Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August 2016 to 23 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects. No.403, "Acute Inhalation Toxicity", September 2009.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Version / remarks:

Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.2. AcuteToxicity (inhalation). Official Journal of the European Communities No. L142, May 2008, including most recent amendments.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

EPA Health Effects Test Guidelines OPPTS 870.1300, Acute inhalation Toxicity. EPA 712-C-98-193, August 1998.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), Notification No 8147.

Version / remarks:

Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000; including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes

Test type:

traditional method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Formaldehyde, oligomeric reaction products with acetone and diphenylamine

EC Number:

500-011-5

EC Name:

Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Cas Number:

9003-80-9

Molecular formula:

UVCB substance - not applicable

IUPAC Name:

N-phenylaniline; formaldehyde; propan-2-one

Test material form:

solid: flakes

Details on test material:

Identification: Formaldehyde, oligomeric reaction products with acetone and diphenylamine
Appearance: Dark brown flakes
Batch: IC5B04P006
Purity/Composition: 100% Unknown or Variable compositions, Complex reaction products and Biological materials
Test substance storage: At room temperature
Stable under storage conditions until: 26 February 2019 (expiry date)
Purity/composition correction factor: No correction factor requiredTest substance handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Formaldehyde, oligomeric reaction products with acetone and diphenylamine (BXA)CAS Number: 9003-80-9

Specific details on test material used for the study:

Test item: 206534/B
Identification: Formaldehyde, oligomeric reaction products with acetone and diphenylamine
Appearance: Dark brown flakes
Batch: IC6D11P001
Purity/Composition: 100% Unknown or Variable compositions, Complex reaction products and Biological materials (UVCB)
Test item storage: At room temperature
Stable under storage conditions until: 27 April 2020 (expiry date)
Chemical name (IUPAC), synonym or trade name: Formaldehyde, oligomeric reaction products with acetone and diphenylamine (BXA)
CAS Number: 9003-80-9

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Rat: Crl:WI(Han) (outbred, SPF-Quality). Recognised by international guidelines as the recommended test system (e.g. OECD, EC). Source: Charles River Deutschland, Sulzfeld, Germany.
Number of animals: 5 males and 5 females (females were nulliparous and nonpregnant) per exposure level. Two exposure levels
Age and body weight: Young adult animals were selected (approximately 10-11 weeks old). Animals used within the study were of approximately the same age and body weight variation did not exceed +/- 20% of the sex mean.
Identification: Individual unique number by tattoo on hind leg.
Health inspection: At least prior to exposure. It was ensured that the animals were healthy and without any abnormality that might affect the study integrity.

Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.
Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation
Group housing of five animals per sex per cage in labelled Makrolon cages (type IV; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS -J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cageenrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) except during exposure to the test item.
Water
Free access to tap water except during exposure to the test item.

Animal husbandry on the day of exposure
The animals were moved to the inhalation area to in order to perform the exposure. During the exposure, there was no access to food and water. After exposure, the animals were returned to their cages which were placed in a fume cupboard for a short time period to allow test item remnants to evaporate. A sheet of filter paper was used to cover the bedding material to prevent suffocation in case of bad health condition and in order to recover and to aid the clinical observations. The sheet was removed and before the end of the exposure day, the surviving animals were returned to the animal room.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 3.9 - <= 4.3 µm

Geometric standard deviation (GSD):

>= 1.9 - <= 2.1

Remark on MMAD/GSD:

The particle size distribution was characterized twice during each exposure period. The samples were drawn (2 L/min) from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor containing fiber glass filters (TE-290-GF. Tisch Environmental, Cleves, Ohio, USA) and a fiber glass back-up filter (SEC-290-F1, Westech, Upper Stondon, Bedfordshire, England). Amounts of test item collected were measured gravimetrically. Subsequently the Mass Median Aerodynamic Diameter (MMAD) and the Geometric Standard Deviation (GSD) were determined.

Details on inhalation exposure:

Exposure Chamber
The design of the exposure chamber is based on the flow past nose-only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983). The chamber (volume approximately 150 mL) consisted of three animal sections with eight animal ports each.
Each animal port had its own atmosphere inlet and exhaust outlet. The animals were placed in restraining tubes and connected to the animal ports. The number of animal sections and number of open inlets were adapted to the air flow in such a way that at each animal port the theoretical air flow was at least 1 L/min, which ensures an adequate oxygen supply to the animals. The main inlet of the test atmosphere was located at the top section and the main outlet was located at the bottom section. The direction of the flow of the test atmosphere guaranteed a freshly generated atmosphere for each individual animal.
All components of the exposure chamber in contact with the test material were made of stainless steel, glass, rubber or plastic. To avoid exposure of the personnel and contamination of the laboratory the exposure chamber was placed in a fume hood, which maintained at a slight negative pressure.

Test Atmosphere Generation
The test atmosphere generation was based on the method developed during extensive trial generations.
Administering the test item to a stream of pressurized air using a combination of a brush feeder and micronizing jet mill generated (Bernstein, D.N., Aerosols, pp 721- 723, 1984) generated a dust. The dust was passed through a series of three cyclones, allowing larger particles to settle, and diluted with pressurized air before it entered the exposure chamber. For the exposure at 1 mg/L, the mean total air flow was 23 L/min. and at 5 mg/L the mean total airflow was 20 L/min.
From the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.

Test Atmosphere Characterization
Nominal Concentration
The nominal concentration was calculated by dividing the amount of test item used by the volume of pressurized air (average air flow times exposure time) entering the exposure chamber used for exposure of the animals. Due to the small volume of the exposure chamber the equilibrium time was negligible. The volume of air was calculated from the average air flow (measured by means of thermal mass flow meters and was recorded regularly, preferably in 30 minute intervals) and the exposure time.

Actual Concentration
A total of 21 and 28 representative samples were taken for determination of the actual concentrations during exposure at 5 and 1 mg/L, respectively. Samples were drawn from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. Samples were drawn through a glass fiber filter (type APFC04700, Millipore, Billerica, MA, USA). The collected amount of the test item in the air sample was measured gravimetrically. Sample volumes were measured by means of a dry gas meter (type G 1.6, Actaris Meterfabriek B.V., Dordrecht, The Netherlands). Subsequently the timeweighted mean concentrations with the standard deviations were calculated.

Analytical verification of test atmosphere concentrations:

no

Duration of exposure:

4 h

Concentrations:

The study was performed following a stepwise exposure scenario. Target concentrations were based on the cut off concentration values specified in the UN and EC classification guidelines for dusts and mists (0.05, 0.5, 1 or 5 mg/L.).

No. of animals per sex per dose:

Toxicity data of the test item indicated that severe effects may be expected at the highest target concentration as indicated above. Therefore, as a first step the study was initiated with the exposure of five animals of each sex to 1 mg/L for 4 hours. Based on the results, five animals of each sex were exposed to 5 mg/L

Control animals:

no

Details on study design:

Stability Monitoring
It was considered that the opacity of the test atmosphere could not be reliably monitored by means of an aerosol monitoring system. An indication of the stability of the test atmosphere was obtained from the concentration measurements, which were equally distributed over time.

Temperature and Relative Humidity
The temperature and relative humidity were measured with a humidity and temperature indicator (E+E Elektronik, Engerwitzdorf, Austria) and were recorded after the animals were placed in the experimental set-up and at 30 minute intervals after initiation of the exposure.
The probe was inserted in a tube mounted in one of the free animal ports of the middle section of the exposure chamber.
The temperature of the atmosphere during the exposures were between 21.1 and 22.8°C and relative humidity was between 16 and 39%. These conditions were considered appropriate for this relatively short 4 hours exposure duration.

Treatment
Prior to each exposure, both eyes of each rat were instilled with Opthosan (AST Farma BV, Oudewater, The Netherlands) to protect the eyes against potential irritation by the test item.
Prior to exposure the animals were restrained in polycarbonate restraining tubes; these tubes were connected to the exposure chamber. Sixteen or eighteen minutes after the last animal was placed the generation of the test atmosphere was started. The exposure time was 4 hours.

Observations
Mortality/Viability: Twice daily.
Clinical signs: During exposure
Three times during exposure for mortality, behavioural signs of distress and effects on respiration.
Clinical signs: After exposure
On Day 1, one and three hours after exposure and once daily thereafter until Day 15. The clinical signs were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 4: grading slight (1) to very severe (4)
Maximum grade 3: grading slight (1) to severe (3)
Maximum grade 1: presence is scored (1).
Body weights: Days 1 (pre-administration), 2, 4, 8 and 15.
Necropsy: All animals were sacrificed at the end of the observation period by an intraperitoneal injection with Euthasol® (AST Farma BV, Oudewater, The Netherlands). All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded. Particular attention was given to any changes in the respiratory tract.

Statistics:

No statistical analysis was performed.

Results and discussion

Preliminary study:

For the 1 mg/L exposure group, the time-weighted mean actual concentration was 1.1 ± 0.03 mg/L.

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 mg/L air (nominal)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality occurred.

Clinical signs:

other: At 1 mg/L, quick breathing was seen during exposure. After exposure, one male showed hunched posture on Day 1. Brown staining by test item remnants of the skin and fur was seen between Days 1 and 6. At 5 mg/L, quick breathing was seen during exposure. Aft

Body weight:

Overall body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity.

Gross pathology:

Macroscopic post mortem examination revealed several purple foci on the left side of the thymus of one female exposed to 1 mg/L. Macroscopic examination of the other animals did not reveal further abnormalities.

Other findings:

Concentration
For the 1 mg/L exposure group, the time-weighted mean actual concentration was 1.1 ± 0.03 mg/L. The nominal concentration (amount of test item used divided by the volume of pressurized air used) was 9.7 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 11%.
The concentration was measured at time points (n=28) that were equally distributed over the exposure period, the results of which demonstrated that it was difficult to maintain the test item stable. The generation was interrupted on two occasions in order to make adjustments to the system. To compensate for these interruptions, the generation time was elongated by 9 minutes in order to achieve an actual exposure time of 240 minutes. By calculating the time weighted mean concentration, the effect of the interruptions and variations were taken into account. Overall, it was considered that the generation represented the intended exposure to the 1 mg/L target concentration for 4 hours.
For the 5 mg/L exposure group, the time-weighted mean actual concentration was 5.3 ± 0.4 mg/L. The nominal concentration was 64.1 mg/L and the generation efficiency was 8%.
The concentration was measured at time points (n=21) that were equally distributed over the exposure period, the results of which showed that it was difficult to maintain a stable concentration. The generation was interrupted twice and the set-up was changed within the first 50 minutes after start. The generation time was elongated with 12 minutes in order to achieve an actual exposure time of 240 minutes. By calculating the time weighted mean concentration, the effect of the interruptions and variations were taken into account. Overall, it was considered that the generation represented the intended exposure to the 5 mg/L target concentration for 4 hours.

Particle Size
The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice during the exposure period. At 1 mg/L, the MMAD was 3.9 μm (gsd 1.9) and 4.0 μm (gsd 2.1). At 5 mg/L, the MMAD was 4.0 μm (gsd 2.1) and 4.3 μm (gsd 2.0).

Any other information on results incl. tables

Gravimetric Concentration of Test Item

1 mg/L exposure group

Start of generation of test atmosphere:         09:55

End of generation of test atmosphere:           14:04

Time (hh:mm)	Action	Sample volume (L)	Mass sampled (mg)	Concentration (mg/L)	% of total exposure time	Weight concentration (mg/L)
9:55 9:59 10:03 10:10 10:18 10:24 10:31 10:48 11:02 11:08 11:14 11:20 11:32 11:51 12:09 12:24 12:31 12:37 12:43 12:49 12:54 13:00 13:03 13:10 13:11 13:17 13:23 13:28 13:34 13:38 13:40 13:42 13:59 14:04	Start of generation Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Stop generation Start of generation Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Stop generation Start of generation Start of sampling Start of sampling End of generation	n.a. 5 5 5 5 6 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 n.a. n.a. 5 5 5 5 5 n.a. n.a. 5 5 n.a.	n.a. 3.90 4.62 4.02 4.84 6.34 5.40 6.07 7.45 8.60 8.08 5.91 5.02 5.97 6.71 6.98 7.53 7.62 4.45 3.52 1.91 0.83 n.a. n.a. 8.34 8.80 4.34 4.85 4.09 n.a. n.a. 5.55 4.48 n.a.	n.a. 0.780 0.924 0.804 0.968 1.057 1.080 1.214 1.490 1.720 1.616 1.182 1.004 1.194 1.342 1.396 1.506 1.524 0.890 0.704 0.382 0.166 0.166^1) n.a. 1.668 1.760 0.868 0.970 0.818 0.818^1) n.a. 1.110 0.896 0.896^1)	0.0 1.7 1.7 2.9 3.3 2.5 2.9 7.1 5.8 2.5 2.5 2.5 5.0 7.9 7.5 6.3 2.9 2.5 2.5 2.5 2.1 2.5 1.3 n.a. 0.4 2.5 2.5 2.1 2.5 1.7 n.a. 0.8 7.1 2.1	n.a. 0.013 0.015 0.023 0.032 0.026 0.032 0.086 0.087 0.043 0.040 0.030 0.050 0.095 0.101 0.087 0.044 0.038 0.022 0.018 0.008 0.004 0.002 n.a. 0.007 0.044 0.022 0.020 0.020 0.014 n.a. 0.009 0.063 0.019
Time-weighted mean concentration Standard deviation Number of samples					1.115 0.028 28

1) Assumed concentration, based on the last sample

2) n.a. = not applicable

 

Gravimetrical Concentration of Test Item

5 mg/L exposure group

Start of generation of test atmosphere:         09:14

End of generation of test atmosphere:           13:26

Time (hh:mm)	Action	Sample volume (L)	Mass sampled (mg)	Concentration (mg/L)	% of total exposure time	Weight concentration (mg/L)
9:14 9:17 9:22 9:28 9:31 9:34 9:35 9:42 9:48 9:58 10:07 10:08 10:11 10:19 10:21 10:29 10:35 10:41 10:46 10:54 11:16 11:20 11:37 11:59 12:21 13:20 13:26	Start of generation Start of sampling Start of sampling Start of sampling Stop generation Start of generation Start of sampling Start of sampling Start of sampling Stop generation Start of generation Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling Start of sampling End of generation	n.a. 5 5 5 n.a. n.a. 5 5 6 n.a. n.a. 5 5 5 5 5 5 5 5 5 5 5 5 5 5 8 n.a.	n.a. 12.74 8.56 3.98 n.a. n.a. 14.91 1.00 0.49 n.a. n.a. 41.86 5.51 79.38 40.47 8.27 33.03 39.39 9.42 38.13 10.64 36.97 32.38 39.08 37.05 49.66 n.a.	n.a. 2.548 1.712 0.796 0.796^1) n.a. 2.982 0.200 0.082 0.082^1) n.a. 8.372 1.102 15.876 8.094 1.654 6.606 7.878 1.884 7.626 2.1228 7.394 6.476 7.816 7.410 6.208 6.208^1)	0.0 1.3 2.1 2.5 1.3 n.a. 0.4 2.9 2.5 4.2 n.a. 0.4 1.2 3.3 0.8 3.3 2.5 2.5 2.1 3.3 9.2 1.7 7.1 9.2 9.2 24.6 2.5	n.a. 0.032 0.036 0.020 0.010 n.a. 0.012 0.006 0.002 0.003 n.a. 0.035 0.014 0.529 0.067 0.055 0.165 0.197 0.039 0.254 0.195 0.123 0.459 0.716 0.679 1.526 0.155
Time-weighted mean concentration Standard deviation Number of samples					5.331 0.351 21

1) Assumed concentration, based on the last sample

2) n.a. = not applicable

 

Aerodynamic Particle Size Distribution in the Test Atmosphere

1 mg/L exposure group

Start of generation of test atmosphere:         09:55

End of generation of test atmosphere:           14:04

Sampling speed (L/min):                    2

Measurement 1:

Sampling time:                     10:56

Sample volume (L):             8

Stage	Cut point (μm)	Mass sampled (mg)	Relative mass (%)	Cumulative mass (% of total sampled)
1 2 3 4 5 6 7 8 Back up	21.0 15.0 10.0 6.0 3.5 2.0 0.9 0.5 0.25	0.01 0.07 1.00 1.89 3.25 1.32 0.48 0.15 0.14	0.12 0.84 12.03 22.74 39.11 15.88 5.78 1.81 1.68	99.88 99.04 87.00 64.26 25.15 9.27 3.49 1.68 0.00
MMAD^1 (μm): gsd^2:		3.9 1.9

¹Mass Median Aerodynamic Diameter

²Geometric standard deviation

Measurement 2:

Sampling time:                     13:47

Sample volume (L):             9

Stage	Cut point (μm)	Mass sampled (mg)	Relative mass (%)	Cumulative mass (% of total sampled)
1 2 3 4 5 6 7 8 Back up	21.0 15.0 10.0 6.0 3.5 2.0 0.9 0.5 0.25	0.00 0.07 1.23 1.66 2.34 1.17 0.38 0.09 0.26	0.00 0.97 17.08 23.06 32.50 16.25 5.28 1.25 3.61	100.00 99.03 81.94 58.89 26.39 10.14 4.86 3.61 0.00
MMAD^1 (μm): gsd^2:		4.0 2.1

¹Mass Median Aerodynamic Diameter

²Geometric standard deviation

 

Aerodynamic Particle Size Distribution in the Test Atmosphere

5 mg/L exposure group

Start of generation of test atmosphere:         09:14

End of generation of test atmosphere:           13:26

Sampling speed (L/min):                    2

Measurement 1:

Sampling time:                     11:54

Sample volume (L):             2

Stage	Cut point (μm)	Mass sampled (mg)	Relative mass (%)	Cumulative mass (% of total sampled)
1 2 3 4 5 6 7 8 Back up	21.0 15.0 10.0 6.0 3.5 2.0 0.9 0.5 0.25	0.00 0.00 0.24 0.48 0.47 0.66 0.26 0.06 0.00	0.00 0.00 11.06 22.12 21.66 30.41 11.98 2.76 0.00	100.00 100.00 88.94 66.82 45.16 14.75 2.76 0.00 0.00
MMAD^1 (μm): gsd^2:		4.0 2.1

¹Mass Median Aerodynamic Diameter

²Geometric standard deviation

Measurement 2:

Sampling time:                     13:24

Sample volume (L):             2

Stage	Cut point (μm)	Mass sampled (mg)	Relative mass (%)	Cumulative mass (% of total sampled)
1 2 3 4 5 6 7 8 Back up	21.0 15.0 10.0 6.0 3.5 2.0 0.9 0.5 0.25	0.00 0.00 0.84 0.95 1.76 0.79 0.72 0.14 0.01	0.00 0.00 16.12 18.23 33.78 15.16 13.82 2.69 0.19	100.00 100.00 83.88 65.64 31.86 16.70 2.88 0.19 0.00
MMAD^1 (μm): gsd^2:		4.3 2.0

¹Mass Median Aerodynamic Diameter

²Geometric standard deviation

 

Mortality

TEST DAY HOURS AFTER TREATMENT	1 1	1 3	2	3	4	5	6	7	8	9	10	11	12	13	14	15
MALES 1 MG/LITER FEMALES 1 MG/LITER MALES 5 MG/LITER FEMALES 5 MG/LITER	- - - -	- - - -	- - - -	- - - -	- - - -	- - - -	- - - -	- - - -	- - - -	- - - -	- - - -	- - - -	- - - -	- - - -	- - - -	- - - -

 

Clinical Signs

TEST DAY HOURS AFTER TREATMENT	MAX GRADE	1 1	1 3	2	3	4	5	6	7	8	9	10	11	12	13	14	15
MALES 1 MG/LITER
ANIMAL 1 Skin / fur                Brown staining (Ear left)                Brown staining (Ear right)                Brown staining (General)	(1) (1) (1)	- - 1	- - 1	1 1 -	1 1 -	1 1 -	1 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
ANIMAL 2 Skin / fur                Brown staining (Ear left)                Brown staining (Ear right)                Brown staining (General)	(1) (1) (1)	- - 1	- - 1	1 1 -	1 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
ANIMAL 3 Posture                Hunched posture Skin / fur                Brown staining (Ear left)                Brown staining (Ear right)                Brown staining (General)	(1)   (1) (1) (1)	-   - - 1	1   - - 1	-   1 1 -	-   1 1 -	-   1 1 -	-   1 1 -	-   - - -	-   - - -	-   - - -	-   - - -	-   - - -	-   - - -	-   - - -	-   - - -	-   - - -	-   - - -
ANIMAL 4 Skin / fur                Brown staining (Ear left)                Brown staining (Ear right)                Brown staining (General)	(1) (1) (1)	- - 1	- - 1	1 1 -	1 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
ANIMAL 5 Skin / fur                Brown staining (Ear left)                Brown staining (Ear right)                Brown staining (General)	(1) (1) (1)	- - 1	- - 1	1 1 -	1 1 -	1 1 -	1 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
FEMALES 1 MG/LITER
ANIMAL 6 Skin / fur                Brown staining (Ear left)                Brown staining (Ear right)                Brown staining (General)	(1) (1) (1)	- - 1	- - 1	1 1 -	1 1 -	1 1 -	1 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
ANIMAL 7 Skin / fur                Brown staining (Ear left)                Brown staining (Ear right)                Brown staining (General)	(1) (1) (1)	- - 1	- - 1	1 1 -	1 1 -	- 1 -	- 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
ANIMAL 8 Skin / fur                Brown staining (Ear left)                Brown staining (Ear right)                Brown staining (General)	(1) (1) (1)	- - 1	- - 1	1 1 -	1 1 -	1 1 -	1 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
ANIMAL 9 Skin / fur                Brown staining (Ear left)                Brown staining (Ear right)                Brown staining (General)	(1) (1) (1)	- - 1	- - 1	1 1 -	1 1 -	1 1 -	1 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
ANIMAL 10 Skin / fur                Brown staining (Ear left)                Brown staining (Ear right)                Brown staining (General)	(1) (1) (1)	- - 1	- - 1	1 1 -	1 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
MALES 5 MG/LITER
ANIMAL 11 Skin / fur                Green staining (General)                Brown staining (Ears)	(1) (1)	1 -	1 -	- 1	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -
ANIMAL 12 Skin / fur                Green staining (General)                Brown staining (Ears)	(1) (1)	1 -	1 -	- 1	- 1	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -
ANIMAL 13 Skin / fur                Green staining (General)                Brown staining (Ear right)                Brown staining (Ears)	(1) (1) (1)	1 - -	1 - -	- - 1	- 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
ANIMAL 14 Skin / fur                Green staining (General)                Brown staining (Ear right)                Brown staining (Ears)	(1) (1) (1)	1 - -	1 - -	- - 1	- 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
ANIMAL 15 Skin / fur                Green staining (General)                Brown staining (Ears)	(1) (1)	1 -	1 -	- 1	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -
FEMALES 5 MG/LITER
ANIMAL 16 Skin / fur                Green staining (General)                Brown staining (Ears)	(1) (1)	1 -	1 -	- 1	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -
ANIMAL 17 Skin / fur                Green staining (General)                Brown staining (Ears)	(1) (1)	1 -	1 -	- 1	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -
ANIMAL 18 Skin / fur                Green staining (General)                Brown staining (Ear right)                Brown staining (Ears)	(1) (1) (1)	1 - -	1 - -	- - 1	- 1 -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
ANIMAL 19 Skin / fur                Green staining (General)                Brown staining (Ears)	(1) (1)	1 -	1 -	- 1	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -
ANIMAL 20 Skin / fur                Green staining (General)                Brown staining (Ears)	(1) (1)	1 -	1 -	- 1	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -

 

Body Weights (Gram)

SEX/DOSE LEVEL	ANIMAL	DAY 1	DAY 2	DAY 4	DAY 8	DAY 15
MALES 1 MG/LITER	1 2 3 4 5   MEAN ST.DEV. N	299 334 318 313 326   318 13 5	294 328 316 307 320   313 13 5	300 336 321 314 325   319 13 5	312 350 332 320 335   330 15 5	331 376 356 340 360   353 18 5
FEMALES 1 MG/LITER	6 7 8 9 10   MEAN ST.DEV. N	195 198 196 200 214   201 8 5	194 200 201 209 211   203 7 5	191 198 208 211 213   204 9 5	194 202 207 209 215   205 8 5	216 219 221 220 224   220 3 5
MALES 5 MG/LITER	11 12 13 14 15   MEAN ST.DEV. N	279 285 276 290 264   279 10 5	266 264 263 276 252   264 9 5	277 276 273 289 259   275 11 5	291 297 292 308 273   292 13 5	315 319 313 335 290   314 16 5
FEMALES 5 MG/LITER	16 17 18 19 20   MEAN ST.DEV. N	184 206 187 181 194   190 10 5	178 195 184 180 186   185 7 5	182 195 188 179 196   188 8 5	193 204 199 185 209   198 9 5	198 207 212 202 215   207 7 5

 

Macroscopic Findings

ANIMAL	ORGAN	FINDING	DAY OF DEATH
MALES 1 MG/LITER 1   2   3   4   5		No findings noted   No findings noted   No findings noted   No findings noted   No findings noted	Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment
FEMALES 1 MG/LITER 6   7   8   9   10	Thymus	No findings noted   No findings noted   Left side: focus/foci, several, purple   No findings noted   No findings noted	Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment
MALES 5 MG/LITER 11   12   13   14   15		No findings noted   No findings noted   No findings noted   No findings noted   No findings noted	Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment
FEMALES 5 MG/LITER 16   17   18   19   20		No findings noted   No findings noted   No findings noted   No findings noted   No findings noted	Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results, the inhalatory LC50, 4h value of Formaldehyde, oligomeric reaction products with acetone and diphenylamine in Wistar rats was considered to exceed 5 mg/L.

Executive summary:

Assessment of acute inhalation toxicity with Formaldehyde, oligomeric reaction products with acetone and diphenylamine in the rat (nose-only)

 

The study was carried out based on the guidelines described in:

OECD Guidelines, Section 4, Health Effects. No.403, "Acute Inhalation Toxicity", Sep 2009.

Commission Regulation (EC) No 440/2008, B.2. Acute Toxicity (inhalation), L142, May 2008.

EPA OPPTS 870.1300, Acute inhalation Toxicity. EPA 712-C-98-193, August 1998.

JMAFF Guidelines (2000), including the most recent revisions.

 

Formaldehyde, oligomeric reaction products with acetone and diphenylamine was administered as a dust by nose-only inhalation for 4 hours to two groups of five male and five female Wistar rats. Mortality and clinical signs were observed daily during the observation period and body weights were determined on Days 1, 2, 4, 8 and 15. Macroscopic examination was performed after terminal sacrifice (Day 15).

 

For the 1 mg/L exposure group, the time-weighted mean actual concentration was 1.1 ± 0.03 mg/L. The nominal concentration (amount of test item used divided by the volume of pressurized air used) was 9.7 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 11%. The concentration was measured at time points (n=28) that were equally distributed over the exposure period, the results of which demonstrated that it was difficult to maintain the test item stable. Overall, it was considered that the generation represented the intended exposure to the 1 mg/L target concentration for 4 hours.

 

For the 5 mg/L exposure group, the time-weighted mean actual concentration was 5.3 ± 0.4 mg/L. The nominal concentration was 64.1 mg/L and the generation efficiency was 8%. The concentration was measured at time points (n=21) that were equally distributed over the exposure period, the results of which showed that it was difficult to maintain a stable concentration. Overall, it was considered that the generation represented the intended exposure to the 5 mg/L target concentration for 4 hours.

 

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice during the exposure period. At 1 mg/L, the MMAD was 3.9 μm (gsd 1.9) and 4.0 μm (gsd 2.1). At 5 mg/L, the MMAD was 4.0 μm (gsd 2.1) and 4.3 μm (gsd 2.0).

 

No mortality occurred.

 

At 1 mg/L, quick breathing was seen during exposure. After exposure, one male showed hunched posture on Day 1. Brown staining by test item remnants of the skin and fur was seen between Days 1 and 6.

At 5 mg/L, quick breathing was seen during exposure. After exposure, no systemic clinical signs were seen. Brown and green staining by test item remnants of the skin and fur was seen between Days 1 and 4.

 

Overall body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity.

 

Macroscopic post mortem examination revealed several purple foci on the left side of the thymus of one female exposed to 1 mg/L. Macroscopic examination of the other animals did not reveal further abnormalities.

 

The generation of high dust concentrations is technically challenging. In this study, the concentration varied considerably during the first 2 hours. Over the total 4 hour exposure period, the time weighted mean concentration exceeded 5 mg/L. Considering the absence of mortality and severe signs, no significant mortality would be expected after exposure to 5 mg/L with less variation.

 

Based on the results, the inhalatory LC50, 4h value of Formaldehyde, oligomeric reaction products with acetone and diphenylamine in Wistar rats was considered to exceed 5 mg/L.

 

Based on this study, Formaldehyde, oligomeric reaction products with acetone and diphenylamine does not have to be classified and has no obligatory labelling requirement for acute inhalation toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments.