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Description of key information

Acute toxicity oral : No data (study waived based on corrosivity of the test substance)
Acute toxicity inhalation: In A GLP-compliant study conducted according to OECD Guideline 403 with an aqueous solution of 70% nitric acid, a vapor atmosphere containing ca. 0.8% liquid aerosol was tested in rats via nose-only inhalation. The LC50 was found to be >2.65 mg/L (referring to pure nitric acid).
Acute toxicity dermal: No data (study waived based on corrosivity of the test substance)

Key value for chemical safety assessment

Acute toxicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant study conducted according to OECD Guideline 403
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes
Test type:
other: One concentration selected on the basis of technical pre-tests was tested.
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst, NIEDERLANDE
- Age at study initiation: Young adult animals (male animals approx. 8 weeks, female animals approx. 10 weeks)
- Weight at study initiation: Animals of comparable weight (± 20% of the mean weight)
- Housing: single housing in Typ III polycarbonate cages (floor area about 800 cm2), enrichment in form of wooden gnawing blocks (Typ NGM E-022)
- Diet: ad libitum, Kliba laboratory diet, mouse/rat maintenance "GLP", 12 mm pellets, Provimi Kliba SA, Kaiseraugst, Basel Switzerland.
- Water: Tap water ad libitum
- Bedding: Dust-free wooden bedding
- Acclimation period: for at least 5 days before exposure.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70% relative humidity
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h (6 a.m.- 6 p.m. / 6 p.m. - 6 a.m.)

IN-LIFE DATES: First exposure 2015-03-16, day of last observation 2015-03-30
Route of administration:
other: The atmosphere consisted mainly of vapor with a small aerosol fraction of ca. 0.8%.
Type of inhalation exposure:
nose only
Vehicle:
other: The test substance was dosed unchanged.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose-only inhalation system INA 20 (glass steel construction, BASF SE)
- Exposure chamber volume: ca. 55 L
- Method of holding animals in test chamber: Animals were restrained in glass tubes and their snouts projected into the inhalation system.
- Rate of air: Supply air flow: 1.5 m3/h, ca. 27 air changes per hour.
- Method of conditioning air: Cold air (ca. 15 °C) provided by central air conditioning system passed through an activated charcoal filter, was adjusted to room temperature of 20-24°C and passed through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- Compressed air: Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through a second ultra-filter (SMF 5/3, 108 mm, Donalson), the
compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 6 bar. In the laboratory, the compressed air can be taken as required.
- Supply air flows (compressed air): 1.5 m3/h (flow was adjusted and continuously measured with a flowmeter (Yokawa)).
- Exhaust air flows: 1.35 mg3/h (flow was adjusted and continuously measured with a flowmeter (Yokawa))
- Method of particle size determination: aerodynamic particle sizer (APS 3321, TSI, USA)
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: Ion chromatography (881 Compact IC pro, Metrohm); Precolumn: Metrosep A Supp 4/5, S-Guiard, Metrohm; Analytical column: Metrosep A Supp 7, 4x250 mm, Metrohm; Eluent: Na2Co3, 3.6 mmol/L; Flow rate: 0.7 ml/min; Column temp. 45 °C; Detector temp. 40 °C; Injection volume 100 µl; Suppressor: MSM Roto A (Metrohm); Detection: conductivity; Evaluation: External calibration (peak area)
- Samples taken from breathing zone: yes
- Particle size analysis: Due to the volatility of nitric acid, no detectable amount of aerosols could be measured on the stages of a cascade impactor during the technical trial. Thus, particle size distribution was determined by an aerodynamic particle sizer (APS 3321, TSI, USA). Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were obtained directly by APS 3321. During exposure, 3 measurements with 3 repeats each were performed (sample volume: 5 L). However, two erroneous measurements were considered erroneous due to extremely low particle count concentration
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 1.66-2.33 µm (2.35-3.13 GSD)

RATIONALE FOR SELECTION OF CONCENTRATION:
Based on available data (see description of technical pre-tests in section "any other information on material and methods incl. tables"), an atmospheric concentration of 2 mg/L nitric acid was tested.

DETERMINATION OF CONCENTRATION OF THE TEST SUBSTANCE IN INHALATION ATMOSPHERE:
The sample volume was adjusted to achieve suitable amounts of the test substance in the samples of the test groups in reference to the calibration of the analytical method. An air sampler GS 312 (DESAGA) was used.
2 sampling devices were used to determine the test substance concentration in the atmosphere:
1) a sampling probe (diameter 7 mm) with quartz wool plug, and
2) 4 tandem impingers filled with 0.5 M NaOH as sorption solvent.
The quartz wool plug was used to trap the aerosol fraction in the test atmosphere. It was suspended in sorption solvent and was analyzed for each sample. The content of the first 3 impingers, which reflexed the vapor fraction, was pooled and analyzed for each sample. The fourth impinger was used to control the effectiveness of the sorption for all samples of the atmosphere and was analyzed separately at the end of the sampling campaign.
Sampling position: immediately adjacent to the animals' noses at a separate spare port
Sampling flow: 3 L/min
Sampling velocity: 1.25 m/s
Sampling frequency: 4 samples at about hourly intervals
Sample volume: 10 L
For the quantitative determination of the aerosol and vapor concentration, an ion chromatographic method was used. The concentration was determined on the basis of nitrate.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
ion chromatography
Duration of exposure:
4 h
Concentrations:
Mean atmospheric concentration: 2.65 mg/L (analytical, referring to pure nitric acid)
Nominal concentration: 51% of analytical concentration (referring to 70% nitric acid); 73% of analytical concentration (referring to pure nitric acid)
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: feed and water (2x daily on workdays, 1x daily on weekends and public holidays); individual body weights (1x during acclimatization period, shortly before exposure on day 0 and at least on days 1, 3 and 7, and before sacrifice at the end of the observation period. Additionally, body weight was measured in animals that died from study day 1 onwards); clinical observations (several times during exposure, at least 1x daily on the pre-exposure day and during the observation period); mortality (2x daily on workdays, 1x daily on weekends and public holidays);
- Pathology: At the end of the observation period the surviving animals were sacrificed with CO2-inhalation in a chamber with increasing concentration over time, and were subjected to gross-pathological examination as well as the animal which died before. To clarify the gross-pathological findings, selected organs of individual animals were examined histopathologically.
Statistics:
In this study, only one concentration was tested, at which only one animal died. The result belonged to the type ”LC50 greater than”. Therefore, the binomial test was used for statistical evaluation Steel R.G.D., Torrie J.H. (1984):Principles and procedures of statistics a biometrical approach. McGraw - Hill.
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 2.65 mg/L air (analytical)
Based on:
act. ingr.
Remarks:
pure nitric acid
Exp. duration:
4 h
Mortality:
One of five male animal died on study day 9. No lethality was observed in females.
Clinical signs:
other: A series of clinical signs indicating the irritating and corrosive property of the test substance was observed: visually depressed respiration during exposure, intermittent and or abdominal respiration after exposure, gasping, respiration sounds, nose dis
Body weight:
The mean body weights of the surviving male animals decreased on the first post exposure observation days and thereafter increased. The mean body weights of the female animals decreased on the first post exposure observation day and thereafter increased.
Gross pathology:
Gross necropsy of the male animal that died prematurely revealed yellow discoloration of the nose region, encrusted, red and swelling nose, dilation of the intestines with gaseous content.
To further evaluate the macroscopic findings, histopathological examination was carried out in the respiratory tract and small intestine of animal no. 331, that died prematurely. Histological examination of the respiratory tract revealed focal desquamation of squamous epithelium in level I of the nasal cavity. This finding was accompanied by a minimal luminal inflammatory exudate of the same region. Moreover, in level I of larynx epithelial alteration, focal, minimal (base of epiglottis) and level III of larynx, minimally increased number of mucus cells, moderate attenuated epithelium were noted. There were no adverse changes in nasal cavity levels II to IV, trachea, lung, pharynx, larynx level II, and small intestine.
Necropsy of the animals sacrificed at termination of the post exposure periods revealed yellow discoloration of the fur in the nose region in all 4 of the 5 males and all of the 5 females. Moreover, 2 of the 5 female animals showed skin lesions in the nose region, 3 of the 5 males and 1 of the 5 females lost the tip of their nose.
For further details refer to section "any other information on results incl. tables.

Table 7. Duration of clinical signs observed during and after exposure.

Test group 1 (2.65 mg/L)

Male animals

Female animals

Lethality (number of animals)

1

-

Animal body, injury, loss of nose tip

d14

d14

Eye, red encrusted

d2–d9

-

Eye, reddened conjunctiva

d0–d2

-

Fur, substance contaminated

d0–d3

d0–d2

Fur, yellow discolored, nose region

d0–d14

d0–d14

General condition, dehydrated

d9

-

General condition, poor

d1–d9

-

Nose, colorless discharge

-

d1–d4

Nose, red discharge

d1–d14

d1–d14

Nose, red encrusted

d2–d14

d2–d14

Nose, swelling

d0–d14

-

No feces

d9

-

Respiration, abdominal

d0–d14

d2; d10–d13

Respiration, depressed

h1–h4

h1–h4

Respiration, intermittent

d0–d4

d0–d9

Respiration, gasping

d0–d9

d1

Respiration, sounds

d1–d14

d1–d7; d10d14

Piloerection

d0–d9

d0–d4

Table 8. Individual body weights [g] of male animals during the in-life phase.

Dose group

Animal Number

Day 0

Day 1

Day 3

Day 7

Day 9

Day 14

Test group 1 /M

2.65 mg/L

 

0331

236.8

205.4

180.1

155.9

139.6

NS

0332

236.2

219.2

230.7

256.9

 

291.2

0333

235.0

221.2

220.4

241.3

 

265.6

0334

233.8

213.8

210.4

224.9

 

225.1

0335

230.0

206.3

202.7

215.6

 

236.0

Mean

234.4

213.2

208.9

218.9

139.6

254.5

S.D.

2.7

7.2

19.2

38.6

 

29.9

N

5

5

5

5

1

4

Table 9. Individual body weights [g] of female animals during the in-life phase.

Dose group

Animal Number

Day 0

Day 1

Day 3

Day 7

Day 14

Test group 1 /F

2.65 mg/L

 

0336

216.8

209.0

219.2

225.5

230.3

0337

205.2

202.0

199.2

205.3

213.8

0338

204.7

196.4

197.8

211.2

224.8

0339

215.5

205.1

207.6

213.0

221.6

0340

204.0

194.2

199.0

205.5

211.9

Mean

209.2

201.3

204.6

212.1

220.5

S.D.

6.3

6.1

9.1

8.2

7.7

N

5

5

5

5

5

Table 10. Pathological findings.

 

Animal No.

Findings

331*

332

333

334

335

336

337

338

339

340

Organs without particular findings

 

 

 

 

 

 

 

 

 

 

Fur: yellow discolored, nose region

x

x

x

x

x

x

x

x

x

x

Nose: swelling, red encrusted

x

 

 

 

 

 

 

 

 

 

Skin lesion, nose region

 

 

 

 

 

 

 

x

 

x

Injury: tip of nose is lost

 

 

x

x

x

 

 

 

x

 

Intestine: dilation with gaseous content

x

 

 

 

 

 

 

 

 

 

* animal dead

Interpretation of results:
other: cat. 3
Remarks:
Criteria used for interpretation of results: OECD GHS
Conclusions:
Under the conditions of this study the LC50 for male and female rats after inhalation exposure to vapor atmosphere of nitric acid containing 0.8 % aerosol fraction is > 2.65 mg/L (referring to pure nitric acid).
Executive summary:

A GLP-compliant acute toxicity study according to OECD Guideline 403 was conducted with a 70% aqueous solution of nitric acid (BASF SE, 2015). A group of 5 male and 5 female Wistar rats was exposed for 4 hours by nose-only inhalation to an atmosphere containing nitric acid at a concentration of 2.65 mg/L (referring to pure nitric acid). The atmosphere consisted mainly of vapor and contained a small fraction of aerosol (ca. 0.8%), which is considered representative for the conditions under which the atmosphere was generated. One male died on day 9, no lethality was observed in females. In the deceased male, yellow discoloration of the nose region, an encrusted, red and swelling nose and dilation of the intestines with gaseous content were found upon gross necropsy. Histopathological examination revealed adverse effects in the nasal area, probably related to the corrosivity of the test substance. No observations related to systemic effects were done. The mean body weights of the surviving animals decreased on the first post-exposure day(s) and thereafter increased again. A series of clinical signs was observed that were indicative of the irritating and corrosive properties of the test substance (visually depressed respiration during exposure, intermittent and or abdominal respiration after exposure, gasping, respiration sounds, nose discharge, red encrusted nose, swelling nose, red encrusted eye, reddened conjunctiva of eyes). Towards the end of the post-exposure period, 3 male and one female animals lost their nose tip. Necropsy of the animals sacrificed at termination of the post exposure periods revealed yellow discoloration of the fur in the nose region in all surviving rats. Two of the 5 female animals showed skin lesions in the nose region. In conclusion, as only one animal died before the end of the observation period, the LC50 after a 4-hour exposure to a vapor of 70% nitric acid containing 0.8% liquid aerosol is considered to be >2.65 mg/L (referring to pure nitric acid).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
2 650 mg/m³
Quality of whole database:
Two reliable studies are available. A technical report is available to account for missing information in one of the two reports. Furthermore, three disregarded studies are available (Klimisch 3).

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

According to the REACH Regulation, an acute toxicity test does not generally need to be conducted if the substance is classified as corrosive to the skin (column 2 adaptation, Annex VIII, section 8.5). Therefore no data on oral and/ or dermal acute toxicity are included in the dossier.

Information regarding the acute inhalation toxicity of the test substance is available and described below:

A GLP-compliant acute toxicity study according to OECD Guideline 403 was conducted with a 70% aqueous solution of nitric acid (BASF SE, 2015). A group of 5 male and 5 female Wistar rats was exposed for 4 hours by nose-only inhalation to an atmosphere containing nitric acid at a concentration of 2.65 mg/L (referring to pure nitric acid). The atmosphere consisted mainly of vapor and contained a small fraction of aerosol (ca. 0.8%), which is considered representative for the conditions under which the atmosphere was generated. One male died on day 9, no lethality was observed in females. In the deceased male, yellow discoloration of the nose region, an encrusted, red and swelling nose and dilation of the intestines with gaseous content were found upon gross necropsy. Histopathological examination revealed adverse effects in the nasal area, probably related to the corrosivity of the test substance. No observations related to systemic effects were done. The mean body weights of the surviving animals decreased on the first post-exposure day(s) and thereafter increased again. A series of clinical signs was observed that were indicative of the irritating and corrosive properties of the test substance (visually depressed respiration during exposure, intermittent and or abdominal respiration after exposure, gasping, respiration sounds, nose discharge, red encrusted nose, swelling nose, red encrusted eye, reddened conjunctiva of eyes). Towards the end of the post-exposure period, 3 male and one female animals lost their nose tip. Necropsy of the animals sacrificed at termination of the post exposure periods revealed yellow discoloration of the fur in the nose region in all surviving rats. Two of the 5 female animals showed skin lesions in the nose region. In conclusion, as only one animal died before the end of the observation period, the LC50 after a 4-hour exposure to a vapor of 70% nitric acid containing 0.8% liquid aerosol is considered to be >2.65 mg/L (referring to pure nitric acid).

An older acute toxicity study conducted with methods similar to OECD Guideline 403 (Acute Inhalation Toxicity) was performed with a 70.76 % nitric acid aqueous solution ( E.I. du Pont de Nemours and Company, Inc., 1987). Groups of 5 male and 5 female Crl:CD BR rats were each exposed for 1 hour by nose-only inhalation to an atmosphere of nitric acid in air at concentrations ranging from 260 -3100 ppm. The composition of the atmosphere with regard to the aerosol or vapor content was not specified for the individual concentrations. Following exposure, rats were weighed and observed for 14 days of recovery. A gross pathological examination was performed on all animals at the time of death or after 14 day recovery period. Clinical signs and gross lesions of external tissues were observed in most rats which were indicative of the test substance’s corrosive nature. However, no significant gross lesions indicative of systemic toxicity or cause of death were observed. The combined 1-hour LC50 for male and female rats was determined to be 2500 ppm (6.25 mg/L).

A technical trial was performed largely according to the study condition of the du Pont study, to determine which fraction (vapor or liquid aerosol) was the predominant one present at the LC50 value. In the first trial, the liquid aerosol and vapor concentration were measured together, and the vapor concentration was determined seperately after trapping of the aerosols by filtration. Liquid aerosol was detected at measured atmospheric concentrations between 0.37 and 1.76 mg/L. The saturated vapor concentration under the test condition was in the range of a few mg/m³, based on which it was concluded that the test substance was classified as aerosol. In an extended trial (study amendment no. 1), the liquid aerosol and vapor concentration were measured seperately. Although the atmospheric concentrations of nitric acid were in a comparable range in both experiments, the test substance concentration of vapor was found to be much higher. Further analysis showed that separating aerosol and vapour pressure by a quartz wool plug (paralleling the original trial) led to loss of vapor concentration of > 80%. Taken all information together, it is concluded that the main fraction of an atmosphere generated from 70% nitric acid by nebulization is vapor, containing a small fraction of aerosol. Based on this information, it is concluded that the two acute inhalation studies are done under comparable atmmosphere. Therefore, conversion of the inhalation toxicity data which have been generated using a 1-hour exposure can be carried out by dividing by a factor of 2 for vapours. This would result in an estimated LC50 (4 hour exposure) of > 3.125 mg/L, leading to the same classification as the LC50 from the key study.

Two additional nitric acid studies were available. However, none of the studies followed a guideline and were deemed unreliable (Klimisch 3).


Justification for selection of acute toxicity – inhalation endpoint
The study was performed according to OECD guideline and GLP principles.

Justification for classification or non-classification

Based on the available studies, the LC50 of pure nitric acid (which is a present as a vapour) is found to be >2.65 mg/L, therefore it is classified cat. 3 for acute inhalation toxicity and should be labelled with H331: Toxic if inhaled according to CLP Regulation EC (No.) 1272/2008. As data are available that indicate that the mechanism of toxicity was corrosivity, in addition to classification for inhalation toxicity, the substance should also be labelled as ‘corrosive to the respiratory tract’ (EUH071).

In absence of data, nitric acid is not classified for acute toxicity via oral and dermal route.