Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13th August 2009 - 28 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
Values for relative humidity above the target range occasionally occurred, usually following room cleaning, and are considered not to have any influence on the study.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
yes
Remarks:
Values for relative humidity above the target range occasionally occurred, usually following room cleaning, and are considered not to have any influence on the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: Benzoic acid isononylester
Description: Colourless liquid
Batch Number: 649/82493 Charge 60980
Purity: 99.96%
Expiry Date (Retest Date) as given by the Sponsor: Apr-2010
Expiry Date (Retest Date) as handled by Harlan Laboratories Ltd.: 30-Apr-2010

Test material used as supplied.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS

Source:
Harlan Laboratories Ltd., Kreuzelweg 53, 5960 AD Horst / Netherlands

Age at study initiation:
9 weeks

Weight at study initiation:
Males: 245.8 to 281.7 g
Females: 177.4 to 205.4 g

Housing:
In groups of five (in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland).

Diet:
Pelleted standard Kliba Nafag 3433 (batch no. 35/09) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.

Water:
Community tap-water from Füllinsdorf was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of respective samples are included in the report.

Acclimation period:
Thirteen days under test conditions after health examination. Only animals without any visible signs of illness were used for the study. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2, and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
Optimal hygienic conditions behind a barrier system. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, higher values usually following room cleaning, and are considered not to have any influence on the study. Therefore, these data are not reported but are retained at Harlan Laboratories Ltd. There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.

IN-LIFE DATES: From: Day 1 To: Day 92

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Group Range of MMAD [µm] Range of GSD
2 1.59 - 2.38 1.93 - 2.76
3 1.79 - 1.92 2.05 - 2.27
4 1.74 - 2.23 2.03 - 2.55
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Exposure apparatus:
A Hudson nebulizer connected to a syringe pump. The polyethylene injector inside the nebulizer was replaced by a stainless steel injector. Flow-past, nose-only exposure chamber.

Exposure chamber volume:
Not applicable (nose-only, flow-past inhalation exposure chamber)

Method of holding animals in test chamber:
The animals were confined separately in restraint tubes which were positioned radially around the flow-past, nose-only exposure chamber. Only the nose of each animal was exposed to the test atmosphere.

Source of air:
Compressed air was supplied into the exposure system.

System of generating particulates/aerosols:
The test aerosol for groups 3 and 4 was generated using a Hudson nebulizer connected to a syringe pump. The polyethylene injector inside model was replaced by a stainless steel injector. The generated test aerosol was diluted as necessary with compressed air to achieve the concentrations required for this study. The aerosol concentrations for group 2 were achieved by serial dilution with compressed, filtered, dry air of the test aerosol generated for group 3 using an air vacuum device.


Method of particle size determination:
Mercer Impactor (Model 02-130, In-Tox. Products Inc., Albuquerque, New Mexico, U.S.A.).

Temperature, humidity, oxygen concentration:
The oxygen concentration, temperature and relative humidity of the test atmosphere were measured continuously during the exposure on test aerosol samples taken at a representative exposure port using a calibrated device. The results were reported twice during each exposure.

Air flow rate
The exposure airflow rate was adjusted as appropriate before the start of the exposure using calibrated flow-meters and / or pressure gauges. The actual airflow rate was recorded at least twice during each exposure.

TEST ATMOSPHERE
Brief description of the method used:
The samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47 mm in-line stainless steel filter sampling device. The duration of sampling was 30 min in groups 2 and 3 and approximately 5 min in group 4. Gravimetric determination of the aerosol concentration was performed in groups 2 to 4 three times per exposure per group.

Samples taken from breathing zone:
yes

VEHICLE
No vehicle used.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not required
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, at 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
Group 1: 0 mg/L air, Group 2:0.10 mg/L air, Group 3: 0.40 mg/L air, Group 4: 4.8 mg/L air
Basis:
other: Gravimetry
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
Details on study design:
Method: Inhalation by nose-only, flow-past exposure.
Rationale for Method: Inhalation is a possible route for human exposure.
Frequency of Administration: 6 hours/day, at 5 days/week
Target Aerosol Concentration: Target Aerosol
Concentration
Group 1: 0 mg/L air
Group 2: 0.10 mg/L air
Group 3: 0.40 mg/L air
Group 4: 5.0 mg/L air

Rationale for Aerosol Concentration Selection: Target aerosol concentration were selected by the Sponsor based on the results from a previous non-GLP 2 week inhalation study performed at Harlan Laboratories (study C47478) and from a previous acute inhalation study performed at SafePharm Laboratories Limited (SPL Project Number 1571/006)

Duration of Treatment Period: 13 weeks
Animals of group 1 were treated with air under the same conditions as animals exposed to the test item.

Examinations

Observations and examinations performed and frequency:

Viability / Mortality:
Twice daily during treatment, once daily during quarantine and acclimatization.

Clinical Signs:
Cage-side clinical observations (once daily during treatment, once weekly during acclimatization). In addition, once weekly careful clinical examination of each animal in a standard arena.

Functional Observation Battery (FOB):
Animals were observed for behavior, reflexes, activity, responsiveness, urine or feces, posture and general abnormalities once at the end of the treatment period. Any abnormal findings were recorded and, where appropriate, graded in severity. Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with a suitable device. Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes.
Dates at the end of treatment:
18-Nov-2009 (Males Group 2 – 4)
19-Nov-2009 (Females Group 2 – 4)
20-Nov-2009 (Males and Females Group 1)

Food Consumption
Was recorded weekly (per cage) during acclimatization and treatment.

Body Weights:
Were recorded weekly (each individual animal) during acclimatization and treatment.

Ophthalmoscopy:
All main study animals, using an appropriate ophthalmoscope.
Dates:
Acclimatization: 14-Aug-2009
Week 13 of treatment: 23-Nov-2009
Clinical Laboratory Investigations:
Blood and Urine Sampling:
After 13 Weeks: 25-Nov-2009 (Males)
26-Nov-2009 (Females)

Sacrifice and pathology:
Necropsy
After 13 Weeks: 25-Nov-2009 (Males)
26-Nov-2009 (Females)


Histopathology:
Groups 1 and 4: all collected organs
Groups 2 and 3: target organs
Other examinations:
None:
Statistics:
The following statistical methods were used to analyze locomotor activity, food consumption, body weight, ophthalmoscopic examinations, macroscopic findings, organ weights and ratios, as well as clinical laboratory data and FOB data where applicable:

• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control group for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to the ophthalmoscopic and macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Hair loss and scabs on the head, the nose and/or the cheek were observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
Hair loss and scabs on the head, the nose and/or the cheek were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight gain was statistically significantly reduced in males and females of group 4.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption was similar across all groups. However, the lowest mean values over the whole treatment period were noted for males and females of group 4 and, therefore, a possible effect on the food intake cannot be excluded.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Relative and total eosinophil levels were increased in males (attaining statistical significance) and females of group 4 after 13 weeks of treatment with the test item. Neutrophils were also increased in females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Sodium and chloride were increased in groups 2 to 4. In addition, urea was increased in group 4 and reduced levels were noted for total cholesterol and phospholipids and increased globulin levels resulted in increased total protein.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Group 4: relative density increased and volume decreased, protein contents, ketone concentrations, leukocyte counts increased and pH-value decreased. Finally, a turbid or cloudy appearance of the urine was observed in all groups.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Unkempt fur was noted in group 3 and group 4 during the open field observations. Chromorhinorrhea was recorded in group 4 during the In-hand observation. Hair loss was observed in the same animals already mentioned in the clinical signs section.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increased absolute and relative organ weights were recorded for the kidneys in males and for the liver in females of group 4.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Skin sores in the nose region were recorded for group 4.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In groups 3/4: Skin: Chronic inflammation with acanthosis and eschars. Nasal cavity: Eosinophilic inclusions in the epithelium. Larynx: squamous metaplasia. Liver: Periportal hepatocellular vacuolation. Kidneys: Hyaline droplets/granules.
Histopathological findings: neoplastic:
no effects observed
Details on results:
Observations
Viability / Mortality
All animals survived the scheduled treatment period.


Clinical Signs
The clinical signs described in this section cover the general observations for clinical signs as well as the detailed clinical signs observed outside the home cage.

Hair loss and scabs on the head, the nose and/or the cheek were observed in male animals of group 4 mainly from weeks 4 to 13. In general, the severity increased up to marked in single animals and with an increasing incidence almost all males were affected after approximately 2 months of treatment. In females, the incidence was slightly lower, the findings were limited to the nose region and they were recorded only from week 7 onwards.

Hair loss which was observed at the anterior dorsum in single animals of the control group as well as the low and high dose groups was considered not to be related with treatment but with the restraining procedure during exposure. No findings were recorded for the animals of the mid dose group.


Functional Observational Battery

Unkempt fur was noted in one female of group 3 as well as in three males and three females of group 4 during the open field observations. Chromorhinorrhea was recorded in seven males and four females of group 4 during the In-hand observation. Hair loss was observed in the same animals already mentioned in the clinical signs section.

Other observations in treated animals like increased lifting or vocalization, increased or decreased rearing or activity, increased urine puddles or feces-balls and discoloration of urine occurred occasionally also in the control animals or showed no dose-relationship and were therefore considered not to be test item-related.


Grip Strength, Landing Foot Splay and Body Temperature
Mean values of grip strength (fore and hind leg), landing foot splay and body temperature measurements show similar values within all treatment groups and demonstrate no effect of the treatment with the test item.


Locomotor Activity
Measurement of the locomotor activity by low beams count after 6, 12, 18, 24 and 30 min in all animals during week 13 of the treatment period gave no indication of a test item related effect.


Food Consumption
The food consumption was similar across all groups. However, the lowest mean values over the whole treatment period were noted for males and females of group 4 and, therefore, a possible effect on the food intake cannot be excluded.


Body Weights
The body weight gain was statistically significantly reduced in males of group 4 during the whole treatment period with an initial body weight loss after 1 week of treatment when compared with air controls. In addition, statistical significance was obtained also for a reduction in the absolute body weight on single occasions including the end of treatment.

The body weight gain in females of group 4 was slightly reduced compared with air controls without attaining statistical significance (the statistically significantly reduced absolute body weight was already present before treatment start.)

There was no significant difference in body weight development in the animals of groups 2 and 3 compared with the control animals.


Ophthalmoscopic Examinations
There were no ophthalmoscopic findings that were considered to be related to the treatment with the test item.


Clinical Laboratory Investigations
Hematology
Relative and total eosinophil levels were increased in males (attaining statistical significance) and females of group 4 after 13 weeks of treatment with the test item. Neutrophils were also increased in females.


Clinical Biochemistry
Sodium and chloride in the plasma were dose-dependently increased in males of groups 2 to 4 and in females of group 4. Statistical significance was achieved when compared with air controls except for chloride levels in females.

In addition, urea was increased in males and females of group 4 (statistical significance obtained only in males). In males of group 4 only, statistically significantly reduced levels were noted for total cholesterol and phospholipids and increased globulin levels resulted in statistically significantly increased total protein.


Urinalysis
A statistically significant increase in the relative density was measured in the males and females of group 4 along with a decreased volume of urine. Protein contents in the urine of males and females of the same group were also statistically significantly increased and a statistically significantly decreased pH-value was noted in males and statistically significantly elevated ketone concentrations and increased leukocyte counts in females only. Finally, a turbid or cloudy appearance of the urine was observed in the most males and females of group 4 but only in single animals of the other groups.

Some further inter-group differences in hematology, clinical biochemistry and urinalysis parameters occasionally achieved statistical significance but were considered not to be related to treatment with the test item as they were reflecting the historical control data, were not consistently present in both sexes and/or did not show a dose-response.


Pathology
Organ Weights
Statistically significant increased absolute and relative organ weights were recorded for the kidneys in males and for the liver in females of group 4.

Statistically significantly increased organ to body weight ratios were considered to mainly related to the statistically significantly reduced terminal body weight in males and females of group 4. These alterations were considered to be without toxicological relevance (with the exception of the liver in males of group 4) in the absence of corresponding histopathological findings. Some further inter-group variations occasionally achieved statistical significance but showing no dose-relationship. Therefore they are considered not to be test item related.


Macroscopic Findings
Skin sores in the nose region were recorded for males (6/10) and females (3/10) of group 4. All other findings are considered to be incidental findings commonly noted in rats of this strain and age and were therefore considered not to be related to treatment with the test item.


Microscopic Findings
The sores noted at the skin in 6 males and 3 females of group 4 corresponded to chronic inflammation with acanthosis and eschars.

Eosinophilic inclusions in the respiratory/olfactory epithelium at different levels of the nasal cavity were noted in 1 male and 4 females of group 4 and squamous metaplasia at level VI of the larynx was noted in 2 further females of group 4.

Periportal hepatocellular vacuolation of the liver was noted in 3 males and 1 female of group 4. An increased incidence and severity of hyaline droplets/granules were noted in the kidneys of male rats of groups 3 and 4.

There were no further microscopic findings that were considered to be related to the treatment with the test item.

Please see attached summary tables for full results.

Effect levels

Dose descriptor:
NOAEC
Effect level:
0.4 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the irritant effects in group 4, the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 0.400 mg/L air.  

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Wistar rats were exposed by nose-only flow past inhalation for 6 hours/day, 5 days/week over a period of at least 13 weeks. Control animals were exposed to air only. Exposure to gravimetrically determined aerosol concentrations of 0.100, 0.400 and 4.8 mg Benzoic acid isononylester/L air was achieved in the different treatment groups and resulted in no premature deaths. Principal findings were limited to the high dose group.

Clinical signs consisted mainly of hair loss and scabs at the head region of animals which were exposed to high concentrations of the test item along with unkempt fur and chromorhinorrhea recorded during the functional observation. These findings corresponded to the sores at the nose which were noted during necropsy as well as to the chronic inflammation with acanthosis and eschars observed histopathologically. These observations were considered to be a local irritant effect of the test item and additionally resulted in the squamous metaplasia in the larynx at the predilection site with the highest impact of the inhaled air and in the eosinophilic inclusions noted in the respiratory/olfactory epithelium of the nasal cavity. This chronic inflammation was considered to be related with a reduced body weight development and an eventually reduced food intake as well as with increased eosinophil and neutrophil levels in the plasma and increased leukocyte counts in the urine.

The increased incidence and severity of hyaline droplets/granules in the kidneys of male rats were considered to be treatment-related and correlated with increased kidney weights. Hyaline droplets/granules is α2-µ-globulin that appears as phagolysosomes in the proximal tubular epithelium of male rats. The toxicological relevance of an increase of these hyaline droplets/granules is unclear.

The minimal periportal hepatocellular vacuolation along with increased liver weights cannot definitively be excluded as a possible adaptation/tolerance effect to the exposure to the test item as compared to the results of the 2-week study (C47478). This finding was considered to be related with the metabolism of the test item and subsequently with alterations in several clinical biochemistry and urinalysis parameters.

Accordingly, the skin at the region of the local exposure, the upper respiratory tract (nasal cavity and larynx), the kidneys and the liver were considered as target organs.


Based on the irritant effects in group 4, the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 0.400 mg/L air.
Executive summary:

General

 

The purpose of this 13-week inhalation study was to assess the cumulative toxicity of Benzoic acid isononylester when administered 5 days per week to rats by inhalation exposure for a period of at least 13 weeks. The results of this study should indicate potential target organs. This study was designed to provide a rational basis for the assessment of the toxicological risk to man.

 

Groups of 10 male and 10 female Wistar rats each were exposed by nose-only flow-past inhalation to target concentrations of 0.100, 0.400 and 5.0 mg Benzoic acid isononylesther/L air in each of groups 2 to 4, respectively. The rats of the control group were exposed to filtered air only.

 

Mortality, clinical signs, body weights, food consumption, functional observation battery, grip strength, landing foot splay, body temperature, locomotor activity, ophthalmoscopic examinations, clinical laboratory investigations, organ weights, macroscopic and microscopic findings were recorded.

 

 

Technical Data

 

Exposure to gravimetrically determined aerosol concentrations of 0.100, 0.400 and 4.8 mg Benzoic acid isononylester/L air were achieved in groups 2 to 4, respectively, and were close to the respective targets. The generated aerosols were considered to be respirable to rats and temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study.

 

 

Mortality / Viability

 

All animals survived the scheduled treatment period.

 

 

Clinical Signs

 

Treatment-related clinical signs observed during general and detailed observations consisted ofhair loss and scabs at the head region of group 4 animals.

 

 

Functional Observational Battery

 

The main findings of the functional observation battery consisted of chromorhinorrhea,hair loss and unkempt fur observed mainly in the animals of group 4.

Grip Strength, Landing Foot Splay and Body Temperature

 

Mean values of grip strength, landing foot splay and body temperature measurements showed no abnormalities when compared to the control group and demonstrate no test item related effect.

 

 

Locomotor Activity

 

There was noindication of a test item related effect for thelocomotor activity.

 

 

Food Consumption

 

The food consumption was similar across all groups although the lowest mean over treatment values were noted for group 4.

 

 

Body Weights

 

Body weight development was mainly reduced in group 4, mainly in males.

 

 

Ophthalmoscopic Examinations

 

There were no ophthalmoscopic findings that were considered to be related to the treatment with the test item.

 

 

Clinical Laboratory Investigations

 

Hematology

 

Eosinophil and/or neutrophil counts were increased in males and females of group 4.

 

 

Clinical Biochemistry

 

Sodium and chloride were dose-dependently increased in males of groups 2 to 4 and in females of group 4. In addition, urea was increased in males and females of group 4 and reduced levels of total cholesterol and phospholipids and increased globulin and total protein levels were noted in males of group 4.

Urinalysis

 

In both sexes of group 4, an increase in the relative density along with a decreased volume of urine as well as an increased protein content was measured and aturbid or cloudy appearance of the urine was recorded. Further on, a decreased pH-value in males and elevated ketone concentrations and increased leukocyte counts in females was noted.

 

 

Organ Weights

 

A treatment-related increase in the organ weights was recorded for the kidneys in males and for the liver in females of group 4.

 

 

Macroscopic / Microscopic Findings

 

Skin sores in the nose region were recorded for the animals of group 4.

 

The sores noted at the skin in group 4 animals corresponded histopathologically to chronic inflammation with acanthosis and eschars. Eosinophilic inclusions in the respiratory/olfactory epithelium of the nasal cavity and squamous metaplasia in the larynx was noted mainly in females of group 4.

 

Periportal hepatocellular vacuolation of the liver was noted in a few animals of group 4.

 

An increased incidence and severity of hyaline droplets/granules were noted in the kidneys of male rats of groups 3 and 4.

 

There were no further findings that were considered to be related to treatment with the test item.

 

Accordingly, the skin at the region of the local exposure, the upper respiratory tract (nasal cavity and larynx), the kidneys and the liver were considered as target organs.

 

Conclusion

 

Based on the irritant effects in group 4, the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 0.400 mg/L air.