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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 October 2017 to 05 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
OECD Guideline for the Testing of Chemicals No. 403 “Acute Inhalation Toxicity”, adopted: 07 September 2009.
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Version / remarks:
EC 440/2008, Annex Part B, (2008) Method B2
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
US Environmental Protection Agency (EPA) Health Effects Division Test Guidelines, OPPTS 870.1300, Acute Inhalation Toxicity, (August 1998)
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Test type:
traditional method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
EC Number:
201-044-5
EC Name:
2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
Cas Number:
77-62-3
Molecular formula:
C29H40O2
IUPAC Name:
2-{[2-hydroxy-5-methyl-3-(1-methylcyclohexyl)phenyl]methyl}-4-methyl-6-(1-methylcyclohexyl)phenol
Test material form:
solid: particulate/powder
Details on test material:
Name: 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
CAS Number: 77-62-3
Batch/Lot number: WWP4J0001
Appearance: White to cream powder
Purity: Considered as 100% (>95%)
Manufacture date: 04 September 2014
Expiry date: 03 September 2018
Storage conditions: Controlled room temperature (15-25 °C, below 70 RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female Wistar Crl:WI rats (from SPF colony) were obtained from Charles River Laboratories, Research Model and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany).
After arrival, the animals’ health was certified by the resident veterinarian.
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Justification of strain: Recognized by international guidelines as a recommended test system
Animal room: 245/3
Number of animals: 12 (6 animals/sex)
Sex: Male and female, the females were nulliparous and non-pregnant
Age and body weight range of animals on the exposure day (Day 0) were:
Sighting exposure (Group 0.1): approximately 8 weeks old, 335 g (male) and 234 g (female)
Main study (Group 1): approximately 9 weeks old, 365-392 g (males) and 234-249 g (females).

Justification
Rats are the preferred species as they have been used historically for safety evaluation studies and they are specified by the appropriate regulatory authorities. Citoxlab Hungary Ltd. also has considerable experience with this species and historical control data in which to set the findings in context.

Husbandry
Housing: Group caging (5 animals, by sex, per cage), individual caging during the sighting study
Cage type: Polycarbonate solid floor cages (type II or III) with stainless steel mesh lids.
Light: 12 hours of continuous artificial light in each twenty-four hour period (from 6.00 a.m. to 6.00 p.m.)
Temperature: 17.0 – 26.3 °C (245/3)
Relative humidity: 37 – 54 % (245/3)
Ventilation: At least 15 air exchanges per hour.
The temperature and relative humidity were recorded twice daily during the study.

Diet and Water
The animals were provided with ssniff SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, D-59494 Soest Germany) ad libitum, and tap water from the municipal supply, as for human consumption from 500 ml bottles ad libitum.
Water quality control analysis is performed once every 3 months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36, Hungary). The quality control results are retained in the archive of Citoxlab Hungary Ltd.
The food and drinking water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Bedding
Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co. KG (Holzmühle 1, D-73494 Rosenberg, Germany), was used. The quality of the bedding material was guaranteed by the supplier. Nest building material (ARBOCEL crinklets natural (produced by J. Rettenmaier & Söhne GmbH & Co.KG, Germany)) was also added to the cages. Copies of the certificates are retained in the archives of Citoxlab Hungary Ltd.
The bedding and the nest building material were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Acclimation and randomization
Animals were acclimated to laboratory conditions for 5 (sighting group) or 13 days (main study) prior to involvement in the study. Animals were also acclimatised to the test apparatus (restraint procedures) for a short period prior to testing in order to lessen the stress during exposure. Animals were randomly allocated to exposure groups before animal exposure based on the actual body weights.

Identification
Animals were individually identified with numbers written on the tail with an indelible marker. The numbers were given on the basis of Citoxlab Hungary Ltd.'s master file.
Cages were identified by cards holding information on the study code, the sex of animals, the dose group, the cage number and the individual animal number.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: 50% acetone formulation of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
Remarks:
It was not technically feasible to produce an atmosphere without the use of a vehicle; water was not a suitable vehicle.
Mass median aerodynamic diameter (MMAD):
2.89 µm
Geometric standard deviation (GSD):
1.94
Details on inhalation exposure:
Preparation
The test item was not suitable for atmosphere generation in the form supplied, therefore a trial formulation was performed in acetone as the Test Item is not soluble in water. Based on the results of the trial formulation a 50% formulation in acetone (VWR., Batch: 17B284019, Expiry: 28 February 2022) was used in both study groups.

Inhalation Exposure
Technical Trials
Prior to animal exposures, test material atmospheres were generated within the exposure chamber. Due to the physical properties of the test item, it was established that a powder atmosphere was not technically feasible. Further trials were made with formulations in acetone. During these technical trials, air-flow settings, test material input rates and test item formulation of the material were varied to achieve the required aerosol concentration of particles with a mass median aerodynamic diameter (MMAD) between 1 to 4 µm and a geometric standard deviation (GSD) in the range of 1.5 to 3.0. Measurements of aerodynamic particle size were performed from the animal’s breathing zone using a cascade impactor.

Atmosphere generation
The test item formulation was aerosolized using a stainless steel concentric jet nebulizer (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chamber. The rate of test item usage was controlled by a syringe pump.
Compressed air was supplied by means of an oil-free compressor and passed through a suitable filter system prior to introduction to the nebuliser.

Animal exposure system
The animals were exposed, nose-only, to an atmosphere of the test item using a TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany). This system comprises of two concentric anodised aluminium chambers and a computer control system incorporating pressure detectors and mass flow controllers.
The exposure unit was placed in closed hood in order to avoid cross-contamination and contamination of the laboratory environment.
Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where, under positive pressure, it was distributed to the individual exposure ports. The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animal’s nostrils to enter the exposure port. After passing through the animal’s breathing zone, used aerosol entered the outer cylinder from where it was exhausted through a suitable filter system. Atmosphere generation was therefore dynamic.
Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). The flow of air through each port was at least 0.5 L/min. This flow rate was considered adequate to minimize re-breathing of the test atmosphere as it is approximately twice the respiratory minute volume of a rat.
Homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was not specifically determined during this study. However, considerably larger chambers of this design have been fully validated and have shown to produce evenly distributed atmospheres in the animals’ breathing zones.

Sighting Exposure
Sighting exposures were performed in order to estimate the item’s inhalation toxicity, identify sex differences in susceptibility and assist in selecting exposure concentration levels for the main study.

Exposure procedure
Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber. Only the nose of each animal was exposed to the test atmosphere.
Following an equilibration period of at least the theoretical chamber equilibration time (T99), each group of rats was exposed to an atmosphere of the test material for a period of 4 hours.
No control animals were used in the study; the effects of the acetone vehicle are well known.
Analytical verification of test atmosphere concentrations:
not specified
Duration of exposure:
4 h
Concentrations:
Sighting exposure: 1.94 mg/L (maximum attainable)
Main Study: 3.18 mg/L (maximum attainable)
No. of animals per sex per dose:
Sighting exposure: 2 (1 male/1 female)
Mian study: 10 (5 male/5 female)
Control animals:
not specified
Details on study design:
Study design
The animals were exposed to an atmosphere of the test item for a single, continuous four-hour period, generated according to the system and flow rates determined during the technical trials.

Exposure Monitoring
Test atmosphere concentrations
The test atmosphere was sampled at regular intervals during the exposure period. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone) by pulling a suitable volume of test atmosphere through weighed GF10 glass fibre filters (Whatman®, Whatman GmbH, Germany, Ref./Lot no.: G9768136).
The difference in the pre and post sampling (dried) weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.
Filter samples (17) were collected at the breathing zone (approximately every 10-20 minutes) during each 4-hour exposure period and measured.
The nominal concentration was calculated by dividing the mass of test material disseminated into the chamber by the total volume of air that flow through the chamber during the same period.

Particle size analysis
The particle size of the test atmosphere was determined three times during the exposure period using a 7-stage impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles in the discrete aerodynamic size ranges. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone).
The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by this difference.
The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than < 0.550, 0.550, 0.960, 1.550, 2.105, 3.555, 6.655 and 10.550 m was calculated.
From these data, using the software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated. In addition, the proportion (%) of aerosol less than 4 m (considered to be the inhalable portion) was determined.

Chamber environmental conditions
The following variables were monitored continuously and recorded at regular intervals during each exposure period by a validated monitoring system integrated into the exposure system:
-Chamber airflow rates
-Test atmosphere temperature
-Test atmosphere relative humidity
-Test atmosphere carbon dioxide concentration
-Test atmosphere oxygen concentration

Observations
Morbidity/mortality
Animals were checked hourly during exposure, 1 hour after exposure and twice daily (early and late in the working day) during the 14-day observation period for morbidity and/or mortality.

Clinical signs
All animals were observed for clinical signs at hourly intervals during exposure whilst the animals were still restrained. Following exposure, clinical observations were performed twice on the day of exposure (following removal from the restrainer and approximately one hour after completion of the exposure) and subsequently once daily for 14 days.
Observations included changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somato-motor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body weight
Individual body weights were recorded prior to treatment on the day of exposure (on Day 0) and on Days 1, 3, 7 and 14.

Necropsy
At the end of the 14-day observation period, the surviving animals were sacrificed by exsanguination under anaesthesia (intra-peritoneal injection of pentobarbital solution – Euthanimal 40%; Lot No.: 1609291-03; Expiry: October 2019; Produced by Alfasan International B. V., Woerden, Netherlands) and gross macroscopic examination was performed.
All rats were subject to a gross necropsy which included a detailed examination of the abdominal and thoracic cavities. Special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity.
Statistics:
Only a limit test was performed, the four-hour inhalation LC50 was not calculated.

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 3.18 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: maximum achievable amount in air
Mortality:
There was no mortality in the study.
Clinical signs:
other: In each group, red-brown staining or fur staining, by the test item was commonly recorded on the day of the exposure and in the observation period, which were considered to be related to the restraint and exposure procedures but not to be toxicologically
Body weight:
In Group 0.1, slight body weight loss (8.5 and 10.4 %) was recorded on the day of treatment. In the observation period, normal body weight gain was recorded in both animals.
In Group 1, slight body weight loss (3.0 – 11.5 %) was recorded on the day of treatment. In the observation period, normal body weight gain was recorded in the surviving animals from Day 1 (9/10) or Day 3 (1/10).
Gross pathology:
A single four hours nose-only exposure of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] to Wistar Crl:WI rats dosed at 1.94 mg/L in Sighting exposure or 3.18 mg/L in the Main study was not associated with any test item-related gross changes.
Other findings:
No further findings reported.

Any other information on results incl. tables

Mean achieved actual and nominal aerosol concentration

Part of Study

Target Concentration (mg/L)

Mean Achieved Concentration (mg/L)

Standard Deviation of Achieved Concentration (mg/L)

Nominal Concentration (mg/L)

Sighting exposure: Group 0.1

Maximum attainable

1.94

0.22

49.73

Main study: Group 1

Maximum attainable

3.18

0.27

48.15

 

Mean achieved particle size distribution data (MMAD and GSD)

Part of Study

Target Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (MMAD) (μm)

Geometric Standard Deviation (GSD)

Inhalable Fraction

(%<4μm)

Sighting exposure: Group 0.1

Maximum attainable

3.10

1.96

64.7

Main study: Group 1

Maximum attainable

2.89

1.94

68.9

 

Mortality Data

Day Number

Number of Deaths

Group 0.1

(Sighting study)

(1.94 mg/L)

Group 1

(Main study)

(3.148 mg/L)

Male

Female

Male

Female

0

(During Exposure)

0

0

0

0

0

(After Exposure)

0

0

0

0

1

0

0

0

0

2

0

0

0

0

3

0

0

0

0

4

0

0

0

0

5

0

0

0

0

6

0

0

0

0

7

0

0

0

0

8 – 14

0

0

0

0

Total Deaths

0/1

0/1

0/5

0/5

Grand Total Deaths

0/2

0/10

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of the study, no death occurred in a group of 10 Wistar Crl:WI rats (main study) that were exposed to a test atmosphere concentration of 3.18 mg/L for 4 hours. The acute inhalation median lethal concentration (LC50) of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] in Wistar Crl:WI rats was therefore considered to be above 3.18 mg/L (the maximum achievable dose concentration).
Executive summary:

Wistar Crl:WI rats were exposed to a test atmosphere of 50% acetone formulation of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] at the concentration of 3.18 mg/L of test item, which was considered to be the highest attainable dose concentration. It was not technically feasible to produce an atmosphere without the use of a vehicle; water was not a suitable vehicle.

 

The study was performed in multiple steps. A sighting exposure was performed first, where a test atmosphere of 50% acetone formulation of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] at 1.94 mg/L concentration of test item was tested on single animals of both sexes (Group 0.1). As both animals survived the treatment, the main study group (Group 1) of 5 male and 5 female Crl:WI Wistar strain rats were exposed to an aerosol atmosphere of 50% acetone formulation of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] at the concentration of 3.18 mg/L.

 

In all study phases, the animals were exposed to the test atmosphere for 4 hours using a nose-only exposure system. Aerosol concentration was measured gravimetrically 17 times during each 4-hour exposure in both parts of the study and the particle size distribution of the test aerosol was determined 3 times. The day of exposure was designated as Day 0 followed by a 14-day observation period.

 

Clinical observations were performed on all animals during exposure at hourly intervals, following removal from restraint, approximately 1 hour after exposure, and daily for 14 days thereafter, as applicable. Body weight was measured on Days 0 (before the exposure), 1, 3, 7 and 14. Gross necropsy was performed on all animals sacrificed on Day 14.

 

No control group was exposed in this study.

 

Results

The quality of the test atmosphere fully complied with criteria documented in the respective guidelines: OECD 403, EPA OPPTS 870.1300 and Council Regulation (EC) No 440/2008.

 

The mean achieved atmosphere concentrations in the study were 1.94 mg/L in Group 0.1 (sighting study), and 3.18 mg/L in Group 1 (main study). The mass median aerodynamic diameters (MMAD) were 3.10 µm and 2.89 µm with geometric standard deviations (GSD) of 1.96 and 1.94 in the Sighting Group and the Main Group respectively.

 

Mortality

There was no mortality in the study.

 

Clinical observations

In each group, red-brown staining or fur staining, by the test item was commonly recorded on the day of the exposure and in the observation period, which were considered to be related to the restraint and exposure procedures but not to be toxicologically significant. In one male alopecia was observed during the observation period, this was considered non-treatment related.

 

The following observations were considered to be related to exposure to the atmospheres:

Group 0.1 (1.94 mg/L with 1 male + 1 female): Laboured respiration (slight to severe), gasping respiration, incoordination (slight) were observed on the day of the treatment. The animals were symptom free from Day 1.

 

Group 1 (3.18 mg/L with 5 males + 5 females): Laboured respiration (slight to severe), noisy respiration (slight), decreased activity (slight to severe) and prone position were recorded. The animals were symptom free from Day 2.

 

These clinical signs observed in both groups are typical of the transient effects of acetone and are not considered as clearly being effects of the test item.

 

Body weight

In Group 0.1, slight body weight loss (8.5 and 10.4 %) was recorded on the day of treatment. In the observation period, normal body weight gain was recorded in both animals.

In Group 1, slight body weight loss (3.0 – 11.5 %) was recorded on the day of treatment. In the observation period, normal body weight gain was recorded in the animals from Day 1 (9/10) or Day 3 (1/10).

 

Necropsy

A single four hours nose-only exposure of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] to Wistar Crl:WI rats dosed at 1.94 mg/L in Sighting exposure or 3.18 mg/L in the Main study was not associated with any test item-related gross changes.

 

Conclusion

Under the experimental conditions of this study, no death occurred in a group of 10 Wistar Crl:WI rats (main study) that were exposed to a test atmosphere concentration of 3.18 mg/L for 4 hours. The acute inhalation median lethal concentration (LC50) of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] in Wistar Crl:WI rats was therefore considered to be above 3.18 mg/L (the maximum achievable dose concentration).

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