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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Aug - 12 Oct 1984
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: EPA 560/6-82-001
Deviations:
yes
Remarks:
see section "Any other information on material and methods"
Qualifier:
according to guideline
Guideline:
other: Buehler dermal sensitization test. LAIR Standard Operating Procedure OP-STX-82, Letterman Army Institute of Research, Presidio of San Francisco, CA. 18 May 1984
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
Study from 1988 is readily available.

Test material

Constituent 1
Chemical structure
Reference substance name:
1-nitroguanidine
EC Number:
209-143-5
EC Name:
1-nitroguanidine
Cas Number:
556-88-7
Molecular formula:
CH4N4O2
IUPAC Name:
N-nitroguanidine
impurity 1
Chemical structure
Reference substance name:
Sodium sulphate
EC Number:
231-820-9
EC Name:
Sodium sulphate
Cas Number:
7757-82-6
Molecular formula:
Na2O4S
IUPAC Name:
disodium sulfate
impurity 2
Chemical structure
Reference substance name:
Sodium nitrate
EC Number:
231-554-3
EC Name:
Sodium nitrate
Cas Number:
7631-99-4
Molecular formula:
HNO3.Na
IUPAC Name:
sodium nitrate
impurity 3
Chemical structure
Reference substance name:
4,6-diamino-1,3,5-triazin-2(1H)-one
EC Number:
211-455-1
EC Name:
4,6-diamino-1,3,5-triazin-2(1H)-one
Cas Number:
645-92-1
Molecular formula:
C3H5N5O
IUPAC Name:
4,6-diamino-1,3,5-triazin-2(1H)-one
impurity 4
Chemical structure
Reference substance name:
6-amino-1,3,5-triazine-2,4(1H,3H)-dione
EC Number:
211-456-7
EC Name:
6-amino-1,3,5-triazine-2,4(1H,3H)-dione
Cas Number:
645-93-2
Molecular formula:
C3H4N4O2
IUPAC Name:
6-amino-1,3,5-triazine-2,4(1H,3H)-dione
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Test substance supplier Sunflower AAP

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Age at study initiation: approx. 4 weeks
- Weight at receipt: 140-233 g
- Housing: individual housing in stainless steel wire mesh cages in racks equipped with automatically flushing dump tanks; no bedding was used in any of the cages
- Diet (e.g. ad libitum): Certified Pig Chow Diet 5026 (Ralstom Purina Company, Checkerboard Square, St. Louis, MD 63188)
- Water: continous drip from a central line
- Acclimation period: approx. 3 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 -25.6
- Humidity (%): 28-50 (occasional spikes as high as 70% > room washing)
- Air changes (per hr):
- Photoperiod (hrs dark/hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
NiGu was applied as a 10 % solution. A pilot study, using 100 %, 10 %, 1 %, and 0.1 % concentrations, indicated 10 % solution to be the highest non-irritating concentration under the conditions of this test.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
NiGu was applied as a 10 % solution. A pilot study, using 100 %, 10 %, 1 %, and 0.1 % concentrations, indicated 10 % solution to be the highest non-irritating concentration under the conditions of this test.
No. of animals per dose:
Ten animals were assigned to each of four groups by a stratified randomization technique based on their body weights.
Details on study design:
RANGE FINDING TESTS:
A pilot study, using 100 %, 10 %, 1 %, and 0.1 % concentrations, indicated the 10 % solution to be the highest non-irritating concentration under the conditions of this test.

MAIN STUDY
A. INDUCTION EXPOSURE
- Site: no details reported; the same application site was used for each induction dose; to distinguish between reactions from repeated insult and sensitization, duplicate patches of the challenge dose were applied, one on the old site and one on a new site
- Negative control group: to distinguish between reactions from primary irritation and sensitization, negative control groups were added which received only the challenge dose (nitroguanidine)
- Positive control group: dinitrochlorobenzene (DNCB), a known potent sensitizing agent, was applied to one group, at a 0.1 % concentration
- Vehicle control group: isotonic saline was applied to one group
- Frequency of applications: once a week for three weeks
- Duration: 6 hours under a closed patch
- Experimental, saline control, and positive control groups: dosed with 0.5 ml of the appropriate compound applied topically under a one-inch square guze patch; this procedure was performed for three consecutive weeks (12 Sep, 19 Sep, and 26 Sep 1984); the day before each dosing a three-inch square area on the left side of the animal was clipped with an electric clipper and then shaved with an electric razor
- The patch was taped with with hypoallergenic surgical tape to the same site each time and the animal was wrapped several times with Vetrap
- The patch was left in place for six hours
- When the wrap and patch were removed, the area under the patch was merked off for scoring
- Evaluation (h after each induction dose): skin was scored 24 and 48 h after induction


B. CHALLENGE EXPOSURE
- Animals were challenged 2 weeks following the third induction phase
- Test groups: the experimental group and the positive control group received two 0.5 ml doses, one applied to the old site on the left side and the other to a new site on the right side
- Control group: negative and vehicle control groups only received a single 0.5 ml dose which was applied to the left side
- Exposure period: 6 hours under a closed patch
- Evaluation (h after challenge): skin was scored 24 and 48 h after challenge


OTHER:
A. Compound preparation:
- mixing of 0.5 g NGu with 0.5 ml of isotonic saline to make a paste
- dinitrochlorobenzene dosing solution was prepared by first adding 30 mg DNCB to 1 ml of propylene glycol and this heating until dissolved (approx. 40 °C); to this 29 ml of 0.9% sodium chloride solution were added, to give a final concentration of 0.1 % (w/v); this solution was heated to 65 °C and vortexed before application to keep the DNCB in solution; DNCB solutions were prepared fresh for each application day
Positive control substance(s):
yes
Remarks:
Dinitrochlorobenzene

Results and discussion

Positive control results:
DNCB produced a positive response at all time points. Between 40 % and 100 % of the DNCB-treated animals exhibited a response 24 h following the second and/or thirs induction and challenge doses. These reactions persisted, yielding scorable effects in 20 to 90% of the animals at 48 h after dosing.
Severity scores for these responses to DNCB ranged from 0.2 to 1.0 at the 24-hour scoring period; see table 3). The highest score, 1.0, was observed on the left (induction) side in response to the challenge dose. By 48 h the reactions had subsided somewhat; consequently, the severity range decreased to 0.1 to 0.95 (Table 4).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 % concentration solution
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
not observed
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 % concentration solution. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: not observed.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10 % concentration solution
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
not observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10 % concentration solution. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: not observed.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.1 % (w/v)
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
not observed
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.1 % (w/v). No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: not observed.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1 % (w/v)
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
not observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.1 % (w/v). No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: not observed.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
not observed
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: not observed.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
not observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: not observed.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10 % concentration solution
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
not observed
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10 % concentration solution. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: not observed.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10 % concentration solution
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
not observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10 % concentration solution. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: not observed.

Any other information on results incl. tables

Skin reactions were assigned scores according to Buehler’s grading system: 0 (no reaction), 1 (slight erythema), 2 (moderate erythema) and 3 (marked erythema). The results are expressed both in terms of incidence (the number of animals showing responses of 1 or greater at either 14 or 48 h) and severity (the sum of the test scores divided by the number of animals tested). Results from the left side are compared with the right side and with the negative control group.

Tables 1 and 2 summarize the incidence of reactions 24 and 48 h after each dose. There were no reactions observed in response to nitroguanidine administration, either at 24 or 48 h.

Table 1: Incidences of skin reactions after 24 h

 

Induction

Challenge

Test Group

First

Second

Third

Left

Right

Nitroguanidine

0/10

0/9

0/9

0/9

0/9

Negative Control *

----

----

----

0/9

0/9

Saline Vehicle

1/10

0/10

0/10

1/10

0/10

DNCB

1/10

4/10

4/10

9/10

8/10

* The Negative Control Group received only a challenge dose of the test compound.

 

Table 2: Incidences of skin reactions after 48 h

 

Induction

Challenge

Test Group

First

Second

Third

Left

Right

Nitroguanidine

0/10

0/9

0/9

0/9

0/9

Negative Control *

----

----

----

0/9

0/9

Saline Vehicle

0/10

0/10

0/10

0/10

0/10

DNCB

1/10

2/10

6/10

9/10

6/10

* The Negative Control Group received only a challenge dose of the test compound.

This lack of response is reflected in Tables 3 and 4, which report the severity of skin reactions at 24 and 48 hours. Response severity for each group is calculated by summing the scores of responding animals and dividing by the total number of animals within the group. This produced a severity index of 0.0 for nitroguanidine.

Table 3: Severity of skin reactions after 24 h

 

Induction

Challenge

Test Group

First

Second

Third

Left

Right

Nitroguanidine

0.0

0.0

0.0

0.0

0.0

Negative Control *

----

----

----

0.0

0.0

Saline Vehicle

0.1

0.0

0.0

0.05

0.0

DNCB

0.05

0.3

0.2

1.0

0.95

* The Negative Control Group received only a challenge dose of the test compound.

 

Table 4: Severity of skin reactions after 48 h

 

Induction

Challenge

Test Group

First

Second

Third

Left

Right

Nitroguanidine

0.0

0.0

0.0

0.0

0.0

Negative Control *

----

----

----

0.0

0.0

Saline Vehicle

0.0

0.0

0.0

0.0

0.0

DNCB

0.05

0.3

0.4

0.95

0.75

* The Negative Control Group received only a challenge dose of the test compound.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Nitroguanidine exhibited no potential for inducing dermal sensitization based on a zero percent sensitization rate in this study.
Executive summary:

In a dermal sensitization study with Nitroguanidine in saline solution, nine four weeks old, male guinea pigs (Hartley strain) were tested in accordance with LAIR SOP-OP-STX-82 “Buehler Dermal Sensitization Test” and EPA Guideline 560/6-82-001.

Nitroguanidine was evaluated for its ability to elicit a delayed hypersensitivity reaction via dermal contact. Using the method of Buehler and Griffith, no response indicative of dermal sensitization produced by nitroguanidine could be observed. Therefore, in this study, nitroguanidine showed no evidence of potential to elicit an allergic response.

Any sensitizing produced by nitroguanidine would have been easily detected by this study. An allergic response was reliably elicited by DNCB in the present group of animals. This response to DNCB was characteristic of that observed previously within the institute. Although DNCB is capable of producing primary irritation, the responses observed in this study were indicative of an allergic reaction since the concentration of DNCB used for the induction and challenge doses was too low to produce primary irritation. With the exception of a very slight irritation response in one animal 24 hours after the first induction, responses to DNCB were observed only after two or more exposures and the severity generally increased with the number of previous exposures.

Nitorguanidine, based on a zero percent sensitization rate in this study, exhibited no potential for inducing dermal sensitization.