0	0 0 0   0 0	0 0 0   0 0	9 0 0   0 0
Lungs and Bronchi Alveolar Macrophage Aggregation     Inflammation	No. examined Minimal Total   Minimal Total	10 0 0   0 0	10 1 1   0 0	10 2 2   1 1	10 1 1   0 0	10 2 2   0 0	10 2 2   0 0	10 0 0   0 0	9 1 1   0 0

 

Table 7 (cont): Histopathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Lymph Node, Axillary Lt Plasmocytosis	No. examined Minimal Slight Total	10 0 0 0	0 0 0 0	0 0 0 0	10 0 0 0	10 3 0 3	0 0 0 0	0 0 0 0	9 2 1 3
Lymph Node, Mediastinal Mastocytosis     Plasmacytosis	No. examined Slight Total   Minimal Total	0 0 0   0 0	0 0 0   0 0	0 0 0   0 0	0 0 0   0 0	0 0 0   0 0	0 0 0   0 0	0 0 0   0 0	1 1 1   1 1
Lymph Node, Tracheobronchial Erythrocytes, Intrasinusoidal       Plasmacytosis	No. examined Minimal Slight Total   Minimal Total	10 0 0 0   0 0	10 0 0 0   0 0	10 0 0 0   0 0	10 0 0 0   0 0	10 0 0 0   0 0	9 0 1 1   0 0	10 0 0 0   2 2	9 1 0 1   0 0

Table 7 (cont): Histopathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Mammary	No. examined	10	0	0	10	10	0	0	9
Nerve, Optic	No. examined	10	0	0	10	10	0	0	9
Nerve, Sciatic	No. examined	10	0	0	10	10	0	0	9
Nose/Turbinates	No. examined	10	10	10	10	10	10	10	9
Ovaries	No. examined	-	-	-	-	10	0	0	9
Pancreas	No. examined	10	0	0	10	10	0	0	9
Parathyroids	No. examined	7	0	0	10	10	0	0	9
Pharynx, Nasal	No. examined	10	10	10	10	10	10	10	9
Pituitary	No. examined	10	0	0	10	10	0	0	9

 

Table 7 (cont): Histopathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Prostate Infiltrate, Inflammatory Cell         Inflammation	No. examined Minimal Moderate Marked Total   Marked Total	10 1 0 1 2   0 0	1 0 1 0 1   1 1	0 0 0 0 0   0 0	10 0 0 0 0   0 0	- - - - -   - -	- - - - -   - -	- - - - -   - -	- - - - -   - -
Rectum	No. examined	10	0	0	10	10	0	0	9
Salivary Gland, Mandibular	No. examined	10	0	0	10	10	0	0	9
Salivary Gland, Sublingual	No. examined	10	0	0	10	10	0	0	9
Seminal Vesicles	No. examined	10	0	0	10	-	-	-	-

 

Table 7 (cont): Histopathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Skeletal Muscle	No. examined	10	0	0	10	10	0	0	9
Skin and Subcutis Scab(s)	No. examined Minimal Total	10 0 0	0 0 0	1 1 1	10 0 0	10 0 0	0 0 0	0 0 0	9 1 1
Spinal Cord Cervical	No. examined	10	0	0	10	10	0	0	9
Spinal Cord Lumbar	No. examined	10	0	0	10	10	0	0	9
Spinal Cord Thoracic	No. examined	10	0	0	10	10	0	0	9
Spleen	No. examined	10	0	0	10	10	0	0	9
Stomach	No. examined	10	0	0	10	10	0	0	9
Teeth	No. examined	10	0	0	10	10	0	0	9

 

Table 7 (cont): Histopathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Testes Degenration/Atrophy, Tubular	No. examined Minimal Slight Moderate Total	10 5 1 1 7	1 0 1 0 1	4 1 0 3 4	10 3 6 0 9	- - - - -	- - - - -	- - - - -	- - - - -
Thymus	No. examined	10	0	0	10	10	0	0	9
Thyroids Hypertrophy, Follicular Cells	No. examined Minimal Total	10 1 1	0 0 0	0 0 0	10 5 5	10 0 0	0 0 0	0 0 0	9 0 0
Trachea	No. examined	10	10	10	10	10	10	10	9
Tracheal Bifurcation	No. examined	10	10	10	10	10	10	10	8
Ureters Dilatation	No. examined Total	0 0	0 0	0 0	1 1	0 0	0 0	0 0	0 0

 

Table 7 (cont): Histopathology - group distribution of findings for animals killed after 13 weeks of treatment

Tissue Organs and Findings	Group/Sex No. of animals	Number of animals affected
		1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 9
Urinary Bladder Infiltrate, Inflammatory Cell	No. examined Minimal Total	10 1 1	0 0 0	0 0 0	10 0 0	9 0 0	0 0 0	0 0 0	9 0 0
Uterine Cervix	No. examined	-	-	-	-	10	0	0	9
Uterus	No. examined	-	-	-	-	10	0	0	9
Vagina Diestrus Proestus Metestrus Estrus	No. examined Total Total Total Total	- - - - -	- - - - -	- - - - -	- - - - -	10 6 3 1 0	0 0 0 0 0	0 0 0 0 0	9 4 2 1 2

 

Table 8: Food consumption – individual values (g/animal/week)

Group /Sex	Cage Number	Week P1	Week
			1	2	3	4	5	6	7	8	9	10	11	12	13
1M	001	132	125	139	148	149	151	151	147	151	144	146	146	150	142
	002	136	127	137	138	136	148	141	142	138	143	139	142	141	131
2M	003	126	117	133	142	142	145	148	146	149	149	150	149	147	148
	004	135	125	137	144	142	140	144	138	142	148	143	150	147	138
3M	005	126	121	139	143	141	143	146	146	149	146	146	148	148	143
	006	133	128	142	149	144	147	146	145	147	150	146	148	148	138
4M	007	137	129	137	136	137	141	141	134	147	146	145	156	151	135
	008	132	128	136	135	133	137	135	133	142	146	140	148	149	132

 

Table 8 (cont): Food consumption – individual values (g/animal/week)

Group /Sex	Cage Number	Week P1	Week
			1	2	3	4	5	6	7	8	9	10	11	12	13
1F	010	97	105	106	107	107	112	108	107	107	109	107	106	109	104
	011	104	105	109	114	111	112	112	114	112	113	111	109	109	105
2F	012	95	95	102	103	103	106	106	104	104	104	100	101	102	101
	013	95	99	105	110	106	110	109	111	112	109	105	107	108	101
3F	014	108	106	118	117	118	121	122	121	121	120	117	122	117	113
	015	108	106	111	116	115	121	118	117	115	116	113	116	114	109
4F	016	92	104	101	102	99	109	110	104	106	110	108	107	109	98
	017	104	100	106	108	108	110	111	109	111	110	110	113	113	102

 

 

Table 9: Clinical signs – individual observations

Group /Sex	Animal Number	Week of Death	Phase	Category	Observation	Week(s) observed
1M	0004	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface	10-14
	0006	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Ventral surface	7-14 9-14
	0008	14	Treatment	Coat Skin abrasion Staining	Hair loss, Dorsal surface Dry, Lower dorsal surface Abnormal color, Brown, Dorsal surface	10-14 10-13 10-14
2M	0012	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface	8-14
	0016	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface Abnormal color, Brown, Upper ventral surface	8-14 12-14
	0017	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Upper dorsal surface	12-14 9-11
	0018	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface	13-14
	0020	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface	13-14
3M	0024	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface Abnormal color, Brown, Upper ventral surface	9-14 13-14
	0026	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface	10-14
	0027	14	Treatment	Skin abrasion Staining	Wet, Hindlimb digit(s) Abnormal color, Brown, Upper dorsal surface	3 9-14
	0029	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface	9-14
	0030	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface	13-14
4M	0032	14	Treatment	Coat Skin abrasion	Hair loss, Dorsal surface Dry, Tail	1-2 8
	0035	14	Treatment	Coat Skin abrasion	Hair loss, Dorsal surface Wet, Hindlimb digit(s)	8-14 12-13
	0036	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface	10-14
	0037	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface	9-14
	0040	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface	9-14

 

Only animals with observations are presented

 

Table 9 (cont): Clinical signs – individual observations

Group /Sex	Animal Number	Week of Death	Phase	Category	Observation	Week(s) observed
1F	0101	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface Abnormal color, Brown, Ventral surface	8-14 9-13
	0102	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Ventral surface	7-14 7-14
	0103	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface	7-14
	0104	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Ventral surface	8-14 8-14
	0105	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface Abnormal color, Brown, Ventral surface	8-14 10-14
	0106	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Ventral surface	7-14 8-14
	0108	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Upper dorsal surface	10-14 9
	0110	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Upper dorsal surface	12-14 9-11
2F	0111	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Ventral surface	8-14 8-14
	0112	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Ventral surface	7-14 6, 8-14
	0113	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface Abnormal color, Brown, Ventral surface	10-14 9-14
	0115	14	Treatment	Coat Staining	Hair loss, Dorsal surface Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Ventral surface	12-14 12-14 9-14
	0116	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface	13-14
	0118	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Ventral surface	8-14 10-14
	0119	14	Treatment	Staining	Abnormal color, Brown, Lower ventral surface Abnormal color, Brown, Upper dorsal surface	11-14 9-14
	0120	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface	9-14
3F	0121	14	Treatment	Staining	Abnormal color, Brown, Ventral surface	9-14
	0122	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface	6-14
	0123	14	Treatment	Staining	Abnormal color, Brown, Ventral surface	9-14
	0124	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Upper dorsal surface Abnormal color, Brown, Whole body	13-14 5-8 9-12
	0125	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface	7-14
	0127	14	Treatment	Staining	Abnormal color, Brown, Ventral surface	10-14
	0128	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface Abnormal color, Brown, Upper dorsal surface Abnormal color, Brown, Ventral surface	13-14 9-12 13-14
	0130	14	Treatment	Staining	Abnormal color, Brown, Dorsal surface	8-14
4F	0131	14	Treatment	Coat Staining	Hair loss, Dorsal surface Abnormal color, Brown, Upper dorsal surface Abnormal color, Brown, Ventral surface	1-2 11-14 9-14
	0132	14	Treatment	Coat	Hair loss, Dorsal surface	8-14
	0134DD	7	Treatment	Reason for dispatch	Found dead in a restraint tube	7
	0135	14	Treatment	Coat	Hair loss, Ventral surface	3
	0136	14	Treatment	Coat Staining	Hair loss, Dorsal surface Abnormal color, Brown, Upper dorsal surface Abnormal color, Brown, Ventral surface	13-14 9-14
	0137	14	Treatment	Coat	Hair loss, Head	4-5
	0138	14	Treatment	Staining	Abnormal color, Brown, Ventral surface	10-14
	0139	14	Treatment	Staining	Abnormal color, Brown, Upper dorsal surface	9-14
	0140	14	Treatment	Coat Staining	Hair loss, Dorsal surface Abnormal color, Brown, Dorsal surface	9-14 13-14

DD – Found dead after dosing

Only animals with observations are presented

Applicant's summary and conclusion

Conclusions:

Exposure of Han Wistar rats to ethylal by inhalation for 13 weeks (6 hours per day, 5 days per week) at achieved aerosol concentrations of 505, 1520 or 3860 ppm was well tolerated. Treatment related clinical signs of unsteady gait and/or swaying, decreased activity, salivation and wet rales were intermittent, transient and considered not to be adverse at these concentrations.
Exposure of the highest dose group to an above target ethylal concentration for 3 days in Week 7 (3 day mean concentration 4920 ppm, presented for information only), resulted in a single unscheduled death and an increase in the number and type of clinical signs evident for other rats in this group. These signs were consistent with exposure at 4810 ppm on a previous dose range finding study.
Pathological changes in the thyroid glands and liver in males exposed to 3860 ppm are considered rodent-specific, adaptive and non-adverse.
The No Adverse Effect Level was considered to be 3860 ppm.

Executive summary:

The purpose of this study was to assess the systemic toxic potential of ethylal, an industrial chemical, in a 13 week inhalation study in Han Wistar rats.
The study design was as follows:

Group	Treatment	Target exposure level (ppm)	Number of animals
			Male	Female
1	Air control	0	10	10
2	Ethylal	500	10	10
3	Ethylal	1500	10	10
4	Ethylal	4000	10	10

Animals received the control, air or the test item, ethylal by inhalation for 13 weeks (6 hours daily exposure for 5 days per week).
During the study, clinical condition, body weight, food consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, organ weight, bronchoalveolar lavage, macropathology and histopathology investigations were undertaken.

Results
Summary data are presented below:

Group	Atmosphere Concentration (ppm)
	Target	Achieved
1	0	-
2	500	505
3	1500	1520
4	4000	3860

The mean achieved aerosol concentrations were 101, 101 and 97% of target for Groups 2, 3 and 4, respectively, with little daily variation.
In error, a draining valve (used as part of once weekly cleaning procedures) on the Group 4 exposure chamber, was not closed prior to the start of the first daily exposure in Week 7. The error was detected during exposure on Week.Day 7.4. Failure to close this valve is considered to have resulted in reduced efficiency of the chamber aerosol extract and thus higher achieved aerosol concentrations on these days. Higher than target aerosol concentrations were measured for all chamber aerosol samples collected from Group 4 on Week.Days 7.1, 7.2 and 7.3 and the first sample collected on Week.Day 7.4. Mean achieved aerosol concentrations for Group 4 on Week.Days 7.1, 7.2 and 7.3 were 127%, 112%, 131% of target respectively (3 day mean concentration 4920 ppm). Due to the failure of the analytical standard comparison, the aerosol concentrations from Week.Days 7.1, 7.2 and 7.3 (together with the results from Week.Day 6.1 to 6.5 and Week.Day 7.4 to 7.5) are reported for information purposes only and are excluded from the overall group mean.

 

Animal 134, a female receiving the highest exposure level of ethylal was found dead at the end of exposure on Week.Day 7.1. On Week.Days 7.1 to 7.3, clinical signs evident for rats receiving the highest ethylal exposure level were more marked than those seen after other exposures. Signs seen after exposure, for a proportion of rats, included locomotor and responsiveness effects (decreased activity, unsteady gait, unresponsiveness, flattened posture) and breathing effects (slow and/or shallow breathing and wet rales). Animal 138 (a high exposure level female) was removed from exposure on Week.Day 7.2, after approximately 4.5 hours, due to welfare concerns. Clinical signs evident for this animal included abnormal gait (unsteady and elevated), hunched posture, shallow breathing, decreased activity and pale ears; but it had recovered by the end of the working day.
Unsteady gait was occasionally observed for rats receiving ethylal at all exposure levels. In addition, swaying, decreased activity, lacrimation and wet rales were occasionally observed for a proportion of rats exposed to 3860 ppm. These signs were observed on return to home cage and resolved by the end of the working day.
Pathological examination revealed follicular cell hypertrophy in the thyroid of five males exposed to 3860 ppm and hypertrophy of the centrilobular hepatocytes of the liver in four of the same males. These findings are considered rodent-specific, adaptive and non-adverse.

Conclusion
Exposure of Han Wistar rats to ethylal by inhalation for 13 weeks (6 hours per day, 5 days per week) at achieved aerosol concentrations of 505, 1520 or 3860 ppm was well tolerated.
Treatment related clinical signs of unsteady gait and/or swaying, decreased activity, salivation and wet rales were intermittent, transient and considered not to be adverse at these concentrations.
Exposure of the highest dose group to an above target ethylal concentration for 3 days in Week 7 (3 day mean concentration 4920 ppm, presented for information only), resulted in a single unscheduled death and an increase in the number and type of clinical signs evident for other rats in this group. These signs were consistent with exposure at 4810 ppm on a previous dose range finding study.
Pathological changes in the thyroid glands and liver in males exposed to 3860 ppm are considered rodent-specific, adaptive and non-adverse.
The No Adverse Effect Level was considered to be 3860 ppm.