1,2,3-Propanetriol, mono- and diformates

Endpoint summary

Description of key information

Test results for oral and inhalation exposure to formic acid and glycerol (degradation products of 1,2,3-propanetriol, mono- and diformates) are available. Based on respiratory injuries reported in the 90d inhalation study, local long-term effects are used for the risk assessment (section 7).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Secondary literature about the use of potassium diformate (a degradation product of 1,2,3-propanetriol, mono- and diforamte) as food additive.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

not specified

GLP compliance:

not specified

Limit test:

no

Species:

pig

Route of administration:

oral: feed

Remarks:

Doses / Concentrations:
up to 18.000 mg/kg in the feedingstuff
Basis:

Dose descriptor:

NOAEL

Effect level:

18 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Critical effects observed:

not specified

Conclusions:

The European Food Safety Authority evaluated the use of potassium diformate in pigs. It was concluded that different potassium diformate can permitted up to a maximum level in complete feedingstuff of 18000 mg per kg complete feedingstuff for weaned piglets and
12000 mg per complete feedingstuff for sows and pigs for fattening.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

18 000 mg/kg bw/day

Study duration:

subchronic

Species:

pig

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 1987 - Mar 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, tested with the source substance formic acid (CAS No. 64-18-6). For details on read-across refer to the attached read-across report.

Reason / purpose for cross-reference:

reference to same study

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; additional examinations of effects on respiratory tract and reproductive organs.

GLP compliance:

yes

Limit test:

no

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

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

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

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.

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.

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.

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

Critical effects observed:

not specified

There were no mortalities nor clinical signs or systemic toxicity in male and female rats exposed to 8, 16, 32, 64, or 128 ppm for 13 weeks (5 days/week, 6 h/day). There were no unusual gross lesions noted during necropsy, organ weights were not affected by treatment. Male and female reproductive parameters (sperm motility, density, and testicular or epididymal weight; length of the estrous cycle) were not affected.

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

Conclusions:

Inhalation exposures to formic acid for 13 weeks in F344/N rats produced minimal systemic toxic effects. A NOAEL of 64 ppm was derived on local effects.

Executive summary:

In a subchronic inhalation toxicity study formic acid was administered to 10 F344 rats/sex/concentration by whole body inhalation exposure at concentrations of 0, 8, 16, 32, 64 and 128 ppm (15.3, 30.6, 61.2, 122.4 and 244.7 mg/m³) for 6 hours per day, 5 days/week for a total of 13 weeks.

Inhalation exposures to formic acid for 13 weeks in F344/N rats produced minimal systemic toxic effects. Hematologic and biochemical changes were mild . Gross and microscopic changes were confined to the upper respiratory tract. Effects on the respiratory and olfactory epithelium consisted of squamous metaplasia and degeneration. The NOAEL is 64 ppm based on local effects.

This subchronic inhalation toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic inhalation study OPPTS 870.3465; OECD 413 in the rodent. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

244 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 1987 - Mar 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, tested with the source substance formic acid (CAS No. 64-18-6). For details on read-across refer to the attached read-across report.

Reason / purpose for cross-reference:

reference to same study

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; additional examinations of effects on respiratory tract and reproductive organs.

GLP compliance:

yes

Limit test:

no

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

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

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

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.

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.

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.

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

Critical effects observed:

not specified

There were no mortalities nor clinical signs or systemic toxicity in male and female rats exposed to 8, 16, 32, 64, or 128 ppm for 13 weeks (5 days/week, 6 h/day). There were no unusual gross lesions noted during necropsy, organ weights were not affected by treatment. Male and female reproductive parameters (sperm motility, density, and testicular or epididymal weight; length of the estrous cycle) were not affected.

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

Conclusions:

Inhalation exposures to formic acid for 13 weeks in F344/N rats produced minimal systemic toxic effects. A NOAEL of 64 ppm was derived on local effects.

Executive summary:

In a subchronic inhalation toxicity study formic acid was administered to 10 F344 rats/sex/concentration by whole body inhalation exposure at concentrations of 0, 8, 16, 32, 64 and 128 ppm (15.3, 30.6, 61.2, 122.4 and 244.7 mg/m³) for 6 hours per day, 5 days/week for a total of 13 weeks.

Inhalation exposures to formic acid for 13 weeks in F344/N rats produced minimal systemic toxic effects. Hematologic and biochemical changes were mild . Gross and microscopic changes were confined to the upper respiratory tract. Effects on the respiratory and olfactory epithelium consisted of squamous metaplasia and degeneration. The NOAEL is 64 ppm based on local effects.

This subchronic inhalation toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic inhalation study OPPTS 870.3465; OECD 413 in the rodent. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

61 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The hazard assessment of 1,2,3-propanetriol, mono- and diformates is based on its degradation products glycerol and formic acid/formates. In 2-week studies, groups of 5 F344/N rats and 5 B6C3F1mice of each sex were exposed to formic acid for 6 hours a day, 5 days a week, at concentrations of 0, 31, 62.5, 125, 250, or 500 ppm. Deaths occurred in animals exposed to 500 ppm (rats and mice) and 250 ppm (1 female mouse). Microscopic lesions in the respiratory and olfactory epithelia occurred in rats and mice exposed to 62.5 ppm and higher concentrations, with severity related to the exposure concentration. The lesions consisted of squamous metaplasia, necrosis, and inflammation. Exposures had minimal or no effects on coagulation times, blood pH and electrolytes, or on concentrations and activities of urine analytes in rats during the 2-week studies. In 13-week studies, groups of 10 animals of each species and sex were exposed to formic acid at concentrations of 0, 8, 16, 32, 64, and 128 ppm for 6 hours a day, 5 days a week. Two mice, 1 male and 1 female, died in the 128 ppm groups. Body weight gains were significantly decreased in mice exposed to 64 and 128 ppm formic acid. Microscopic changes in rats and mice ranged from minimal to mild in severity and generally were limited to animals in the 128 ppm groups. Lesions related to exposure to formic acid consisted of squamous metaplasia and degeneration of the respiratory and olfactory epithelia, respectively. Haematologic and serum biochemical changes at interim and terminal time points were minimal to mild and, generally, were consistent with haemoconcentration. Overall, the effects of formic acid were consistent with those of irritant chemicals administered by inhalation exposure. The no-observed-adverse-effect level (NOAEL) for respiratory injury was 32 ppm in rats and mice. There was no significant evidence of systemic toxicity in these studies. From available data it was concluded, that that formic acid/formates is more relevant for the risk assessment compared to glycerol. Refer to the read across report for further details.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The degradation products of 1,2,3-propanetriol, mono- and diformate (formate and glycerol) have been evaluated in a variety of laboratory studies. 1,2,3-Propanetriol and several formates (e.g. potassium diformate) have been evaluated for the use as food additives and are recognized safe (GRAS) when applied by the oral route. Thus, the oral route is not considered relevant for the assessment of systemic effects.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Hazard assessment is based on the degradation products of 1,2,3-propanetriol, mono- and diformates (formate and glycerol, refer to the read across report for further details). The NOAEC refers to the raw data (128 ppm formic acid).

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Hazard assessment is based on the degradation products of 1,2,3-propanetriol, mono- and diformates (formate and glycerol, refer to the read across report for further details). The NOAEC refers to the raw data (32 ppm formic acid).

Justification for classification or non-classification

Based on the assessment of degradation products, 1,2,3-propanetriol, mono- and diformates does not meet the classification criteria according to Regulation (EC) 1272/2008 or according to Directive 67/548/EEC.