Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
2009
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Females nulliparous and non-pregnant: yes
- Weight at study initiation: 248.5-250.2 g (males) and 199.5-200.2 g (females)
- Housing: housed in pairs in cages with softwood granulate bedding and environmental enrichment was provided
- Diet: gamma-irradiated pellet diet, ad libitum
- Water: sterilized drinking water, ad libitum
- Acclimation period: ≥ 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 52-55
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: phosphate-buffered saline (PBS)
Mass median aerodynamic diameter (MMAD):
ca. 1.5 µm
Geometric standard deviation (GSD):
1.75
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past nose-only exposure chambers
- System of generating particulates/aerosols: Aerosols were generated by Collison Nebulizers and diluted with filtered conditioned fresh air.
- Air flow rate: continuous flow rate
- Method of particle size determination: Aerodynamic particle size (APS) spectrometer

TEST ATMOSPHERE
- Brief description of analytical method used: Levels of pyruvate were measured by enzymatic means by a commercial Pyruvate Detection kit (Cayman Chemicals, Ann Arbor, MI) in total particulate matter (TPM) samples of the aerosols collected on Cambridge filter pads.

VEHICLE
- Composition of vehicle: phosphate-buffered saline
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Levels of pyruvate were measured by enzymatic means by a commercial Pyruvate Detection kit (Cayman Chemicals, Ann Arbor, MI) in total particulate matter (TPM) samples of the aerosols collected on Cambridge filter pads. Sampled were taken at least 3 times a day with a sampling time of 20 minutes. The analytical method used was an indirect enzymatic fluorescence assay.
Duration of treatment / exposure:
6 hours / day
Frequency of treatment:
5 days / week
Doses / concentrations
Dose / conc.:
0.034 mg/L air
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
yes, sham-exposed
Details on study design:
- Dose selection rationale: a range finding study was performed

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included check for mortality and health status

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: at least twice a week

FOOD CONSUMPTION:
- Food consumption was determined by gravimetry once a week.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 28
- Anaesthetic used for blood collection: Yes, pentobarbital
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 28
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: once during the study
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: No
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see table 4 and table 5)

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
All rats had an increase in body weight throughout the exposure period. No effects compared with sham groups were observed.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No exposure-related effects were observed in male and female rats.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No exposure-related effects were observed in male and female rats.
Urinalysis findings:
not specified
Description (incidence and severity):
A fecal contamination occured. Therefore, data were not reported.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Spleen weights in female rats elicited a different response, with higher weights after exposure to sodium pyruvate. This result in females, however, appeared to be due to the low spleen weight in the sham group. No exposure-related effects were observed in male and female rats.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were several macroscopic observations of organs at necropsy; but they were specifically related to neither treatment nor exposure concentration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In the mid-base of epiglottis a slight increase in ventral-squamous hyperplasia was observed in males exposed to sodium pyruvate. Overall cytoplasmic vacuolation of hepatocytes was observed in the livers of most rats of the study, and increased vacuolation could be observed in rats exposed to sodium pyruvate. Vacuolation was increased in the livers of female rats that inhaled sodium pyruvate compared with sham and PBS control females, but this difference was small. A similar effect was not seen in males, and sodium pyruvate was not considered to have contributed to this change.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
0.034 mg/L air
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effects observed up to the highest concentration tested.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 6: Organ weights in male rats

 

 

Sham

PBS

Sodium Pyruvate

Liver

g

11.77 ± 0.391

12.19 ± 0.455

12.08 ± 0.360

X10E-4

348.97 ± 7.986

371.22 ± 7.420

362.39 ± 7.275

N

10

10

10

Spleen

g

0.69 ± 0.040

0.73 ± 0.099

0.65 ± 0.031

X10E-4

20.61 ± 1.173

22.40 ± 3.130

19.53 ± 0.942

N

10

10

10

Left adrenal

g

0.03 ± 0.009

0.02 ± 0.002

0.03 ± 0.001

X10E-4

0.97 ± 0.257

0.75 ± 0.078

0.78 ± 0.036

N

10

8

10

Right adrenal

g

0.02 ± 0.002

0.03 ± 0.001

0.02 ± 0.001

X10E-4

0.73 ± 0.045

0.77 ± 0.036

0.71 ± 0.030

N

9

9

10

Left kidney

g

1.07 ± 0.032

1.04 ± 0.055

1.03 ± 0.021

X10E-4

31.89 ± 0.722

31.95 ± 1.688

30.92 ± 0.713

N

10

10

10

Right kidney

g

1.09 ± 0.031

1.08 ± 0.056

1.03 ± 0.026

X10E-4

32.20 ± 0.738

33.10 ± 1.812

31.07 ± 0.629

N

10

10

10

Thymus

g

0.31 ± 0.021

0.31 ± 0.023

0.34 ± 0.026

X10E-4

9.12 ± 0.598

9.43 ± 0.583

10.23 ± 0.776

N

10

10

10

Lung/Larynx/

Trachea

g

1.40 ± 0.025

1.44 ± 0.047

1.46 ± 0.047

X10E-4

41.76 ± 1.064

44.21 ± 1.480

44.08 ± 1.950

N

10

9

10

Brain

g

2.05 ± 0.023

2.04 ± 0.072

2.08 ± 0.033

X10E-4

60.94 ± 1.146

62.21 ± 2.088

62.39 ± 0.955

N

10

10

10

Heart

g

1.07 ± 0.034

1.08 ± 0.025

1.07 ± 0.020

X10E-4

31.82 ± 0.760

33.08 ± 0.491

32.18 ± 0.802

N

10

10

10

Left testis

g

1.63 ± 0.046

1.36 ± 0.140

1.66 ± 0.025

X10E-4

48.25 ± 1.204

41.74 ± 4.378

50.00 ± 1.204

N

10

10

10

Right testis

g

1.61 ± 0.051

1.38 ± 0.142

1.61 ± 0.034

X10E-4

47.94 ± 1.358

42.12 ± 4.429

48.62 ± 1.433

N

10

10

10

Absolute and relative body weight, mean ± SD

Table 7: Organ weights in female rats

 

 

Sham

PBS

Sodium Pyruvate

Liver

g

9.17 ± 0.387

10.02 ± 0.507

9.50 ± 0.293

X10E-4

388.30 ± 11.643

418.08 ± 21.681

406.83 ± 8.813

N

10

10

10

Spleen

g

0.46 ± 0.027

0.61 ± 0.069+

0.55 ± 0.028*

X10E-4

19.80 ± 1.060

25.50 ± 3.115

23.64 ± 1.095*

N

9

9

10

Left adrenal

g

0.04 ± 0.013

0.04 ± 0.001

0.03 ± 0.001

X10E-4

1.85 ± 0.486

1.53 ± 0.045

1.42 ± 0.048

N

9

9

10

Right adrenal

g

0.03 ± 0.002

0.03 ± 0.001

0.03 ± 0.001

X10E-4

1.27 ± 0.105

1.31 ± 0.056

1.42 ± 0.048

N

8

9

10

Left kidney

g

0.77 ± 0.016

0.85 ± 0.059

0.78 ± 0.019

X10E-4

33.01 ± 0.629

35.69 ± 2.555

33.37 ± 0.585

N

9

9

10

Right kidney

g

0.78 ± 0.017

0.83 ± 0.037

0.82 ± 0.017

X10E-4

33.35 ± 0.618

34.89 ± 1.603

35.02 ± 0.688

N

9

9

10

Thymus

g

0.35 ± 0.034

0.45 ± 0.103

0.33 ± 0.024

X10E-4

14.81 ± 1.412

18.24 ± 4.025

14.22 ± 0.900

N

10

9

10

Lung/Larynx/

Trachea

g

1.27 ± 0.036

1.56 ± 0.175

1.19 ± 0.026

X10E-4

53.86 ± 1.382

64.77 ± 6.851

51.10 ± 1.212

N

9

9

10

Brain

g

1.85 ± 0.091

2.00 ± 0.034

1.89 ± 0.034

X10E-4

79.00 ± 4.427

83.52 ± 1.590

81.25 ± 2.316

N

10

8

10

Heart

g

0.91 ± 0.073

0.83 ± 0.062

0.82 ± 0.021

X10E-4

38.46 ± 2.629

34.59 ± 2.830

35.40 ± 0.876

N

10

10

10

 Absolute and relative body weight, mean ± SD, *p<0.05

Applicant's summary and conclusion

Conclusions:
No treatment-related effects were observed in Sprague-Dawley rats after inhalative nose-only exposure to sodium pyruvate (33.9 µg/L) for 28 days, 6 h/day, 5 days/week.
Executive summary:

A sub-chronic inhalation study according to OECD guideline 412 was performed with Sprague-Dawley rats. The rats were nose-only exposed, 6 h/day, 5 days/week for 28 days to filtered air, saline, nicotine (50 µg/L), sodium pyruvate (33.9 µg/L) or equimolar nicotine/pyruvic acid mixtures (18, 25 and 50 µg nicotine/L).

After exposure to sodium pyruvate no exposure-related effects on body weight, haematological parameters and clinical parameters were observed. Spleen weights in female rats elicited a different response, with higher weights after exposure to sodium pyruvate. This result in females, however, appeared to be due to the low spleen weight in the sham group. No further changes in organ weights were observed after exposure to sodium pyruvate. No treatment-related gross pathological findings were revealed after inhalation of sodium pyruvate. In the mid-base of epiglottis a slight increase in ventral-squamous hyperplasia was observed in males exposed to sodium pyruvate. Overall cytoplasmic vacuolation of hepatocytes was observed in the livers of most rats of the study, and increased vacuolation could be observed in rats exposed to sodium pyruvate. Vacuolation was increased in the livers of female rats that inhaled sodium pyruvate compared with sham and PBS control females, but this difference was small. A similar effect was not seen in males, and sodium pyruvate was not considered to have contributed to this change. These results suggest that aerosols of sodium pyruvate did not result in substantial toxicity.