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Repeated dose toxicity: inhalation

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Endpoint:
sub-chronic toxicity: inhalation
Remarks:
Repeated-dose toxicity inhalation study in rats for 90 days (according to OECD 413)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-24 to 2018-04-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD 413 guideline with GLP
Justification for type of information:
Study was conducted according ECHA Decision TPE-D-2114313721-61-01/F Helsinki, 07 January 2016
Cross-reference
Reason / purpose for cross-reference:
other: related to DRF study
Remarks:
DRF
Reference
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
This study is a 3-Week dose range finding study (similar to OECD 412). The results of the study will be used for dose level selection for a forthcoming 90-day subchronic inhalation toxicity study.
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017-07-03 to 2017-08-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Duration: 3 weeks
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Spain S.L. Ctra. St. Miquel del Fai, Km. 3.5 08182 - Sant Feliú de Codines, Barcelona, – Spain
- Strain: Sprague Dawley, Specific Pathogen Free (SPF)
- Age: Young adult rats, Approx. age of the animals on arrival: 7 weeks
- Animals: 24 were used in the study (6 animals per group) and 4 spare animals (2 males and 2 females)
- Acclimatisation: 5 days
Acclimatisation to the nose-only restraining tubes was performed with fresh air for 3 days before the beginning of the treatment phase with incremental durations (1.5 h, 3 h and 4.5 h, respectively
- Body Weight at study initiation: male: 219.7 - 300.0 g, female: 166.3 - 210.5 g
- Housing: Group caging (in groups of 3 by sex)
- Diet: ad libitum, Global diet, 2914C, Envigo Teklad, Station Road, Blackthorn, Bicester Oxon, OX25 1TP United Kingdom
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 – 23.9°C
- Humidity: 28 - 66 %
- Illumination: 12 hours of continuous artificial light in each twenty-four period (from 6.00 a.m. to 6.00 p.m.)
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- A vapour was generated from the liquid test item. Vapors of test item were generated using a VOC (Volatile organic compound) vapour generator (TSE-systems).
- The test item was inside of the generator vessel in liquid state. It was heated at a controlled temperature (100 ºC approximately) by means of recirculating water bath equipped with a temperature probe performing the vapour phase.
- In addition, the liquid was stirred by means of magnetic stirrer in order to keep an even evaporation surface.
- At the same time a source of compressed air at a controlled temperature of 30 ºC was delivered into the VOC generator (30 L/min).
- This compressed air was mixed with the vapour phase of test item extracting the formulation of the product in a controlled amount of air (L/min) and concentration.
- The vapour was conveyed via glass tubing, from the generator to the exposure chambers.
- To obtain the target concentration levels the test item vapour was diluted with filtered, compressed air downstream from the generator.
- The dose levels, 1.0 mg/m3, 0.3 mg/m3 and 0.1 mg/m3, were achieved by dilution with compressed filtered air from a high concentration chamber (3.0 mg/m3) using an air vacuum pump in a serial dilution system.
- The flow rate through the exposure chamber was adjusted as necessary.
- In order to prevent exposure of animals to test item droplets originated from condensation, an appropriate filter was placed immediately before the serial dilution of the high concentration chamber.
- Previous to exposure onset, a complete setup of the system and characterisation of test atmosphere were performed
- Inhalation exposure was performed using a flow-past, nose-only exposure system.
- The animals were confined separately in restraint tubes which are positioned radially around the exposure chamber.
- The position of the rats in each group within the exposure chamber was changed daily according to an insertion schedule.
- Exposure chambers type EC-FPC-232 (anodized aluminium, volume inside compartment: approximately 3 L, equipped with glass exposure tubes) were used, see picture below.
- The rats were individually exposed in glass tubes matching their size.
- Before treatment start the homogeneity for the different levels of the exposure chamber was confirmed.
- The exposure system ensures a uniform distribution and provides a constant flow of test material to each exposure tube.
- The flow of air at each tube was between 0.6 to 1.5 L/min, which was sufficient to minimize re-breathing of the test item vapour as it was more than twice the respiratory minute volume of rats.
- The temperature and relative humidity of the test atmosphere in the exposure chamber were maintained as required by experimental conditions.
- Air flow was monitored regularly.
- Oxygen concentration in the exposure chamber was at least 19% and carbon dioxide did not exceed 1%.

TEST ATMOSPHERE
Characterisation of test atmospheres
- In order to characterize the test atmospheres and to check the reproducibility of vapour generation, several analytical parameters were determined at defined intervals.
Determination of the nominal vapour concentration
- The test item usage was determined by weighing the amount in the reservoir of the vapour generator and adjacent lines before and after exposure to determine the quantity of test item used. The weight used was then divided by the total air-flow volume to give the nominal concentration. The nominal concentration for different groups was calculated from the nominal concentration of high dose chamber and the dilution factor for each group, respectively. These data were used for the purpose of monitoring the performance of the generation system.
Analytical determination of the vapour concentration
- Analytical determination of the vapour concentration was performed six times during the exposure period for each test item dose by Swiss BioQuant AG. Additional samples were analysed by Vivotecnia for monitoring purposes only.
- Vapour samples were captured in a solvent trap containing a defined volume of a working solution of 1-(2-mehoxyphenyl)piperazine in acetonitrile (cooled with an appropriate ice bath) positioned in an empty port of the inhalation chamber. The sampling flow rate matched the air flow rate per port. The defined volume of working solution in the solvent trap was refilled to the start volume after sampling completion for dose groups 0.3 and 1.0 mg/m3 until the 6th day of exposure. The remaining exposure days for these dose groups and all exposure days for dose group 0.1 mg/m3, the defined volume of working solution in the solvent trap was measured after sampling completion and was not refilled to the start volume. The duration of sampling was sufficient to ensure reliable results according to the outcome from the technical trials.
- Aliquots of samples of the resulting solution of test item in working solution were appropriately labelled and stored at -20 ± 5 °C until shipment for analysis at cool conditions.
- After the beginning of treatment, two samples of test item captured in solvent trap from two exposures (for dose groups B and C) were analyzed within 3 days after sampling.
- The determination of the derivate of test item in working solution was performed by HPLC UV at Swiss BioQuant AG.
- Calibration levels and quality controls are mentioned in the raw data.
The analysis of additional aliquots of solvent trap samples by Vivotecnia for monitoring purposes collected as stated above consisted of:
• 3.0 mg/m3: At least 3 samples per day
• 1.0 mg/m3: At least 2 samples per day
• 0.3 mg/m3: At least 2 samples per day
• 0.1 mg/m3: At least 1 samples per day
The frequency of sampling was reduced at 1.0, 0.3 and 0.1 mg/m3 when no animals were exposed in these groups. Only samples from groups with actually exposed animals are reported.
Determination of the temperature
- The temperature in the vapour was measured hourly in each group during each day of exposure using a thermohygrometer (TFA 30.5036.13). The target range was 22 ± 3ºC. Daily results are reported.
Determination of relative humidity
- The relative humidity (%) in the vapour was measured hourly in each group during each day of exposure using a thermohygrometer (TFA 30.5036.13). A target of approximately 10 % for the dose groups was selected to minimise hydrolysis of the test item. Daily results are reported.
Determination of oxygen and CO2 concentration
- The oxygen concentration of the test atmosphere was measured at least once during each day of exposure using a portable gas detector (PGAS-24-O2/CO2, PGAS-24). The oxygen and CO2 concentration was maintained above 19% and below 1% respectively, during the exposure period. Daily results are reported.
Airflow rate
- The exposure airflow rate was adjusted as appropriate before the start of the exposure using the pressure difference over a Venturi tube. The actual airflow rate was monitored hourly in each group during each exposure. Additional measurements were performed if considered necessary.

VEHICLE (if applicable)
- For sham exposure, filtered, compressed fresh air was provided to the exposure chamber.
- Sham-exposed rats were exposed to filtered, compressed air, the exposure conditions being the same as those for the test item-exposed rats.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical determination of the vapour concentration was performed six times during the exposure period for each test item dose by Swiss BioQuant AG. Additional samples were analysed by Vivotecnia for monitoring purposes only.
Duration of treatment / exposure:
5 days per week for 3 weeks
Frequency of treatment:
5 days/week, 6 hours/day
Dose / conc.:
0 mg/m³ air (nominal)
Remarks:
Sham filtered air, Group A
Dose / conc.:
0.1 mg/m³ air (nominal)
Remarks:
Group D, Exposure to the third dose level (animals from group D) was started two weeks after groups A-C were commenced.
Dose / conc.:
0.3 mg/m³ air (nominal)
Remarks:
Group B
Dose / conc.:
1 mg/m³ air (nominal)
Remarks:
Group C
No. of animals per sex per dose:
3 males and 3 females per dose, 4 doses (incl. control)
4 spare animals (2 males and 2 females)
Control animals:
yes, concurrent vehicle
Details on study design:
The inhalation route is considered to be the predominant route for human exposure.
The target concentrations were selected in agreement with the sponsor based on available toxicity information.
Recitals:
From older studies (Kimmerle_1972_rats II) it was known that target concentration should be expected between 0.34 and 4 mg/m3 with vehicle DMSO. Based on new experimental tests in this DRF Study, there was chosen a staggered study design with 0; 0,3 and 1 mg/m3 but without DMSO as vehicle.
The target concentration of the forth dose level was selected considering the outcome after two weeks of exposure in dose groups 0,3 mg/m3 (B) and 1 mg/m3 (C). Based on a strong body weight loss after 2 exposition weeks in 1 mg/m3 group, it was decided to add a lower (forth) concentration 0,1 mg/m3 (D). It has to be expected that the observed effects in the high dose group 1 mg/m3 should become more relevant within 90 day exposure. As so far the highest concentration for the 90 day study was selected with 1 mg/m3.
Positive control:
not nesessary
Observations and examinations performed and frequency:
MORBIDITY/MORTALITY:
- The animals were examined twice daily by cage-side observation for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS:
- Clinical observations were recorded daily until sacrifice within 60 minutes after finishing exposure in all animals.
- Any visible clinical signs, discomfort and mortality were recorded. Monitoring was adjusted accordingly when the condition of the animal gave cause for concern. Observations included changes in skin, eyes, and mucous membranes. Alterations in respiratory pattern or behaviour as well as changes in posture and response to handling and the presence of abnormal movements were also recorded.

BODY WEIGHT:
- Body weight was recorded in all animals once during the acclimatization period, before the first exposure day, twice weekly thereafter and prior to sacrifice.

FOOD CONSUMPTION:
- Food consumption throughout the study period of all animals was calculated approximately once weekly by cage, estimating the average food intake for each animal.
Sacrifice and pathology:
TERMINAL PROCEDURES AND MACROSCOPIC EVALUATION
- On the day of sacrifice all surviving animals were exsanguinated via abdominal aorta under sodium pentobarbital anaesthesia (75 mg/kg i.p.).

Gross necropsy
- A gross necropsy consisting in a macroscopic evaluation of all the external body orifices and the examination of the abdominal and thoracic cavities and contents was performed. Any organs with gross lesions were collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant.

Organ collection and organ weight
- Organs listed below were collected upon gross necropsy from all animals. Lungs with trachea and larynx, heart and kidneys were weighed (after trimming off of fat and other surrounding tissue). Organs were preserved in 4 % neutral-buffered formaldehyde except for lungs, trachea, larynx and nasal cavities (including four levels and nasopharyngeal duct) that were fixed in ethanol glycerol acetic acid formaldehyde solution (EGAFS) during 48h and finally in 70% ethanol.
Tissue Weighed Fixed
Whole lungs with trachea and larynx* X X
Tracheobronchial lymph nodes - X
Heart X X
Kidneys X X
Nasal cavities
( including four levels and nasopharyngeal duct) - X
*Lungs Fixation : instillation via the trachea under a constant pressure of 20 cm water column for approximately 1 minute until lungs are completely filled and subsequent immersion

Histopathology
- Collected organs were sent to the Test Site, AnaPath GmbH, AnaPath Services, (Hammerstrasse 49, 4410 Liestal, Switzerland) for histological processing. Trimming, blocking, embedding, cutting, staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test site.
- The histopathological evaluation was performed at AnaPath GmbH (Buchsweg 56, 4625 Oberbuchsiten, Switzerland).

Other examinations:
no other examinations
Statistics:
Provided that the sample size was considered adequate, the following parameters were subjected to statistical analysis:
- Body weight
- Absolute and relative organ weights

Analysis of data from two groups
Test of normal distribution Yes Un-paired t-test
No Mann-Whitney test
Test of equal variance No Un-paired t-test with Welch’s correction

Analysis of data from more than two groups
Test of normal distribution Yes One-way ANOVA Dunnett’s post test (comparison versus control group)
Tukey post test (comparison of all groups)
No Kruskal-Wallis Dunn’s post test
Test of equal variance No Kruskal-Wallis Dunn’s post test

A value of P < 0.05 was considered statistically significant.

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The most frequent clinical signs present in animals from both sexes belonging to all experimental groups were the presence of chromodacryorrhea, chromorhinorrhea, soiled coat and piloerection immediately after the end of the daily exposure period. These clinical signs are frequently observed in inhalation studies at the day of treatment and are considered to be mainly related with the stress of the procedure.
No clinical signs were observed in any animal just before each exposure day and on exposure-free days, with the exception of piloerection that was present in one male and one female from the low dose group (D, 0.1 mg/m3 test item) one day after exposure (study day 13). Nevertheless, this clinical sign was transient and it was no longer visible on the following day.
Mortality:
no mortality observed
Description (incidence):
No mortality was recorded during the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In males and females, the body weight of animals from group D (exposed to 0.1 mg/m3 Test item, low dose) was in general statistically significant higher than in animals from control group throughout the study period. These differences could be due to the fact that animals from group D started the exposure period two weeks later than the other groups and hence the initial body weight was already significantly higher. This could also be the reason why body weight gain in these animals was lower than in the rest of the groups as they were older and consequently in a slower phase of the growth curve.
In males, mean body weight of animals from group C (1.0 mg/m3 Test item, high dose) tended to be slightly lower than in the sham-exposed control group over the study period, with the differences being statistically significant on study day 18. No differences between the mid dose (0.3 mg/m3 Test item) and control groups were found.
When considering body weight gain, a decrease was found in males from all groups, including control, between study days 8 and 11, corresponding to the second exposure week. The same trend was observed in the third exposure week for the groups B and C (exposed to 0.3 mg/m3 and 1.0 mg/m3, respectively), which presented a significant body weight decrease with respect to the control group between study days 15 and 18. Nevertheless, at the end of the study period, all experimental groups increased their body weight compared to basal weight before starting treatment, with the highest increase being recorded in control group (A), followed by groups B, C and D (21.1 %, 19.8 %, 14.6 % and 7.3 % increase, respectively). The differences between total body weight gains recorded in experimental groups C and D reached statistical significance when compared with control group.
In females, body weight was similar among the experimental groups A (sham), B (mid dose) and C (high dose) throughout the study period. Regarding body weight gain, all experimental groups, excluding group B (exposed to 0.3 mg/m3 Test item), showed a decrease of the body weight in the third exposure week (between study days 15-18) as it was observed in males. This decrease was also recorded in the high dose group (group C) during the second exposure week (between days 8-11). Nevertheless, the differences were not statistically significant when compared to control.
At the end of the study, all groups of females presented an increase of the body weight with respect to basal weight recorded before starting treatment, with this increase being significantly lower in the group D (0.1 mg/m3 Test item) than in the control group (14.3 % for the group A, 11.21 % for the group B, 12.1 % for the group C and 5.8 % for the group D).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In males, the estimated food consumption was similar among all four experimental groups, with a trend to slightly lower values being observed in animals from the high dose group (C, 1.0 mg/m3 Test item), mainly during the first study days.
Also in females food consumption was similar in all groups but in this case the highest values were found in the high dose group (C, 1.0 mg/m3 Test item) during the first four exposure days.
Nevertheless, the statistical analysis could not be performed due to the small sample size (n=1 cage per group).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not specified
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute and relative organ weights of heart and kidneys were similar among all experimental groups in both sexes, whereas absolute and relative weights of lungs with trachea and larynx were significantly higher in animals from group D (0.1 mg/m3 Test item) than in controls also in males and females. The higher body weight of animals from group D over the whole study period could account for these differences in lung weights (see body weight).
Gross pathological findings:
no effects observed
Description (incidence and severity):
No necropsy macroscopic findings were present in any of the animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no findings that distinguished controls from test item-exposed animals.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
no other effects
Details on results:
The exposure to the test item was well tolerated as no mortality was recorded and the main clinical signs, observed immediately after the exposure, were considered to be mainly due to the stress related to the nose-only exposure procedure.
Body weight of all animals at the end of the study period was higher than before starting exposure, although a trend to slightly lower weight was observed in males exposed to the high dose of test item (1.0 mg/m3) when compared to sham-exposed control animals.
At terminal sacrifice, neither gross lesions nor any relevant effects of the exposure on organ weights were observed.
There were no histological findings indicative of toxicity induced by exposure to the test item in any of the organs and tissues examined microscopically.
Key result
Dose descriptor:
NOAEC
Effect level:
1 mg/m³ air
Based on:
test mat.
Sex:
male/female

Determination of the nominal vapour concentration

The overall mean nominal concentrations are summarized below:

Experimental group

A

B

C

D

Treatment

Sham filtered air

Test item

Test item

Test item

Target concentration(mg/m3)

0

0.3

1.0

0.1

Nominal concentration (mg/m3)

Mean ± SD

NA

1.63 ± 0.57

3.51 ± 1.10

0.63 ± 0.40

 

Analytical determination of the vapour concentration

Throughout the study, deviations between 10 - 15 % approximately from the target concentration were observed in the means of the analytical vapour concentrations determined bySwiss BioQuant AG. In addition, data of the vapour concentrations analysed by Vivotecnia for monitoring purpose showed a stability of the atmosphere and the generation techniques during the study period since study mean deviations from target values were between 4 - 10 % approximately.

The overall mean analyzed concentrations are summarized below:

Experimental group

A

B

C

D

Treatment

Sham filtered air

Test item

Test item

Test item

Target concentration(mg/m3)

0

0.3

1.0

0.1

Analytical concentration (mg/m3)
by Swiss BioQuant AG

Mean ± SD

NA

0.35 ± 0.06

1.10 ± 0.08

0.09 ± 0.01

Deviation from target

NA

15.5 %

10.1%

14.4%

Analytical concentration (mg/m3)
by Vivotecnia

Mean ± SD

NA

0.33 ± 0.06

1.05 ± 0.14

0.10 ± 0.02

Deviation from target

NA

10.6%

5.1%

4.3%

Determination of the atmosphere temperature

Mean chamber temperature during the exposure was maintained within the recommended range of 22 ± 3 °C for the group A.

However, mean daily temperature was above the target range for several days in the groups B and C and over all days of the exposure period for the group D, see table below.

Experimental group

Treatment

Temperature (°C)

Relative humidity (%)

Oxygen (%)

CO2(%)

 

A

Sham filtered air

24.2 ± 0.70

7.8 ± 1.31

21.0 ± 0.15

0.04 ± 0.01

B

Test item 0.3 mg/m3

25.5 ± 0.88

6.4 ± 0.76

21.0 ± 0.10

0.05 ± 0.01

C

Test item 1.0 mg/m3

25.6 ± 0.86

7.2 ± 1.31

21.0 ± 0.10

0.05 ± 0.01

D

Test item 0.1 mg/m3

26.1 ± 0.40

6.1 ± 0.39

21.1 ± 0.06

0.04 ± 0.01

Determination of the atmosphere relative humidity

Chamber relative humidity was maintained around 10 % or below throughout the exposure period to minimise hydrolysis of the test item.

Determination of oxygen and CO2concentration

Throughout the exposure period the oxygen concentration was maintained above 19 % (values ranged between 20.8 - 21.3 %) while the CO2concentration remained below 1 % (0.04 - 0.09 %). These values are considered satisfactory for inhalation studies.

Airflow rate

The airflow rate was maintained at 23 ± 2 L/min for sham air chamber over the entire exposure period. However, the airflow rate for the dose exposure chambers was adjusted over each day in order to achieve the target concentration.

Conclusions:
The dose of 1.0 mg/m3 could be established as NOAEC (No Adverse Effect Concentration) for test item under the conditions tested in this study.
Executive summary:

Introduction and study outline

The present dose range finding (DRF) study was designed to provide general information concerning possible toxic effects of the test item after repeated administrations of different concentrations through the inhalation route (nose-only) to male and female Sprague Dawley rats during 3 weeks (5 days per week, 6 hours per day).

The results from this study will be used to select the target concentrations for a subsequent 90-day inhalation study.

For those purposes, 24 animals (12 males and 12 females) were distributed into 4 experimental groups (A to D). Animals were exposed to diluted vapour from the test item (groups B to D) or to filtered compressed fresh air (group A) 6 hours/day during 5 days per week for 3 weeks. The starting doses for group B and C were selected on available data at 0.3 and 1 mg/m3 respectively. Animals from group D were exposed with the same administration regimen, beginning 14 days after dosing start of the initial groups B and C. The decision about the dose to be tested in the group D was taken considering the presence or absence of signs of toxicity and/or mortality observed during the first two weeks of groups B and C exposure. Finally, animals from group D were exposed at 0.1 mg/m3, based on body weight loss in the first days after exposure and after weekends.

The safety assessment relied on observed mortality, local and systemic clinical signs, food consumption and body weight recorded from day 1 of exposure until sacrifice. At the end of the administration period, all surviving animals were sacrificed and subjected to a gross necropsy consisting in a macroscopic evaluation of all external body orifices and the examination of the abdominal and thoracic cavities and contents. Moreover, heart, kidneys and respiratory tract including nasal cavity (four levels and nasopharyngeal duct, lungs, trachea, larynx and tracheobronchial lymph nodes, were collected for histopathological evaluation. Selected organs (lungs with trachea and larynx, heart, and kidneys) were weighed.

Results

The composition and characteristics of test atmosphere were rather stable throughout the study period. The deviations of the analytical vapour concentrations from the target in dose groups ranged 10 - 15 % approximately. In addition, the relative humidity was maintained below 10 % to minimise hydrolysis of the test item.

No mortality was recorded. Animals from all experimental groups, including sham, presented chromodacryorrhea, chromorhinorrhea, piloerection and soiled coat at the end of the daily exposure period. These signs were transient and considered to be related to the stress of the nose-only exposure procedure.

The body weight of the animals from group D exposed at the low dose (0.1 mg/m3), both sexes, was significantly higher than the control group in practically the whole study period and the initial body weight was already significantly higher than in the rest of experimental groups, likely due to the fact that these animals started the exposure two weeks later and therefore they were two weeks older.

Males from the high dose group (C, 1.0 mg/m3 test item) showed a slightly lower body weight than the control animals over the whole study period although statistical significance of the differences was only found on study day 18. Total body weight gain in this high dose group was also statistically significantly lower than in control. No differences between the mid dose (0.3 mg/m3 test item) and control groups were found.

In females, when compared to sham-exposed control animals, no differences in body weight or body weight gain were found in the high and mid dose groups.

Food consumption was similar among the four experimental groups although a trend to lower values was observed in males exposed to the high dose.

No macroscopic findings at necropsy were recorded in any of the animals. The absolute/relative organ weights were similar in all experimental groups. Only, the weight of lungs with trachea and larynx in the low dose-exposed group (D, 0.1 mg/m3) was statistically significant higher than in the control group.

There were no histological findings that could be attributed to test item exposure.

Conclusions

It can be concluded that under the assayed experimental conditions:

The composition and characteristics of test atmosphere were stable and reproducible throughout the study period.

The exposure to the test item was tolerated as no mortality was recorded and the main clinical signs, observed immediately after the exposure, were considered to be mainly due to the stress related to the nose-only exposure procedure.

Body weight of all animals at the end of the study period was higher than before starting exposure, although a trend to slightly lower weight was observed in males exposed to the high dose of test item (1.0 mg/m3) when compared to sham-exposed control animals.

At terminal sacrifice, neither gross lesions nor any relevant effects of the exposure on organ weights were observed.

There were no histological findings indicative of toxicity induced by exposure to the test item in any of the organs and tissues examined microscopically.

The dose of 1.0 mg/m3 could be established as NOAEC (No Adverse Effect Concentration) for test item under the conditions tested in this study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
7 Sptember 2009
Deviations:
yes
Remarks:
Temperature and relative humidity in the room where animals were housed were sporadically outside the optimal range during the study period. This deviation was not considered to have any impact.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction mass of 2,2,4-trimethylhexa-1,6-diyl diisocyanate and 2,4,4-trimethylhexa-1,6-diyl diisocyanate
EC Number:
915-277-1
Cas Number:
32052-51-0
Molecular formula:
C11H18N2O2
IUPAC Name:
Reaction mass of 2,2,4-trimethylhexa-1,6-diyl diisocyanate and 2,4,4-trimethylhexa-1,6-diyl diisocyanate
Test material form:
gas: vapour

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Albino and specific Pathogen Free animals from Envigo RMS Spain S.L.;Ctra. St. Miquel del Fai, Km. 3.5; 08182 - Sant Feliú de Codines; Barcelona – Spain
The Sprague Dawley rat is deemed a suitable rodent species / strain, acceptable to regulatory authorities as an indicator of potential hazards, and for which extensive background data are available
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Total number of animals: 130: 120 used in the study (10 male and 10 female animals per group and allocation) and 10 spare animals (5 males and 5 females)
- Approx. age of the animals: 7-8 weeks old at randomisation
- Animals per cage (after distribution): 5
- Acclimatisation period: Males: 7 days. Females: 14 days
- Acclimatisation to the nose-only restraining tubes was performed with fresh air for 3 days before the beginning of the treatment phase with incremental durations (approximately 1.5 h, 3 h and 4.5 h, respectively)
- Animal identification: Digit ink and ear marks

Bedding material: Sodispan (SR-CHOPO-T) (autoclavable)
Change of cages: At least once a week and when deemed necessary throughout the study period
Inclusion criteria on arrival: Veterinary inspection

Animal Welfare: Whenever possible animals were kept in groups. Enrichment devices (nesting material, tubes, and chew blocks) were provided as a default husbandry practice

ANIMAL DIET:
Food (Global Diet, Supplier: Envigo Teklad) ad libitum
Water : Tap Water at libitum

ENVIRONMENTAL CONDITIONS:
Light cycle: 12h light : 12h dark
Temperature: 17.4 – 25.6ºC
Relative humidity: 25 – 58%

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
A vapour was generated from the liquid test item, TMDI, using a Volatile Organic Compound (VOC) generator (TSE Systems).

The vapour was conveyed via glass tubing, from the generator to the exposure chambers. To obtain the target concentration levels the TMDI vapour was diluted with filtered, compressed air downstream from the generator.
For sham exposure, filtered, compressed fresh air was provided to the exposure chamber.

Inhalation exposure was performed using a flow-past, nose-only exposure system.
The animals were confined separately in restraint tubes which were positioned radially around the exposure chamber.
No diet or drinking water were available during exposure.

DETERMINATION OF THE NOMINAL VAPOUR CONCENTRATION
Nominal concnetrations of the test item were calulated, analytical concnetrations were determined as monitoring data at VVT Spain and confirmed via SBQ Switzerland.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYTICAL DETERMINATION OF THE VAPOUR CONCENTRATION
Analytical determination of the vapour concentration was performed fourteen times (once per week) during the exposure period for each test item concentration by Swiss BioQuant AG. In general, two samples each were analysed at 1.0 and 0.3 mg/m3 and one sample at 0.1 mg/m3 on each occasion. Additional samples were analysed if considered necessary. This includes analyses by Vivotecnia for monitoring purposes.
Vapour samples were captured in a solvent trap containing a defined volume of a working solution of 1-(2-mehoxyphenyl)piperazine in acetonitrile (cooled with an appropriate ice bath) positioned in an empty port of the inhalation chamber. The sampling flow rate matched the air flow rate per port. The final volume of working solution in the solvent trap was measured after sampling completion as some acetonitrile evaporated. The duration of sampling was sufficient to ensure reliable results.
Aliquots of samples of the resulting solution of test item in working solution were appropriately labelled and stored at -20 ±5 °C until shipment at cool conditions. TMDI from the test atmosphere reacted with 1-(2-Methoxyphenyl) piperazine in the trapping solution to a stable derivate. The determination of the derivate of TMDI in working solution was performed by HPLC UV according to Test Site SOP reference M-124 using a validated method. Calibration levels and quality controls are mentioned in the raw data, and the acceptance criteria for the validity of an analytical batch were defined in the Test Site SOP M-124 and M-002. The analytics were performed under the phase number SBQ-17214.
The samples were sent in regular intervals, depending on the stability as demonstrated in the validation study conducted by SBQ.
The actual shipment dates were as follows:
- First shipment (6 Feb 2018; end of study week 2): Samples from Weeks 1 and 2
- Second shipment (27 Feb 2018; end of study week 5): Samples from Weeks 3, 4 and 5
- Third shipment (03 April 2018; end of study week 10): Samples from Weeks 6, 7, 8 and 9
- Fourth shipment (14 May 2018; study week 16): Samples from Weeks 10, 11, 12, 13 and 14
Samples were stored by the Principal Investigator at -25 ± 10 °C until analysis (see contact details at Section 2. The phase report with the results of the analyses (concentrations of the test item in the solvent trap samples) provided by the Principal Investigator is included in Annex IV. The atmosphere concentration was calculated from the concentrations of the test item in the solvent trap samples, the final volume in the solvent trap, the sampling duration and the sampling flow rate.
The analysis of additional aliquots of solvent trap samples by Vivotecnia for monitoring purposes collected as stated above consisted of:
- 3.0 mg/m3: At least 3 samples per day (no animal exposure)
- 1.0 mg/m3: At least 2 samples per day (Group D)
- 0.3 mg/m3: At least 2 samples per day (Group C)
- 0.1 mg/m3: At least 1 samples per day (Group B)
The samples following the day of sample collection for Swiss BioQuant AG were kept for at least 8 weeks as reserve samples. After evaluation of the actual results from Swiss BioQuant AG, these samples were used for additional analyses (if necessary) or discarded at the discretion of the study director.
Duration of treatment / exposure:
92 days (13-weeks)
Frequency of treatment:
6 h/day, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/m³ air (nominal)
Remarks:
Sham filtered air; control group, Group A
Dose / conc.:
0.1 mg/m³ air (nominal)
Remarks:
Group B low dose group
Dose / conc.:
0.3 mg/m³ air (nominal)
Remarks:
Group C middle dose group
Dose / conc.:
1 mg/m³ air (nominal)
Remarks:
Group D high dose group
No. of animals per sex per dose:
10 males and 10 females per dose group; additional 10 males and 10 females for recovery group (only control and high dose group)
Control animals:
yes, sham-exposed
Details on study design:
Treatment start and administration of males was on day 1. Treatment start and administration of females was on day 8 of the study period. Accordingly, all animals were exposed to the same conditions and readouts, but the procedures were performed at different time points. As a consequence, all the follow up readouts were planned accordingly.
Doses for this main study were selected based on the VVT_2017_21 day_DRF Study. The highest dose were established to be 1 mg/m3 because in the DRF study strong changes (degrease in body weight gain) were observed during the first 2 weeks of exposure. It has to be expected that the observed effects in the high dose group 1 mg/m3 should become more relevant within 90 day exposure. As so far the highest concentration was selected with 1 mg/m3 in the 90 day study.
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
MORTALITY
The animals were examined three times daily for mortality and morbidity. Monitoring was adjusted accordingly when the condition of the animal gave cause for concern. During weekends and during the exposure-free recovery period, only one observation per day was performed.


DETAILED CLINICAL OBSERVATIONS
On exposure days, clinical observations were recorded before, during (only grossly abnormal signs as the animals were restrained in the exposure tubes) and within 60 minutes after finishing exposure in all animals. During the exposure-free recovery period and during weekends, clinical observations were recorded once daily.

BODY WEIGHT
Body weight was recorded in all animals once during the acclimatization period, before the first exposure day, twice weekly thereafter and prior to sacrifice (fasted animals).

FOOD CONSUMPTION
Food consumption throughout the study period of all animals was calculated on a weekly basis by cage, estimating the daily food intake for each animal.

OPHTHALMOLOGICAL EXAMINATIONS
All animals were examined twice during the study: before starting the exposure and during the last exposure week. As no changes were observed at the end of the 13-week exposure period, examinations at the end of the exposure-free recovery period were not required.

CLINICAL PATHOLOGY
Complete clinical pathology analyses, comprising haematology, clinical biochemistry, coagulation and urinalysis, were performed in all animals at the end of exposure period and, additionally, at the end of the recovery period in the corresponding animals. For terminal blood samplings, the animals, fasted overnight (only food will be withheld, not water), were bled under sodium pentobarbital anaesthesia (75 mg/kg i.p.) by retro-orbital sinus puncture for haematology, differential leukocyte count (DLC) and coagulation determinations. Immediately thereafter, blood samples were taken for clinical biochemistry analysis by exsanguination via abdominal aorta.
For the intermediate blood sampling in animals from the Recovery allocation at the end of the exposure period blood was collected by retro-orbital sinus puncture under isoflurane anaesthesia.


-HAEMATOLOGY
Approximately 0.5 mL of EDTAK3-uncoagulated blood was used directly for the evaluation of haematological parameters using a haematology counter (Sysmex XS-800i) and the following parameters were analyzed:
- WBC White blood cell count
- RBC Red blood cell count
- HGB Haemoglobin
- HCT Haematocrit
- MCV Mean corpuscular volume
- MCH Mean corpuscular haemoglobin
- MCHC Mean corpuscular haemoglobin concentration
- PLT Platelet count
- MPV Mean platelet volume
Air dried blood smears were stained with May-Grünwald Giemsa, differential leukocyte count (DLC) was assessed by light microscopy and the percentage the following cells was recorded: lymphocytes, monocytes, basophils, eosinophils and neutrophils. The absolute counts were calculated. Reticulocyte percentage was also determined in blood smears stained with brilliant Cresyl blue.


-COAGULATION
Plasma from citrated blood, isolated after centrifugation at approximately 1000 g for 10 min at room temperature, was analysed by means of a coagulation analyser (Diagnostica START4) and the following parameters were determined:
- PT Prothrombin time (extrinsic pathway)
- APTT Activated partial Thromboplastin Time (intrinsic pathway)


-BIOCHEMISTRY
Plasma from heparinised blood, isolated after centrifugation at approximately 1000 g for 10 min at 4ºC, was analysed by means of a biochemistry analyser (COBAS Integra, 400 plus) and the following parameters were determined:
Substrate
ALB Albumin
TP Total proteins
GLOB Globulins*
CHOL Cholesterol
TRIGL Triglycerides
GLUC Glucose
URE Urea
BIL Total bilirubin
CRE Creatinine

Enzymes
ALP Alkaline phosphatase
ALT Alanine aminotransferase
AST Aspartate aminotransferase
GGT Gamma glutamyltransferase

Electrolytes
Ca2+ Calcium
Cl- Chloride
PO43- Inorganic phosphorus
K+ Potassium
Na+ Sodium


- URINALYSIS
All animals were housed individually in metabolic cages with free access to water. The urine was collected overnight with volume recorded the next morning. The urine samples were analysed by means of reactive strips (Henry Schein, USA).
The following parameters were evaluated: pH, glucose, ascorbic acid, proteins, nitrites, ketones, urobilinogen, bilirubin, blood and leucocytes. Specific gravity was determined by means of a refractometer and sediment analysis was assessed by light microscopy.
Sacrifice and pathology:
TERMINAL INVESTIGATIONS

-DETAILED NECROPSY
On the day of sacrifice (study days 93 and 100 for main allocation and study days 121 and 128 for recovery animals) all surviving animals fasted overnight (only food was withheld, not water) were exsanguinated via abdominal aorta under sodium pentobarbital anaesthesia (75 mg/kg i.p.). Immediately thereafter, a macroscopic evaluation of the external surface of the body, all orifices and the cranial, abdominal and thoracic cavities and their contents was performed. Subsequently, a detailed necropsy with the tissue collection and weighing detailed below was conducted.
Organs / tissues listed below were collected upon gross necropsy from all animals. All organs were preserved in 4 % neutral buffered formaldehyde exept for testes and Eyes (incl. optic nerve and Harderian glands) which were Fixed in Davidson’s solution for approximately 24 hours before being transferred to 70% ethanol.

The following organs/tissues were evaluated:
Adrenal glands
Aorta
Brain together with cerebellum and medulla oblongata
Bronchial lymph nodes
Caecum, colon , duodenum, ileum, jejunum (incl. Peyer’s patches), rectum, stomach
Epididymides
Eyes (incl. optic nerve and Harderian glands)
Femoral bone with knee joint
Extraorbital Lacrimal glands
Heart
Kidneys
Liver
Whole lungs together with larynx and trachea*
Mandibular lymph nodes
Mammary gland
Mediastinal lymph nodes
Mesenteric lymph nodes
Nasal cavities incl. pharynx and nasopharyngeal duct
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Salivary glands (mandibular, sublingual)
Sciatic nerve
Seminal vesicle
Skeletal muscle
Skin
Spinal cord (thoracic, lumbar, cervical)
Spleen
Sternum (incl. bone marrow)
Teeth
Testes
Thymus
Thyroids (incl. parathyroids)
Tongue
Urinary bladder
Uterus (with cervix and oviducts)
Macroscopic lesions


- ORGAN WEIGHTS
Organs / tissues indicated in the list below were weighed after trimming off of fat and other surrounding tissue. Paired organs were weighed together.
Relative organ weights (percentage respect to terminal body weight) were calculated.
Adrenal glands
Brain together with cerebellum and medulla oblongata
Epididymides
Heart
Kidneys
Liver
Whole lungs together with larynx and trachea*
Ovaries
Pituitary
Spleen
Testes
Thymus
Thyroids (incl. parathyroids)
Uterus (with cervix and oviducts)


- HISTOPATHOLOGY
A histopathological evaluation was performed for all main study animals of the control- and the high concentration-exposed groups. In case of treatment-related findings, these particular organ(s) or tissue(s) of the intermediate groups and the recovery groups were examined.
Collected organs were sent to the Test Site, AnaPath GmbH, AnaPath Services, (Hammerstrasse 49, 4410 Liestal, Switzerland) for histological processing. Trimming, blocking, embedding, cutting, staining and microscopical quality check of the stained slides will be performed according to the corresponding SOP’s of the test site.
Vivotecnia sent to Anapath the collected organs from all concentration groups in case further examinations were needed.
The histopathological evaluation was performed at AnaPath GmbH (Buchsweg 56, 4625 Oberbuchsiten, Switzerland).
The histopathology results were provided to the study director by e-mail and a pathology phase report was sent to the study director upon completion of the study.
Other examinations:
no other informations
Statistics:
Provided that the sample size was considered adequate, the following parameters were subjected to statistical analysis:
- Body weight
- Food consumption
- Haematology
- Coagulation
- Biochemistry
- Urine volume, pH and density
- Absolute and relative organ weights

Analysis of data from two groups
If data passed the Normal Distribution test, the statistical significance of the differences between both groups was determined by the Un-paired t-test or by the Un-paired t-test with Welch´s correction if the variances of the groups were not the same.
In the case that data did not follow a normal distribution, the statistical significance of the differences between both groups was measured by the Mann-Whitney test.
Analysis of data from more than two groups
If data passed the Normal Distribution test, a One-way ANOVA analysis was performed to determine the existence of differences among the groups, followed by the Dunnett´s post hoc test to evaluate the magnitude of the differences respect to the control group.
If data did not pass the Normal Distribution test or group variances were not the same, the Kruskal-Wallis test was used followed by the Dunn´s post hoc test to determine the magnitude of the differences respect to the control group.
A value of P < 0.05 was considered statistically significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The most frequent clinical signs present in animals from both sexes belonging to all experimental groups were the presence of chromodacryorrhea, chromorhinorrhea, wet fur and piloerection immediately after the end of the daily exposure period. These clinical signs are frequently observed in inhalation studies on the days of treatment and are considered to be mainly related with the stress of the procedure.
From the study week 4 onwards the presence of mild wounds on the hind limbs and the tail base was observed in animals from all experimental groups with the exception of females from the high dose group.
Furthermore, alopecia around the eyes was observed in 3 males belonging to the high dose group from study week 11 and in 2 and 3 females from the mid and high dose groups (Week 7), respectively, and also in one female from the low dose and control groups (Week 10) . Additionally, dirty fur was observed in animals from all groups from study week 7 onwards.
At the end of the recovery period wounds, dirty fur and alopecia around the eyes were still observed in animals from control and high dose groups.
These findings (alopecia, mild wounds and dirty fur) are commonly seen in long term nose-only inhalation studies.
Mortality:
no mortality observed
Description (incidence):
No mortality was recorded throughout the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
During the exposure and exposure-free recovery periods, no statistically significant differences were found in either males or females when mean body weight values of animals exposed to the different concentrations of test item were compared to Sham-exposed control animals.
Regarding body weight gain, in spite of some statistically significant differences compared to control animals on isolated occasions during the exposure period, mean total body weight gain over the exposure period was similar in all experimental groups and both sexes. No statistically significant differences were found either in total body weight gain during the exposure-free recovery period between control and high dose groups for both sexes.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Calculated food consumption was similar in all experimental groups throughout the exposure and exposure-free recovery periods (see Tables 17-20).
Food efficiency:
no effects observed
Description (incidence and severity):
For body weight gain there were a few statistically significant differences to the control animals on isolated days during exposure period, however, mean total body weight gain was similar in all dose groups and both sexes.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No changes attributable to test item were observed in any animals at the end of the 90-day exposure period. Consequently, examinations at the end of the exposure-free recovery period were not performed.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of exposure period, a small but statistically significant increases of mean corpuscular haemoglobin concentration (MCHC) and mean platelet volume (MPV) were found in males from the mid dose group when compared to controls.
White blood cell (WBC) counts tended to decrease in males exposed to TMDI and the differences were statistically significant relative to control in the mid and high dose groups. Small decreases in lymphocyte counts and eosinophils (percentage and counts) were also recorded.
At the end of the recovery period MCHC and eosinophils were significantly lower in males from the high dose group than in controls and WBC counts were no longer decreased.
In females, the only significant change respect to control at the end of the exposure period was a mild decrease of MPV, whereas after the exposure-free recovery period, a significant increase of WBC counts relative to control was found in animals from the high dose group together with an increase in neutrophils and a decrease in lymphocytes. Platelet counts were also significantly increased.
Nevertheless, in spite of the changes and trends above described, the values were generally within the normal range (see section 19.1 in the report) and there were no corresponding histopathological findings. Therefore, the variations were not considered to be of toxicological relevance.

Coagulation
The exposure to TMDI did not affect the coagulation parameters tested and values were within the normal ranges and similar among all experimental groups in both sexes during the exposure and the recovery periods.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, at the end of the exposure period some small changes, although statistically significant relative to control animals, were found in a number of the parameters tested: lower levels of glucose, total proteins, albumin and triglycerides in the mid dose group and of urea in the low dose group, as well as of alkaline phosphatase in the low and mid dose groups. On the contrary, higher levels of bilirubin in the low and mid dose groups and of chloride in the mid dose group were also recorded.
At the end of the exposure-free recovery period in males, the only significant change in the high dose group when compared to control was a minor increase of cholesterol levels.
In females, at the end of the 90-day exposure period, statistically significant lower levels of glucose, urea, and total proteins were found in low and mid dose groups and of albumin in the low dose group, together with higher levels of phosphorus in the low and mid dose groups and also of sodium in the mid dose group.
At the end of the exposure-free recovery period in females, significantly lower levels of urea and creatinine and higher levels of chloride and calcium were found in high dose group when compared to control.
Nevertheless, in both males and females, all the differences above described were of numerically minor magnitude, did not present a dose-response relationship and are within biologically variation. Therefore, these changes were not considered to be of toxicological relevance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No relevant changes in urine density and pH were found in any sex either after the 90-day exposure to TMDI or at the end of the exposure-free recovery period.
Regarding the parameters determined by the reactive strip and the results from the urinary sediment evaluation, they were similar in all experimental groups, both sexes and both allocations, and no effects of test item exposure were recorded.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
A small but statistically significant decrease of absolute and relative liver weights in males from the mid dose group and of liver relative weight in males from the high dose group were recorded at the end of the exposure period when compared to control. Also absolute and relative thymus weights were lower in males from the low dose group than in controls after the exposure period.
The above changes in organ weights did not have any microscopic or clinical pathology correlates. Since no dose-related effect to test item exposure was recorded, they were considered incidental.
In females, the only finding was the presence of higher absolute and relative weights of uterus in the high dose group at the end of the exposure-free recovery period when compared to control. This finding may be related to the presence of hydrometra in a number of animals from the high dose group and, therefore, it was considered incidental.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The presence of hydrometra (a common finding in rats from this strain and age) in some females from the three experimental groups exposed to test item was considered to be incidental No other relevant macroscopic changes related to test item exposure were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathology report see Appendix 3
A minimal degeneration/disorganization of the olfactory (dorsal meatus) epithelium was noted in two males and three females at a dose of 1.0 mg/m3 (nasal cavity level 2 only). There were no further findings that distinguished controls from test item-treated animals.
In a few high dose animals, there was an eosinophilic precipitate in the urinary bladder. This alteration was not associated with an inflammatory or degenerative tissue change in the urinary bladder and kidneys. This alteration of unknown composition was not associated with an inflammatory or degenerative tissue change in the urinary bladder and kidneys or any relevant changes in biochemical or urinalysis parameter. Therefore, a relationship to treatment was considered to be unlikely.


Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Minor histological findings that could be attributed to test item exposure were observed. Under the conditions of this study, the test item caused a minor lesion in a very low incidence in one nasal cavity level only that was characterized by degeneration/disorganization of the olfactory epithelium. Such degeneration is occasionally observed as a spontaneous lesion. However, in this study, no control animal was affected. Therefore, a treatment-relationship is likely. No findings were noted in intermediate group and recovery animals. It is therefore concluded that the minor lesion at the high dose in the nasal cavity, if induced, was fully reversible. As this lesion is a normal background finding and in the absence of any other effect in the nasal cavity, this finding was considered as non-adverse.
Details on results:
The concentrations of test atmosphere were stable and reproducible throughout the study period.
The exposure to the test item TMDI was well tolerated as no mortality was recorded and the main clinical signs were considered to be unrelated to test item exposure.
No effects of the exposure to TMDI on body weight, food consumption, clinical pathology parameters and eye structures were observed.
At terminal sacrifice, neither gross lesions nor any relevant effects of the exposure on organ weights were observed.
Minor and fully reversible histological findings indicative of toxicity induced by exposure to the test item TMDI were observed in the nasal cavity from single high dose animals examined microscopically.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
1 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOAEC
Effect level:
0.98 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Analysis

Analytical determination of the vapour concentration was performed fourteen times (once per week) during the exposure period for each test item concentration by Swiss BioQuant AG. In general, two samples each were analysed at 1.0 and 0.3

mg/m3and one sample at 0.1 mg/m3on each occasion. Additional samples were analysed if considered necessary. This includes analyses by Vivotecnia for monitoring purposes.

The relative humidity (%) in the vapour was measured hourly in each group during each day of exposure using an appropriate device. A target of approximately 10 % for the exposure (and control) groups was selected to minimise hydrolysis of the test item. Daily results are reported.

Study mean values of TMDI concentrations in the test atmosphere.

SBQ and VIVOTECNIA (non-validated method) data

 

Group

Target concentration (mg/m3)

Vivotecnia Data*

 

 

SBQ Data

 

Mean

SD

% deviation from target

Mean

SD

% deviation from target

B

0.1

0.10

0.04

3.0

0.11

0.02

5.0

C

0.3

0.32

0.08

6.6

0.34

0.06

13.0

D

1.0

0.94

0.21

-5.9

0.98

0.16

-2.5

* Mean values reported by Vivotecnia (non-validated method for monitoring purposes) consider the daily determinations of the concentrations and it may differ from the Vivotecnia mean values described in the phase report since weekly samples were analyzed at SBQ test site.

Applicant's summary and conclusion

Conclusions:
Taken together the results obtained in the study and disclosed in this report, it can be concluded that under the present experimental conditions:
The concentrations of test atmosphere were stable and reproducible throughout the study period. The exposure to the test item TMDI was well tolerated as no mortality was recorded and the main clinical signs were considered to be unrelated to test item exposure. No effects of the exposure to TMDI on body weight, food consumption, clinical pathology parameters and eye structures were observed.
At terminal sacrifice, neither gross lesions nor any relevant effects of the exposure on organ weights were observed. Minor and fully reversible histological findings indicative of toxicity induced by exposure to the test item TMDI were observed in the nasal cavity from single high dose animals examined microscopically.
The achieved concentration of 0.98 mg/m3 could be established as NOAEC (No Adverse Effect Concentration) for TMDI under the conditions tested in this study.

Executive summary:

Introduction and study outline

The objective of this study was to evaluate the repeated dose toxicity of the test item 2,2,4 (or 2,4,4)-trimethylhexane-1,6-diisocyanate (hereinafter TMDI) in male and female Sprague Dawley rats when exposed through the inhalation route (nose-only) for 90 days (5 days per week, 6 hours per day).

The reversibility, persistence or delayed occurrence of eventual adverse effects was also studied during a 28-day treatment-free recovery period after the last exposure in animals from control and high concentration-exposedgroups.

For those purposes, 120 rats (60 males and 60 females) were distributed into four experimental groups (A to D) separated in two different allocations (main and recovery).

Animals from group A were exposed to filtered compressed fresh air and served as control and animals from groups B to D were exposed to diluted vapour from the test item at target concentrations of 0.1, 0.3 and 1.0 mg/m3, respectively.

The main allocation served to assess the potential toxic effects of TMDI after a 13 week exposure period, (10 males and 10 females per group), whereas animals from the recovery allocation (10 males and 10 females from the air control and high concentration-exposed animals) were subjected to a treatment-free period of 4 weeks.

The safety assessment relied on observed mortality, systemic clinical signs, body weight and food consumption recorded throughout the whole study. In addition, clinical pathology determinations were performed at the end of treatment for all animals as well as at the end of the recovery period. Ophthalmic examinations were also performed before starting exposure and at the end of treatment for all animals. Additionally, the safety assessment was based on the weight of organs collected at termination as well as on the histopathological evaluation of a full list of tissues from all main study animals of the air control and high concentration-exposed groups and two levels of the nasal cavitiesfrom the intermediate and the recovery groups.

 

Results

The concentrations of test atmosphere were stableand reproduciblethroughout the study period resulting in achieved mean concentrations of 0.11, 0.34 and 0.98mg/m3, respectively. The deviations of the mean analytical vapour concentrations from the target in dose groups ranged within 2.5 – 13.3 %. In addition, the mean relative humidity was maintained generally between 6 % and 8  % to minimise possible hydrolysis of the test item.

No mortality was recorded. Some clinical signs were observed in all groups and, therefore, considered not related to treatment with the test item but to the long term procedures of nose-only inhalation.

The exposure to TMDI did not have any relevant effects onbody weight, food consumption, clinical pathology parameters and eye structures.

No macroscopic findings at necropsy were recorded in any of the animals. The absolute/relative organ weights did not show treatment related effects.

Minor histological findings that could be attributed to test item exposure were observed. Under the conditions of this study, the test item caused a minor lesion in a very low incidence in one nasal cavity level only that was characterized by degeneration/disorganization of the olfactory epithelium. Such degeneration is occasionally observed as a spontaneous lesion. However, in this study, no control animal was affected. Therefore, a treatment-relationship is likely. No findings were noted in intermediate group and recovery animals.It is therefore concluded that the minor lesion at the high dose in the nasal cavity, if induced, was fully reversible.As this lesion is a normal background finding and in the absence of any other effect in the nasal cavity, this finding was considered as non-adverse.

Conclusions

Taken together the results obtained in the study and disclosed in this report, it can be concluded that under the present experimental conditions:

-      The concentrations of test atmosphere were stable and reproducible throughout the study period at expected target.

-      The exposure to the test item TMDI was well tolerated as no mortality was recorded and the main clinical signs were considered to be unrelated to test item exposure.

-      No effects of the exposure to TMDI on body weight, food consumption, clinical pathology parameters and eye structures were observed.

-      At terminal sacrifice,neither gross lesions norany relevant effects of the exposure on organ weights were observed.

-      Minor and fully reversiblehistological findingsindicative of toxicityinduced by exposure to the test item TMDIwere observed in the nasal cavity from single high dose animals examined microscopically.

-      The achieved mean concentration of 0.98 mg/m3could be established as NOAEC (No Adverse Effect Concentration) for TMDI under the conditions tested in this study.