Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
Dec 1987 - Mar 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions. No opthalmoscopic, neurobehavioural examinations, no urinalysis, no food and water consumption examined.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
no opthalmoscopic, neurobehavioural examinations, no urinalysis, no food and water consumption
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Formic acid
EC Number:
200-579-1
EC Name:
Formic acid
Cas Number:
64-18-6
IUPAC Name:
formic acid
Details on test material:
- Name of test material (as cited in study report): formic acid
- Molecular formula (if other than submission substance): HCOOH
- Molecular weight (if other than submission substance): 46 g/mol
- Physical state: liquid
- Analytical purity: approximately 95%
- Impurities (identity and concentrations): approximately 5% water
- Stability under test conditions: Repeated purity analyses of samples taken from the formic acid generator indicated that formic acid did not decompose in the generator reservoir over a period of at least 29 days.
- Other: The infrared, ultraviolet/visible, and nuclear magnetic resonance spectra were consistent with the structure of formic acid.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms (Germantown, NY; USA).
- Age at study initiation: 6 - 7 weeks
- Housing: the animals were housed continuously in exposure chambers with chamber doors closed, except during animal husbandry procedures.
- Diet: Pelleted NIH-07 feed (Zeigler Bros., Inc., Gardners, PA), ad libitum except during the daily exposure period.
- Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.9
- Humidity (%): 55 ± 14
- Air changes (per hr): 15 ± 3
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: inhalation chambers (Harford Systems, Inc., Aberdeen, MD, USA)
- Method of conditioning air: dilution air was conditioned to room temperature at approximately 50% relative humidity and was filtered by HEPA and charcoal filters.
- Temperature, humidity, pressure in air chamber: the vaporizer was operated at approximately 97 ± 5°C.

TEST ATMOSPHERE
- Brief description of analytical method used: an infrared spectrometer (Foxboro Miran 980, The Foxboro Co., Foxboro, MA, USA) was used
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the 13-week study, at least 91% of the measured concentrations for each chamber were within ± 10% of the target concentration.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 h/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
8, 16, 32, 64 and 128 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
15.3, 30.6, 61.2, 122.4 and 244.7 mg/m³
Basis:
other: calculated according to the formula Y mg/m³ = (X ppm)(molecular weight)/24.5
No. of animals per sex per dose:
10 (main study animals)
10 (additional animals for clinical pathology)
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: the doses were selected based on a previous 2-week study with the test material in concentrations from 31 - 500 ppm.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: two times per day for mortality/moribundity

CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at study start, at weekly intervals, and at the end of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 3 and 23 (additional animals) and on core study rats at study termination.
- Anaesthetic used for blood collection: Yes (70% CO2; 30% O2)
- How many animals: all animals
- Parameters checked: erythrocyte, leukocyte, and platelet counts, haemoglobin (HGB) concentration, haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC), leukocyte differential, absolute counts of individual leukocytes, relative numbers of reticulocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 3 and 23 (additional animals) and on core study rats at study termination.
- Anaesthetic used for blood collection: Yes (70% CO2; 30% O2)
- How many animals: all animals
- Parameters checked: urea nitrogen (UN), creatinine, total protein, albumin, alanine aminotransferase (ALT), alkaline phosphatase (AP), creatine kinase (CK), amylase, activity of sorbitol dehydrogenase (SDH) and determination of total bile acids.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. A complete necropsy was performed on all animals. Organs and tissues were examined for gross lesions.
HISTOPATHOLOGY: Yes. The following tissues were examined microscopically from all control and high dose groups: adrenal glands, brain, bronchial lymph nodes, cecum, colon, duodenum, epididymis/seminal vesicles/ prostate/testes or ovaries/uterus, esophagus, eyes (if grossly abnormal), femur (including marrow), gallbladder (mice), gross lesions and tissue masses with regional lymph nodes, heart, ileum, jejunum, kidneys, larynx, liver, lungs with mainstem bronchi, mammary gland and adjacent skin, mandibular and mesenteric lymph nodes, mediastinal lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pharynx (if grossly abnormal),pituitary gland, preputial /clitoral glands, rectum, salivary glands, spinal cord and sciatic nerve (if neurologic signs present), spleen, stomach (including forestomach and glandular stomach), thigh muscle, thymus, thyroid gland, trachea, and urinary bladder. In addition to all gross lesions, the following tissues were examined in all other dose groups: nose (three transverse sections), lung, larynx, trachea, bronchial and mediastinal lymph nodes.
Other examinations:
- Organ weights were obtained from all core study animals and include: liver, thymus, right kidney, right testis, heart and lungs.
- Sperm morphology and vaginal cytology were evaluated in rats exposed to 8, 32, and 128 ppm.

Statistics:
Organ and body weight data were analyzed using the parametric multiple comparisons procedures of Williams (A test for differences between treatment means when several dose levels are compared with a zero dose control. 1997, Biometrics 27, 103-117. The comparison of several dose levels with a zero dose control. 1972, Biometrics 28, 519-531.) and Dunnett (A multiple comparison procedure for comparing several treatments with a control. 1955, J. Am. Stat. Assoc. 50, 1095-1121). Clinical chemistry and hematology data were analyzed using the nonparametric multiple comparisons methods of Shirley (A non-parametric equivalent of Williams’ test for contrasting increasing dose levels of a treatment. 1977, Biometrics 33, 386-389.) and Dunn (Multiple comparisons using rank sums. 1964, Technometrics 6,241-252.). Jonckheere's test (A distribution-free k-sample test against ordered alternatives. 1954, Biometrika 41, 133-145.) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test was more appropriate for pairwise comparisons than a test capable of detecting departures from monotonic dose-response. If the P-value from Jonckheere's test was greater than or equal to 0.10, Dunn's or Dunnett's test was used. The outlier test of Dixon and Massey (Introduction to Statistical Analysis. 1951, New York: McGraw Hill, pp. 145-147.) was employed to detect extreme values. Because the vaginal cytology data are proportions, an arcsine transformation was used to bring the data into closer conformance with normality assumptions. Treatment effects were investigated by applying a multivariate analysis of variance (Multivariate Statistical Methods. 1976, New York: McGraw Hill, pp. 170-179.) to the transformed data to test for the simultaneous equality of measurements across dose levels.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
8, 16, 32 and 64 ppm: increased body weight in males
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
minimal to mild changes in different parameters, non-adverse
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
minimal to mild changes in different parameters, non-adverse
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
males: increased liver weights (absolute and relative); all females and males: decreased lung weights
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
high dose groups: changes in the respiratory and olfactory epithelium, control and 32 ppm groups: minimal to mild inflammatory lesions in the lung
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality and no clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
Body weight was significantly increased in male animals in the groups 8, 16, 32 and 64 ppm at the end of the study.

HAEMATOLOGY
Changes in haematologic variables were few and generally minimal to mild in magnitude. Increases in WBC counts in male and female rats at 3 days were produced by mild lymphocytoses. RBC counts were significantly increased in male rats in the 64 and 128 ppm exposure groups at Day 3. Although there were no statistically significant changes in WBC counts at the 13-week time point, neutrophil counts were mildly to moderate decreased in a not-dose related manner in rats (m,f) in all exposure groups. In the female rats at 23 days, mild but significant increases in MCH and MCV were produced by minimal to mild decreases in RBC counts. In female rats at 13 weeks, there were minimal but significant increases in MCHC in animals at all exposure concentrations, produced by increases in HGB concentrations that were occasionally significant. Minimal but significant decreases in MCV in female rats in 2 exposure groups (16 and 128 ppm) at 13 weeks were associated with increases of similar magnitude in RBC counts.

CLINICAL CHEMISTRY
There were mild, significant decreases in concentrations of serum albumin in female rats at Day 3 (32, 64, and 128 ppm exposure groups) and increases in male rats at 13 weeks (8, 16, and 32 ppm exposure groups). In all female exposure groups, concentrations of total serum protein were decreased at Day 3. Male and female rats exposed to 16, 32 (female only), 64, and 128 ppm had significant increases in serum AP at 13 weeks. Additional changes in serum biochemical variables are shown in Table 1 and 2 (under “any other information on results incl. tables”).

ORGAN WEIGHTS
Liver weights were somewhat greater in male rats in all exposure groups and liver-to-body-weight ratios (relative weights) were increased in male rats exposed to 32, 64, and 128 ppm formic acid. Absolute and relative lung weights were decreased in all exposed groups of female rats. In male rats, relative lung weights were decreased in all exposure groups, and absolute weights were decreased in the 64 and 128 ppm groups.

GROSS PATHOLOGY
No unusual gross lesions were noted at necropsy.

HISTOPHATHOLOGY: NON-NEOPLASTIC
Histophathological changes occurred in the respiratory and olfactory epithelium of the nose and were generally were limited to the 128 ppm exposure groups (see Table 3 under “Any other information on results incl. tables”). Changes in the respiratory epithelium included a minimal squamous metaplasia in which the pseudostratified, ciliated columnar cells were replaced by a flattened, non-ciliated epithelium (2 to 5 cells thick). A few inflammatory cells were associated with these areas of metaplasia, but inflammation was not a prominent feature of the nasal lesions. Squamous metaplasia occurred most often in the respiratory epithelium that lines the most dorsal portion of the dorsal meatus in the nose's anterior section. Foci of squamous metaplasia occasionally were present on the anterior nasal septum and/or tips (margins) of the nasoturbinates. In the olfactory epithelium, degenerative changes were minimal to mild and generally limited to the area of the dorsal meatus in the mid-nasal section. Degeneration was characterized by a loss of the usual orderly arrangement of the pseudostratified layer of nuclei and by a slight reduction in the normal thickness of the olfactory epithelium. This decreased thickness was the result of a reduction in the amount of the cytoplasm at the apical portion of the olfactory epithelial cells and a decrease in the number of sensory and sustentacular cell nuclei. An increase in the basophilic staining of some nuclei was seen, and, in a few cells, the nucleus appeared pyknotic, or fragmented; however, necrosis was not a characteristic feature of the olfactory lesion.There was no evidence of metaplasia in the olfactory epithelium or atrophy of the nerve fibers in the olfactory mucosa. In 19/20 male and female rats from the control and 32 ppm exposure groups there were minimal to mild inflammatory lesions in the lung consisting of aggregates of macrophages and/or neutrophils in alveoli and hyperplasia of peribronchiolar lymphoid tissues and alveolar epithelium. These pulmonary lesions, which were generally less severe in females, were limited to the control and mid-dose groups and corresponded to the slightly greater lung weights observed for these groups of rats.

OTHER FINDINGS
No effects on measures of sperm motility, density, or testicular or epididymal weights were observed within the study, and no changes were seen in the length of the estrous cycle during the study.

Effect levels

open allclose all
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
244.7 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histopathology, effects at the upper respiratory tract (nasal respiratory and olfactory epithelium); corresponding to 128 ppm; maximum attainable concentration
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
122.4 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects; corresponding to 64 ppm
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
244.7 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects; corresponding to 128 ppm; maximum attainable concentration

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Significantly changed clinical chemistry parameters in male rats (n = 9 - 10).

Parameter

Dose group

 

Control

8 ppm

16 ppm

32 ppm

64 ppm

128 ppm

Urea nitrogen (mg/dL)

Day 3

313 ± 10

293 ± 5

281 ± 6**

262 ± 8**

238 ± 5**

244 ± 8**

Creatinine (mg/dL)

Day 3

83 ± 4

81 ± 6

76 ± 3

68 ± 4*

62 ± 4**

60 ± 4**

Total protein (g/dL)

Week 13

76 ± 2

73 ± 1

72 ± 1

72 ± 1

72 ± 1

70 ± 2*

Albumin (g/dL)

Week 13

46 ± 1

48 ± 1*

49 ± 1**

49 ± 1*

48 ± 1

47 ± 1

Globulin (g/dL)

Day3

Week 13

22 ± 1

28 ± 1

21 ± 1

26 ± 1**

21 ± 1*

26 ± 0**

22 ± 1

27 ± 0*

21 ± 1*

26 ± 0**

20 ± 1*

25 ± 1**

A/G ratio

Week 13

16 ± 1**

19 ± 1**

19 ± 1**

18 ± 1**

19 ± 0**

19 ± 1**

Alkaline phosphatase (IU/L)

Day 3

Week13

1049 ± 27

325 ± 14

1059 ± 33

334 ± 11

1011 ± 33

356 ± 7*

1042 ± 27

354 ± 11

970 ± 13*

376 ± 7**

921 ± 23**

375 ± 6**

Creatine kinase (IU/L)

Day 3

752 ± 49

634 ± 34

508 ± 38**

394 ± 19**

412 ± 42**

366 ± 54**

Sorbitol dehydrogenase (IU/L)

Day 3

6 ± 0

7 ± 1

9 ± 1**

11 ± 1**

9 ± 1**

10 ± 1**

*: Statistically significantly different (P ≤ 0.05) from the control group by Dunn's test or Shirley's test

**: Statistically significantly different (P ≤ 0.01) from the control group by Dunn's test or Shirley's test

Table 2. Significantly changed clinical chemistry parameters in female rats (n= 9 -10).

Parameter

Dose group

 

Control

8 ppm

16 ppm

32 ppm

64 ppm

128 ppm

Urea nitrogen (mg/dL)

Day 3

334 ± 9

324 ± 9

308 ± 16*

298 ± 8*

289 ± 11**

267 ± 10**

Creatinine (mg/dL)

Day 3

Week 13

85 ± 4

82 ± 2

76 ± 3

71 ± 2**

74 ± 3

74 ± 2

73 ± 3*

78 ± 2

66 ± 2**

77 ± 2

63 ± 4**

73 ± 2**

Total protein (g/dL)

Day 3

75 ± 1

72 ± 1*

71 ± 1*

70 ± 1**

68 ± 1**

67 ± 1**

Albumin (g/dL)

Day 3

52 ± 1

50 ± 1

50 ± 1

49 ± 1**

48 ± 1**

47 ± 0**

Globulin (g/dL)

Day3

23 ± 1

22 ± 1

21 ± 1

22 ± 1

20 ± 0**

20 ± 1**

Amylase (IU/L)

Day 3

Day 23

4230 ± 77

4587 ± 55

4152 ± 58

4290 ± 126

4125 ± 53

4412 ± 70

4129 ± 57

4354 ± 61*

3873 ± 66**

4206 ± 59**

3983 ± 95**

4206 ± 61**

Alkaline phosphatase (IU/L)

Day 3

Week13

995 ± 21

954 ± 28

989 ± 39

931 ± 33

864 ± 33**

837 ± 47**

Creatine kinase (IU/L)

Day 3

Day 23

398 ± 41

168 ± 16

373 ± 34

231 ± 29

285 ± 31

227 ± 19*

270 ± 31*

233 ± 26*

454 ± 75

188 ± 24

305 ± 17

251 ± 27*

*: Statistically significantly different (P ≤ 0.05) from the control group by Dunn's test or Shirley's test

**: Statistically significantly different (P ≤ 0.01) from the control group by Dunn's test or Shirley's test

Table 3. Histopathologic lesions in male and female rats.

Site/Lesion

Dose group

Control

8 ppm

16 ppm

32 ppm

64 ppm

128 ppm

Males

Nose, Respiratory epithelium

Squamous metaplasia

0

0

0

0

0

9(1.0)*

Nose, Olfactory epithelium

Degeneration

0

0

0

1 (1.0)

1 (1.9)

9 (1.2)

Females

Nose, Respiratory epithelium

Squamous metaplasia

0

0

0

0

0

6 (1.4)

Nose, Olfactory epithelium

Degeneration

0

0

0

0

0

5 (1.0)

*: Incidence and severity score ( ) based on a scale of 1 to 4: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. Scores are averages based on the number of animals with lesions from groups of 10.

Table 4. Summary of reproductive tissue evaluations.

Parameter

0 ppm

8 ppm

32 ppm

128 ppm

Males

Necropsy body weight (g)

339 ± 5

358 ± 10

367 ± 6*

334 ± 6

Left epididymis weight (g)

0.449 ± 0.011

0.461 ± 0.011

0.469 ± 0.001

0.460 ± 0.004

Spermatozoal messurements

 

 

 

 

Motility (%)

91 ± 1

91 ± 1

91 ± 1

88 ± 1

Concentration (10E8/g)

658 ± 21

706 ± 21

580 ± 60

651 ± 29

Females

Necropsy body weight (g)

212 ± 4

208 ± 6

207 ± 6

202 ± 6

Estrous cycle length (d)

4.80 ± 0.15

4.75 ± 0.11

4.95 ± 0.05

4.95 ± 0.12

*: Statistically significantly different (P ≤ 0.05 ) from the control group by Dunnett´s test

Applicant's summary and conclusion