2,4-dinitroanisole

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Study completed February 2015

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Objective of study:

absorption

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

2,4-dinitroanisole

EC Number:

204-310-9

EC Name:

2,4-dinitroanisole

Cas Number:

119-27-7

Molecular formula:

C7H6N2O5

IUPAC Name:

2,4-dinitroanisole

Specific details on test material used for the study:

DNAN (CAS # 119-27-7) was produced by BAE Systems, Ordnance Systems, 4509 West Stone Drive, Kingsport, TN 37660. The certificate of analysis provided by the supplier indicated that the DNAN (lot# BAE10H281-008) was 100 percent (%) pure.

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

young adult male and female Sprague-Dawley rats obtained from Charles River Laboratories, Wilmington, Massachusetts.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Number of rats: 42 (30 of the 42 rats received were assigned to the LC50 test groups while the remaining 12 were used for the range finding blood absorption test. A total of 14 rats, 8 weeks old with a subcutaneous femoral artery catheter in place, were ordered for the multi-time point blood absorption test. Twelve of the 14 rats received were assigned to either inhalation or oral gavage test groups (one additional rat of each sex was ordered to ensure that a sufficient number of rats were available for testing in case there were clogging issues with any of the catheters following shipment). The attending veterinarian examined the animals and found them to be in acceptable health. Due to potential catheter clogging issues, rats received with femoral artery catheters did not have the standard 5-day acclimatization period, and therefore, were used for testing 1-2 days following their arrival to the testing facility.)
- Age at study initiation: 8 weeks
- Weight at study initiation:
- Housing: 17-inch (length) x 9-inch (width) x 8-inch (height) solid bottom polycarbonate boxes with ALPHA-dri® bedding and suspended on a cage rack equipped with anautomatic water-nipple system.
- Diet: ad libitum, except during the 4-hour exposure period
- Water: ad libitum, except during the 4-hour exposure period
- Acclimation period: 5 days
- Identification: Each rat was uniquely identified by number using cage cards. In addition, an animal identification number was recorded on the tail of each rat with a water-insoluble marker prior to exposure so that individual rats could be identified after exposure. (Teklad® Certified Rat Diet is a registered trademark of Harlan, Teklad. ALPHA-dri® is a registered trademark with Shepherd Specialty Papers).

ENVIRONMENTAL CONDITIONS
- Temperature (°): 71 ± 0.6
- Humidity (%): 47 ± 2.1
- Photoperiod (hrs dark / hrs light): 12/ 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The animals were not fasted prior to dosing since the inhalation animals were not fasted prior to exposure. The dosing procedure for each animal was staggered by a period of approximately 4 minutes to accommodate the blood sampling schedule and all dosing was performed using a 16 gauge x 2-inch stainless steel gavage needle. A 16 milligram per milliliter (mg/mL) suspension of DNAN in corn oil was used for oral gavage and dosage volumes ranged from 7.0 – 9.8 mL of suspension per kg bodyweight.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

7 mg/kg bw/day (nominal)

Remarks:

min

Doses / concentrations 2

Dose / conc.:

9.8 mg/kg bw/day (nominal)

Remarks:

max

No. of animals per sex per dose / concentration:

3

Control animals:

not specified

Details on study design:

The concentration of DNAN absorbed into the bloodstream, as well as its conversion to DNP, of exposed rats via nose-only inhalation or oral gavage was determined from blood samples collected from each surviving rat at seven selected time points for the inhalation portion and six selected time points for the oral gavage portion. All blood samples were preserved at the time of collection by immediately injecting the 100 microliter aliquot of the blood sample into 1 mL of water. Samples were tightly capped and refrigerated at approximately 4°C immediately after collection until the time of delivery for analysis.

Statistics:

Into the blood of rats exposed via inhalation to that of rats given an equivalent oral dose. In addition the concentration of DNP, a known metabolite of DNAN, was also monitored in the blood of the exposed rats. On the day following the conclusion of the inhalation portion of the test, an average chamber concentration was calculated for the 4-hour exposure using the gravimetric filter samples collected during the exposure. The following formula was employed to estimate an equivalent oral dose for the animals to be gavaged (Rusch, 2009):
Oral Dose (mg/kg) = α * exposure level * minute volume * exposure length/body weight
where:
α = amount retained in respiratory system (assume 0.9 or 90 percent in the absence of additional information)
exposure level = average concentration of inhalation exposure (mg/m3)
minute volume = amount of air inhaled per minute (approx. 0.00016 m3/min for rats)
exposure length = total minutes of inhalation exposure
body weight = animal weight (kg)
All calculations were performed using body weights obtained within one hour of initiating the dosing procedure.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

6.4 ± 1.85 micrograms per milliliter (μg/mL) following 1.1 hours of exposure, INHALATION IN MALE

Main ADME results 2

Type:

absorption

Results:

13.5 ± 5.46 μg/mL following 2.0 to 2.1 hours of exposure, INHALATION IN MALE

Main ADME results 3

Type:

absorption

Results:

16.7 ± 6.11 μg/mL following 4.2 to 4.3 hours of exposure, INHALATION IN MALE

Main ADME results 4

Type:

absorption

Results:

4.3 ± 0.42 μg/mL at 4.1 hours post-exposure, INHALATION IN MALE

Main ADME results 5

Type:

absorption

Results:

2.4 ± 2.44 μg/mL at 8.0 to 8.1 hours post-exposure, INHALATION IN MALE

Main ADME results 6

Type:

absorption

Results:

0.1 ± 0.19 μg/mL at 23.3 to 23.5 hours post-exposure, INHALATION IN MALE

Main ADME results 7

Type:

absorption

Results:

25.3 ± 19.66 μg/mL (n=3) at 1.0 to 1.1 hours post-exposure, ORAL IN MALE

Main ADME results 8

Type:

absorption

Results:

17.0 ± 14.82 μg/mL (n=3) at 2.0 to 2.1 hours postexposure, ORAL IN MALE

Main ADME results 9

Type:

absorption

Results:

16.3 ± 15.44 μg/mL (n=3) at 4.1 to 4.2 hours post-exposure, ORAL IN MALE

Main ADME results 10

Type:

absorption

Results:

9.9 ± 8.63 μg/mL (n=2) at 8.1 hours post-exposure, ORAL IN MALE

Main ADME results 11

Type:

absorption

Results:

0.3 ± 0.06 μg/mL (n=2) at 24.6 hours post-exposure, ORAL IN MALE

Main ADME results 12

Type:

absorption

Results:

8.2 ± 1.65 μg/mL following 1.0 to 1.1 hours of exposure, INHALATION IN FEMALE

Main ADME results 13

Type:

absorption

Results:

13.0 ± 2.65 μg/mL following 2.0 to 2.1 hours of exposure, INHALATION IN FEMALE

Main ADME results 14

Type:

absorption

Results:

12.5 ± 4.80 μg/mL following 4.2 hours of exposure, INHALATION IN FEMALE

Main ADME results 15

Type:

absorption

Results:

4.3 ± 3.01 μg/mL at 4.0 to 4.2 hours post-exposure, INHALATION IN FEMALE

Main ADME results 16

Type:

absorption

Results:

1.6 ± 1.17 μg/mL at 8.0 to 8.1 hours post-exposure, INHALATION IN FEMALE

Main ADME results 17

Type:

absorption

Results:

0.9 ± 1.01 μg/mL at 23.3 to 23.4 hours post-exposure, INHALATION IN FEMALE

Main ADME results 18

Type:

absorption

Results:

36.0 ± 3.00 μg/mL (n=3) at 1.0 to 1.1 hours post-exposure, ORAL IN FEMALE

Main ADME results 19

Type:

absorption

Results:

32.3 ± 5.69 μg/mL (n=3) at 2.0 to 2.1 hours post-exposure ORAL IN FEMALE

Main ADME results 20

Type:

absorption

Results:

48.0 ± 9.54 μg/mL (n=3) at 4.0 to 4.1 hours post-exposure, ORAL IN FEMALE

Main ADME results 21

Type:

absorption

Results:

45.0 ± 0.00 μg/mL (n=1) at 7.4 hours postexposure, ORAL IN FEMALE

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Inhalation
The mean whole blood concentration of DNAN in male rats exposed via inhalation (n=3) was 6.4 ± 1.85 micrograms per milliliter (μg/mL) following 1.1 hours of exposure, 13.5 ± 5.46 μg/mL following 2.0 to 2.1 hours of exposure, 16.7 ± 6.11 μg/mL following 4.2 to 4.3 hours of exposure, 4.3 ± 0.42 μg/mL at 4.1 hours post-exposure, 2.4 ± 2.44 μg/mL at 8.0 to 8.1 hours post-exposure, and 0.1 ± 0.19 μg/mL at 23.3 to 23.5 hours post-exposure. The mean whole blood concentration of DNAN in female rats exposed via inhalation (n=3) was 8.2 ± 1.65 μg/mL following 1.0 to 1.1 hours of exposure, 13.0 ± 2.65 μg/mL following 2.0 to 2.1 hours of exposure, 12.5 ± 4.80 μg/mL following 4.2 hours of exposure, 4.3 ± 3.01 μg/mL at 4.0 to 4.2 hours post-exposure, 1.6 ± 1.17 μg/mL at 8.0 to 8.1 hours post-exposure, and 0.9 ± 1.01 μg/mL at 23.3 to 23.4 hours post-exposure. DNAN was not detected in the pre-exposure (baseline) male and female blood samples taken approximately 2 hours prior to exposure. The mean whole blood concentration of DNP in male rats exposed via inhalation (n=2) was 0.0 ± 0.00 μg/mL following 1.1 hours of exposure, 26.0 ± 4.24 μg/mL following 2.0 to 2.1 hours of exposure, 44.0 ± 5.66 μg/mL following 4.2 to 4.3 hours of exposure, 51.0 ± 7.07 μg/mL at 4.1 hours post-exposure, 43.5 ± 10.61 μg/mL at 8.0 to 8.1 hours post-exposure, and 14.5 ± 2.12 μg/mL at 23.3 to 23.5 hours post-exposure. The mean whole blood concentration of DNP in female rats exposed via inhalation (n=3) was 29.0 ± 8.54 μg/mL following 1.0 to 1.1 hours of exposure, 59.0 ± 11.53 μg/mL following 2.0 to 2.1 hours of exposure, 71.3 ± 9.50 μg/mL following 4.2 hours of exposure, 70.7 ± 17.01 μg/mL at 4.0 to 4.2 hours post-exposure, 56.3 ± 13.58 μg/mL at 8.0 to 8.1 hours post-exposure, and 29.3 ± 4.62 μg/mL at 23.3 to 23.4 hours post-exposure. With the exception of one male rat excluded from analysis, DNP was not detected in the preexposure (baseline) male and female blood samples taken approximately 2 hours prior to exposure.

Oral
The mean whole blood concentration of DNAN in orally-gavaged male rats was 25.3 ± 19.66 μg/mL (n=3) at 1.0 to 1.1 hours post-exposure, 17.0 ± 14.82 μg/mL (n=3) at 2.0 to 2.1 hours postexposure, 16.3 ± 15.44 μg/mL (n=3) at 4.1 to 4.2 hours post-exposure, 9.9 ± 8.63 μg/mL (n=2) at 8.1 hours post-exposure, and 0.3 ± 0.06 μg/mL (n=2) at 24.6 hours post-exposure. The mean whole blood concentration of DNAN in orally-gavaged female rats was 36.0 ± 3.00 μg/mL (n=3) at 1.0 to 1.1 hours post-exposure, 32.3 ± 5.69 μg/mL (n=3) at 2.0 to 2.1 hours post-exposure, 48.0 ± 9.54 μg/mL (n=3) at 4.0 to 4.1 hours post-exposure, and 45.0 ± 0.00 μg/mL (n=1) at 7.4 hours postexposure. All orally-gavaged female rats died prior to the 24-hour post-exposure blood sample. DNAN was not detected in the pre-exposure (baseline) male and female blood samples taken several minutes prior to dosing. The mean whole blood concentration of 2,4-D initrophenol (DNP) in orally-gavaged male rats was 31.0 ± 2.65 μg/mL (n=3) at 1.0 to 1.1 hours post-exposure, 47.3 ± 13.58 μg/mL (n=3) at 2.0 to 2.1 hours postexposure, 51.3 ± 13.80 μg/mL (n=3) at 4.1 to 4.2 hours post-exposure, 50.0 ± 4.24 μg/mL (n=2) at 8.1 hours post-exposure, and 19.5 ± 3.54 μg/mL (n=2) at 24.6 hours post-exposure. The mean whole blood concentration of DNP in orally-gavaged female rats was 33.7 ± 5.69 μg/mL (n=3) at 1.0 to 1.1 hours post-exposure, 50.0 ± 8.72 μg/mL (n=3) at 2.0 to 2.1 hours post-exposure, 58.0 ± 6.00 μg/mL (n=3) at 4 to 4.1 hours post-exposure, and 54.0 ± 0.00 μg/mL (n=1) at 7.4 hours postexposure. All orally-gavaged female rats died prior to the 24-hour post-exposure blood sample. DNP was not detected in the pre-exposure (baseline) male and female blood samples taken several minutes prior to dosing.

Details on distribution in tissues:

Not specified

Details on excretion:

Not specified

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Plesae see Details on absorption

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

Not specified

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:

The results of the multi-time point blood absorption portion indicated that, under the stated study conditions and limitations, acute exposure to DNAN via oral gavage appears to induce higher DNAN whole blood concentrations in laboratory rats compared to those exposed via inhalation.
Blood concentrations of the metabolite DNP were not different between the oral and inhalation routes of exposure. Female rats appear to convert a greater proportion of DNAN to DNP than male rats when exposed via inhalation while male rats convert a greater proportion of DNAN to DNP than female rats when exposed orally.

Executive summary:

1.1 Purpose

This study was conducted to determine the 4-hour, inhalation median lethal concentration (LC50) of 2,4-Dinitroanisole (DNAN) in male and female rats. The LC₅₀ is defined as the calculated atmospheric concentration of test substance expected to cause the death of 50 percent of exposed animals either on the day of exposure or within at least 14 days post exposure. In the event that no deaths occur among rats exposed to the highest-obtainable concentration, the LC₅₀ is considered greater than the given concentration and no further testing is required. A secondary objective was to determine the effect that two different routes of administration (inhalation and oral) had on the absorption of the chemical into the bloodstream. Blood samples were collected from exposed rats at seven different time points for rats exposed via inhalation and at six different time points for rats exposed via oral gavage to measure the absorption of DNAN, as well as its metabolite 2,4 -Dinitrophenol (DNP), into the blood and determine the degree of effect that the exposure route had on the absorption of DNAN into the blood. In addition to blood absorption, rats were also evaluated for body weight changes and clinical observations.

1.2 Conclusions

Rats were exposed nose-only to a 2.4 mg/L aerosol atmosphere of DNAN for a single 4-hour exposure. No test compound-related mortalities occurred in rats exposed during the study and no adverse toxic signs, body weight changes, or gross necropsy findings were observed in exposed rats. The LC₅₀ portion of this study indicates that acute inhalation exposure to the highest achievable concentration of DNAN aerosol (2.4 mg/L) is relatively nontoxic to rats. The results of the multi-time point blood absorption portion indicated that, under the stated study conditions and limitations, acute exposure to DNAN via oral gavage appears to induce higher DNAN whole blood concentrations in laboratory rats compared to those exposed via inhalation. Blood concentrations of the metabolite DNP were not different between the oral and inhalation routes of exposure. Female rats appear to convert a greater proportion of DNAN to DNP than male rats when exposed via inhalation while male rats convert a greater proportion of DNAN to DNP than female rats when exposed orally.