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Administrative data

Description of key information

Inhalation: OECD 422; rats. NOAEC = 50000 ppm (282233 mg/m3).  No test substance-related adverse effects were observed on any neurobehavioural parameter examined at the highest concentration tested. Reliability = 1.

Key value for chemical safety assessment

Effect on neurotoxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: sub-chronic inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422
Deviations:
yes
Remarks:
(Study included FOB and motor activity endpoints)
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals Section 4 (Part 412)
Deviations:
yes
Remarks:
(Study included FOB and motor activity endpoints)
Qualifier:
according to
Guideline:
other: U.S. EPA Health Effects Test Guidelines OPPTS 870.3650
Deviations:
yes
Remarks:
(Study included FOB and motor activity endpoints)
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 70 days
- Weight at study initiation: 317-445 grams (males) and 201-269 grams (females)
- Fasting period before study: No
- Housing: Non-Exposure Periods: Individually in solid bottom caging with bedding, and enrichment as appropriate; sexes on separate racks except during cohabitation, gestation/lactation as described below.
Cohabitation: Mating pairs (females placed in males’ cages) on a 1:1 basis in solid bottom caging with bedding and enrichment. Fourteen days after the first day of cohabitation, females without evidence of copulation were housed singly.
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except when fasted
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30%–70%
- Air changes (per hr): 10-13
- Photoperiod (hrs dark / hrs light): 12-hours light/12 hours dark
Route of administration:
inhalation: gas
Vehicle:
other: filtered and conditioned air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: A stainless steel baffle below the chamber turret inside the chambers promoted uniform chamber distribution of the test atmosphere
Exposure #s 1 to 10, stainless steel and glass (NYU style) with a nominal internal volume of 350 L
Exposure #s 11 to 71, control animals were exposed to air in the same chamber while test substance exposures were performed in stainless steel and glass (NYU style) chamber with a nominal internal volume of 150 L
- Method of holding animals in test chamber: individually placed in stainless steel, wire-mesh modules and exposed whole-body in exposure chamber
- Source and rate of air: filtered and conditioned air and oxygen
- System of generating particulates/aerosols: test substance vapour and supplemental oxygen were metered into a 1-liter 3-neck mixing flask by mass flow controllers. The mixture left the mixing flask and entered the glass transfer tube where chamber air supply was added to the mixture by mass flow controllers. The gas mixture then entered the top of the exposure chamber through a turret. The air-control atmosphere was similarly generated in a different room without test substance.
- Temperature, humidity, pressure in air chamber: 21-25ºC, 45-67%, and 21% oxygen, no data on pressure
- Air flow rate: Exposure #s 1 to 10 - chamber flow 60-64 L/min; Exposure #s 11 to 20 - chamber flow 29-33 L/min
- Air change rate: 10-13 per hour
- Treatment of exhaust air: exhausted from the bottom of each chamber through MSA filters using vacuum pumps and discharged into the fume hood


TEST ATMOSPHERE
- Brief description of analytical method used: vapour concentration was directly injected into a gas chromatograph
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each exposure, the vapour concentration was determined by gas chromatography at least once an hour from the test chambers. The control chamber was sampled at least twice a day. Samples of volumes of chamber atmosphere were directly injected into a gas chromatograph (GC) equipped with an automated gas sample valve and a flame ionization detector. All samples were chromatographed isothermally at 80ºC on a 30 m x 0.53 mm (nominal diameter) RTX-502.2 fused silica glass column. The vapour concentration was determined from a standard curve derived from gas standards.
Duration of treatment / exposure:
An abbreviated neurobehavioural evaluation was conducted on males and females prior to test substance administration in order to obtain baseline measurements. This neurobehavioural evaluation was conducted again following approximately 4 weeks of exposure for one subset of animals and following approximately 2 weeks of exposure, 2 weeks of non-exposure, and 2 weeks of exposure for another subset of animals.
Frequency of treatment:
6 hr/day, 5 day/wk
Remarks:
Doses / Concentrations:
0, 2500, 10000, and 50000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.0 ± 0.0; 2500 ± 11; 10000 ± 74; and 51000 ± 650 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
5 animals/sex/group of the Subchronic with Recovery Subset
6 animals/sex/group in the Reproduction Subset
Control animals:
yes, concurrent vehicle
Observations and clinical examinations performed and frequency:
For additional details regarding repeated dose, reproductive, or developmental toxicity refer to IUCLID sections 7.5.2 (DI.K1. 28Day.InhGas.RD/Repro/Dev.R.D-19695-1422.KD), 7.8.1 (DI.K1.1Gen.Screen.RD/Repro/Dev.R.D-19695-1422.KD), or 7.8.2 (DI.K1.Dev.Screen.RD/Repro/Dev.R.D-19695-1422.KD), respectively.
Neurobehavioural examinations performed and frequency:
Neurobehavioural evaluation, consisting of abbreviated functional observational battery (FOB) assessments and motor activity (MA), was conducted during acclimation (baseline) on 6 animals/sex/group in the Reproduction Subset and 5 animals/sex/group of the Subchronic with Recovery Subset. After approximately 3 weeks of exposure, FOB and MA assessments were conducted on the 5 animals/sex/group in the Subchronic with Recovery Subset. Based on the results of the evaluations conducted on the Recovery and Reproduction subsets, neurobehavioural evaluations were not necessary for animals in the recovery subset. On approximately test days 44 and 45, FOB and MA assessments were conducted on the 6 animals/sex/group in the Reproduction Subset. For the baseline evaluation, FOB and MA assessments were also conducted on all spare animals in the event that replacements were needed during acclimation or on test day 0.

Animals were counterbalanced by sex and treatment and tested in replicates over multiple days to minimize the influence of uncontrolled factors. The testing sequence and replicate designations are kept in the study records and are not included in the final report. The experimenter conducting the FOB was blind with respect to the group designation of the animal. To the extent possible, the testing was performed by the same person each time. FOB and MA evaluations were conducted in a sound-attenuated room equipped with a white-noise-generation system to minimize variations in environmental test conditions. Animals were acclimated for at least 10 minutes in the FOB laboratory prior to initiation of evaluation. Body weights were collected on the day of the FOB assessment but were not compared statistically.

1. The following were measured for each category: Abbreviated Functional Observational Battery; Chatillon® Digital Force gauge and at the end of the MA session (See Table 1).

2. Motor Activity
Animals were individually tested in one of 32 identical, automated activity monitors (Kinder Scientific). Each monitor measures movement by detecting the interruption of photobeams by the animal. The monitoring device enabled the calculation of two dependent variables: duration of movements and number of ambulatory movements. All movements counted towards the duration of movements, regardless of the position of the animal. A movement counted as an ambulatory movement only if the position of the animal changed along the X or Y axis. Each test session was 60 minutes in duration, with results expressed for the total session as well as for 6 successive 10 minute intervals.

3. Test Facility Positive Control Data
Procedures and data describing the effects of multiple positive control substances are available in 4 separate reports. These positive control studies are the basis of training certification for the personnel making judgments in the neurobehavioral and neuropathology tests. The data also document that the equipment and procedures are capable of detecting effects that may be seen in neurotoxicity studies of this type.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Behaviour (functional findings):
no effects observed
Details on results:
Forelimb and Hindlimb Grip Strength
No test substance-related or statistically significant effects occurred on forelimb or hindlimb grip strength for males or females exposed to any concentration of the test substance.

Open Field Observations
No test substance-related or statistically significant effects were observed for any behavioural parameter evaluated in males or females exposed to any concentration of the test substance. The incidences for all groups were similar to the values for the control groups.

Motor Activity
No test substance-related or statistically significant effects were observed on motor activity for males or females exposed to any concentration of the test substance.

At week 4, the mean duration and number of movements for 50000 ppm females during the 2nd–6th intervals as well as for the total session were decreased (not statistically significant) compared to control. However, these decreases, although they showed a trend, were not considered to be adverse because the decreases were not observed in males, were not statistically significant, and were within historical control (range of 1412-1757 seconds), and there were no effects on number of movements in 50000 ppm females. Mean values for duration and number of movements for the 2500 and 10000 ppm males and females and 50000 ppm males were similar to their respective controls.

OTHER:
For additional details on the systemic, repeat dose endpoints, refer to Section 7.5.2 Repeat Dose: DI.K1.28Day.InhGas.RD/REPRO/DEV.R.D-19695-1422.KD
For additional details on the Micronucleus genetic toxicity endpoints, refer to Section 7.6.2 Genetic Toxicity – In Vivo: DI.K1.MicroNuc.R.D-19695-1422.KD
For additional details regarding Reproductive/Developmental endpoints, refer to Section 7.8.1 (DI.K1.1Gen.RD/REPRO/DEV.R.D-19695-1422.KD) and 7.8.2 (DI.K1.DEV.RD/REPRO/DEV.R.D-19695-1422.KD)
Dose descriptor:
NOAEC
Effect level:
50 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: based on the absence of effects in all neurotoxicity endpoints
Remarks on result:
other:
Conclusions:
Under the conditions of the study, no test substance-related or adverse effects were observed on neurobehavioural parameters for the subchronic with recovery subset.
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

The purpose of this study was to determine the potential subchronic and reproductive toxicity from repeated inhalation exposure of male and female rats to 0, 2500, 10000, or 50000 ppm of the test substance.  

Groups of 20 young adult male or nulliparous, non-pregnant female Crl:CD(SD) rats were divided into the following subsets: Subchronic with Recovery (including a neurobehavioural evaluation), and Reproduction (including a neurobehavioural evaluation). The Subchronic and Subchronic with Recovery Subsets were exposed to the test substance 6 hours per day, 5 days per week over a one-month period. The Reproduction Subset was exposed during an exposure period of approximately 2 weeks during which the animals were exposed 6 hours per day, 5 days per week in conjunction with the Subchronic and Subchronic Recovery Subsets. Due to a technical issue, exposures for the Reproduction Subset were suspended for 2 weeks. Exposures for animals in the Reproduction Subset were resumed on test day 29. Following resumption of exposures, animals in the Reproduction Subset were exposed for 6 hours per day, 5 days per week for approximately a 2-week premating period, followed by the cohabitation period of approximately 2 weeks during which exposures were conducted 6 hours per day, 7 days per week. This exposure schedule continued during a gestation period of approximately 3 weeks. Gestating dams were not exposed after gestation day 19 or during the approximately 4 day lactation period. Females without evidence of mating continued to be exposed for 24 days after the end of the cohabitation period. Exposures for parental males continued until the day prior to euthanasia on test days 77-78. 

 

Chamber atmospheres of test substance were generated by dilution of the test substance vapour and supplemental oxygen to achieve an oxygen concentration of 21% in the high level chamber. An air control group was also evaluated using a similar generation method. Vapour concentrations of the test substance were measured by gas chromatography (GC). Temperature, humidity, and airflow were also recorded periodically during each exposure day. 

An abbreviated neurobehavioural evaluation was conducted on the 5 males and 5 females assigned to the Subchronic with Recovery Subset and on 6 male and 6 female rats assigned to the Reproduction Subset prior to test substance administration in order to obtain baseline measurements. This neurobehavioural evaluation was conducted again following approximately 4 weeks of exposure for the animals in the Subchronic with Recovery Subset and near the end of the pre-mating period for the animals in the Reproduction Subset.

 

No test substance-related or adverse effects were observed neurobehavioural parameters.

Under the conditions of the study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity in males and females was 50000 ppm, the highest concentration tested, based on the absence of effects in all endpoints. The NOAEL for systemic, reproductive, neurobehavioural, and genetic toxicity effects was 50000 ppm based on the absence of effects on reproductive endpoints and on offspring at the highest concentration tested.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
282 233 mg/m³
Study duration:
subchronic
Species:
rat

Additional information

Male and female rats were exposed whole body to 0, 2500, 10000, or 50000 ppm of the test substance for 6 hours per day, 5 days per week in an OECD 412 and OECD 422 28-Day Inhalation with One-Generation Reproduction Repeat Dose Study. An abbreviated neurobehavioural evaluation consisting of a functional observational battery and motor activity was conducted in rats (11/sex/group) once during pre-test and again after approximately 3 weeks of exposure. No test substance-related adverse effects were observed on neurobehavioural parameters including forelimb and hindlimb grip strength, open field observations, or motor activity. The NOAEC was 50000 ppm based on the absence of neurobehavioural effects at the highest concentration tested.

Justification for classification or non-classification

The test substance did not adversely affect neurobehavioural function in a combined repeated dose toxicity study with reproductive/developmental toxicity screening test in rats. Therefore, the substance does not need to be classified for neurological effects according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.