Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Repeated dose dermal toxicity study of nano zinc oxide with Sprague-Dawley rats
Author:
Surekha P, Kishore AS, Srinivas A, Selvam G, Goparaju A, Reddy PN and Murthy PB.
Year:
2012
Bibliographic source:
Cutaneous and Ocular Toxicology, 31(1): 26–32

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
(different dose levels, biochemical parameters, and collagen content estimation)
GLP compliance:
not specified
Remarks:
GLP compliance not specified in publication
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc oxide
EC Number:
215-222-5
EC Name:
Zinc oxide
Cas Number:
1314-13-2
Molecular formula:
OZn
IUPAC Name:
oxozinc
Test material form:
solid: nanoform
Details on test material:
- Name of test material (as cited in study report): Nano ZnO
- Average size: 20 nm
- Average size using scanning electron microscopy (SEM): 63 nm
- Size in distilled water using dynamic light scattering: 224.7 nm
- Polydispersity index: 0.305
- Surface area using BET (Brunauer, Emmett,Teller) analysis: 50
- Zeta potential using zetasizer: −30.9 mV

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
The healthy Sprague-Dawley rats of both sex, aged between 6 and 8 weeks and body weights of 180–220 g were used. Female rats were nulliparous and nonpregnant. The animals were procured from breeding facilities of International Institute of Biotechnology and Toxicology. Animals were housed in polypropylene cages with stainless steel grills and gamma irradiated corn cobs were used as bedding. Bedding material, cages, grills, and water bottles were changed on alternate days. Animals were housed individually sex wise. Animals were acclimated for a minimum period of 5 d in the controlled environment (temperature: 22 ± 3°C; relative humidity: 50 ± 20%; and light: 12-h light/dark cycle) and ad libitum supply of reverse osmosis water and a standard rodent pellet food (supplier: M/s. Tetragon Chemie Pvt. Ltd., Bangalore, India). Food alone was withdrawn over-night prior to the blood collection on Days 0, 28, and 42. One hundred animals were distributed randomly into different groups.

Administration / exposure

Type of coverage:
open
Vehicle:
water
Details on exposure:
Approximately 10% of the total body surface area of each rat was closely clipped free of hair once in 5 d. The nano ZnO were moistened with distilled water to prepare a paste and the paste thus prepared was evenly applied at dose levels of 75, 180, and 360 mg/kg bw /day to the groups of male and female rats through dermal route to the clipped skin on a 5 d per week basis for 28 consecutive days. Microsize zinc oxide was applied at a limit dose of 2,000 mg/kg bw/day. Control group of animals were similarly treated but with distilled water alone. Restrainer was used to prevent the ingestion of the test
substance from the application sire. At the end of the 6-h exposure period, the residual nano and microsize zinc oxide were removed with cotton soaked in water without altering the integrity of the skin.
Duration of treatment / exposure:
28 d
Frequency of treatment:
6 h per day for 5 d/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 75, 180, and 360 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Selection of doses: In order to understand the risk associated with the realistic exposure to consumers, the potential daily uptake quantity of nanomaterial mg/kg bw/day was calculated as follows:
Uder pot= (Ader× n)/bw
Ader= Qprod X Fprod; where Uder pot is the potential daily uptake quantity of nanomaterial mg/kg bw/day; Ader the quantity of the active substance on the skin per application (mg); and n number of application times, minimum of single application was considered; bw is the average body weight of the consumer which is 60 kg bw; Qprod is the quantity product (sunscreen) recommended for an adult is 36,000 mg; Fprod concentration of active substance in the product. As per FDA monograph the maximum allowable concentration of zinc oxide is 2 to 20%. Considering the worst-case scenario, 2% was taken as the concentration of active substance in the product. Fprod = 2%; Ader=2/100×36000 = 720; Uder pot = 720×1/60=12 mg/kg bw/day
Keeping in view the above quantity of nanomaterial uptake for an average adult woman, we have calculated dose for rat using rat conversion factor with 1, 2.5, and 5 times.
Low dose = Uder pot × 1 × 6 = 12 × 1 × 6 = 72 rounded to 75
Intermediate dose = Uder pot × 2.5 × 6=12 × 2.5 × 6 = 180
High dose = Uder pot × 5 × 6 = 12 × 5 × 6 = 360

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (Daily)

DETAILED CLINICAL OBSERVATIONS: Yes (Daily)

DERMAL IRRITATION (if dermal study): Yes (Daily)

BODY WEIGHT: Yes (Weekly)

FOOD CONSUMPTION: Yes (Weekly)

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (Blood samples were collected from the orbital sinus of the overnight fasted rats on Day 28 from all groups and satellite groups on Day 42)

CLINICAL CHEMISTRY: Yes

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

COLLAGEN ESTIMATION: At the end of the experimental period (Days 28 and 42), the skin and tail of the animals were collected. The amount of hydroxyproline was estimated and the total collagen content was calculated.
Sacrifice and pathology:
Gross pathology was performed at the end of experimental period (Days 28 and 42). The wet weight of the liver, lungs, kidneys, spleen, brain, and heart were recorded at the end of the experimental period. Histopathology of organs (skin, liver, spleen, kidneys, heart, adrenals, lungs, pancreas, stomach, and representative lymph nodes) was evaluated. Tissues were collected and preserved in 10% buffered formalin. All tissues required for histopathology evaluation were subjected to dehydration procedure and processed in tissue processor, embedded in paraffin wax, and sectioned at 5–8 μm thickness and stained with hematoxylin-eosin stain.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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):
A statistically significant increase in clotting time was observed in all the treatment groups of nano zinc oxide with that of micro size ZnO.
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical biochemistry and hematology: No significant changes in clinical biochemistry parameters were observed in both micro and nano ZnO treated rats. There were no statistically significant changes in the hematologic parameters when compared with control. A statistically significant increase in clotting time was observed in all the treatment groups of nano zinc oxide with that of micro size ZnO.
Collagen content: There was a significant decrease in the collagen content of skin and tail in all the nano-size zinc oxide-treated group of rats compared with the control as well as with the micro-size ZnO treated group. An inverse dose-dependent relationship was observed in the treated groups.
Pathology: No gross pathological lesions were observed in any of the treatment groups. No histopathological lesions were observed in any of the organs observed.

Effect levels

Dose descriptor:
LOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increase in clotting time and decrease in the collagen content of skin and tail in all the nano-size zinc oxide-treated group of rats. However, these effects were reversible in a period of 14 d.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Amount of skin collagen, tail collagen, and percentage loss:

Group

Males

Females

Skin (g/100 g tissue)

% Loss

Skin (g/100 g tissue)

% Loss

Skin collagen

G1/Control

46.73 ± 5.80

37.31 ± 8.66

 

G2/Micro size2000 mg/kg bw

41.68 ± 2.21

10.80

34.21 ± 3.56

8.32

 

G3/Nano size 75 mg/kg bw

16.85 ± 2.27

63.94

20.70 ± 1.03

44.53

 

G4/Nano size 180 mg/kg bw

21.41 ± 3.84

54.19

24.43 ± 3.74

34.53

 

G5/Nano size 360 mg/kg bw

34.91 ± 1.72

25.30

29.19 ± 1.53

21.76

 

G6/Micro size satellite 2000 mg/kg bw

40.00 ± 2.31

10.00

38.63 ± 2.83

0.42

 

G7/Nano size satellite 75 mg/kg bw

21.16 ± 1.01

52.38

31.15 ± 5.50

19.69

 

G8/Nano size satellite 180 mg/kg bw

24.86 ± 3.86

44.07

34.16 ± 3.86

11.94

 

G9/Nano size satellite 360 mg/kg bw

39.11 ± 4.47

12.01

36.56 ± 1.53

5.74

 

G10/Control satellite

44.44 ± 5.70

38.79 ± 5.09

 

Group

Males

Females

Tail (g/100 g tissue)

% Loss

Tail (g/100 g tissue)

% Loss

Tail collagen

G1/Control

78.84 ± 12.40

68.70 ± 12.14

G2/Micro size 2000 mg/kg bw

72.58 ± 4.32

7.94

61.72 ± 3.21

10.16

 

G3/ Nano size 75 mg/kg bw

36.64 ± 4.28

53.53

41.95 ± 6.35

38.94

 

G4/ Nano size 180 mg/kg bw

48.13 ± 2.57

38.95

60.55 ± 2.27

11.87

 

G5/ Nano size 360 mg/kg bw

62.66 ± 2.65

20.52

57.30 ± 1.84

16.60

 

G6/ Micro size satellite 2000 mg/kg bw

70.26 ± 2.08

8.63

60.38 ± 2.34

12.51

 

G7/ Nano size satellite 75 mg/kg bw

52.59 ± 5.03

31.61

65.62 ± 4.07

4.93

 

G8/ Nano size satellite 180 mg/kg bw

63.60 ± 4.49

17.29

59.77 ± 5.41

 

13.40

G9/ Nano size satellite 360 mg/kg bw

70.18 ± 5.58

8.73

60.03 ± 2.95

13.02

 

G10/ Control satellite

76.89 ± 10.84

69.02 ± 5.49

 

Applicant's summary and conclusion

Conclusions:
Based on decrease in collagen content in all the nano ZnO treated groups, the LOAEL for systemic toxicity was at the lowest tested dose of 75 mg/kg bw/day. However, these effects were reversible in a period of 14 d.
Executive summary:

The study was conducted to determine the repeated dose dermal toxicity of nano ZnO according to the OECD guideline 410 with modifications for dose levels, biochemical parameters and measurement of collagen content.

Sprague-Dawley rats (5/sex/dose) were applied with three different doses (75, 180, and 360 mg/kg bw/day) of nano ZnO (20 nm) at 5 d/week basis for a period of 28 d. Animals were observed for mortality/morbidity, clinical signs of toxicity, weekly body weight, and weekly food consumption during the experimental period. Blood samples were collected from the orbital sinus of the overnight fasted rats on Day 28 from all groups and satellite groups on Day 42. Different groups of rats were killed on Days 28 and 42 for gross and histopathology. Skin and tail from all the groups were collected for collagen estimation.

No significant changes were observed in the clinical chemistry parameters in both micro and nano ZnO treated rats. There were no statistically significant changes in the hematologic parameters compared with the control. A statistically significant increase in clotting time was observed in all treatment groups of nano ZnO compared to the micro-sized ZnO. No gross pathology or histopathological lesions were observed in any of the organs investigated. There was a significant decrease in the collagen content of the skin and the tail in all the nano ZnO treated groups of rats compared to the control, as well as with the micro-sized ZnO treated groups. The loss was higher in the skin than in the tail. There was an inverse dose relationship with the higher doses inducing a lower decrease.

Based on increase in clotting time and decrease in the collagen content in all the nano ZnO treated groups, the LOAEL for systemic toxicity was established at the lowest tested dose of 75 mg/kg bw/day. However, these effects were reversible in a period of 14 d.