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Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Thirty-two adult male Wistar rats were used. Their backs were shaved and 2% Evans blue was injected intravenously into the lateral caudal tail vein. Immediately afterwards, 0.1 mL of citric acid solution was injected intradermally into the experimental site. The animals were assigned to 4 groups according to the time period tested. The animals were killed 30 minutes as well as 1, 3 and 6 hours after injection of the solution. The dorsal skin was dissected away and skin lesions were punched out. Each piece of skin containing the lesion was cut into small fragments and the dye was extracted upon immersion in 10 mL formamide. Then, the optical density was measured at 620 μm in a spectrophotometer The total amount of dye extracted from the samples was calculated by means of a standard calibration curve.
GLP compliance:
not specified
Remarks:
not specified in the publication
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: adult
- Weight at study initiation: approx. 350 g
Type of coverage:
open
Preparation of test site:
shaved
Vehicle:
other: deionized water
Controls:
yes
Amount / concentration applied:
not specified
Duration of treatment / exposure:
not applicable
Observation period:
30 minutes as well as 1, 3 and 6 hours after injection of the solutions
Number of animals:
32 male rats (8 animals/observation time point)
Details on study design:
NOTE: three further test item solutions were tested with the same animals as used for testing with the citric acid solution.

DOSAGE PREPARATION:
The tested acid solution was freshly prepared at the laboratory. The salt were weighed and diluted in deionized water, and pH was adjusted (pH meter B371, Micronal).

EXPERIMENTAL METHOD:
The rats were anesthetized with sulfuric ether, their backs were shaved. Their tails were washed and dried to facilitate the injections of 2% Evans blue (20 mg/kg) administered intravenously into the lateral caudal vein. Immediately afterwards, 0.1 mL of the test solution was injected intradermally into the experimental site. The animals were assigned to 4 groups according to the time period tested. The animals were killed 30 minutes, 1, 3 and 6 hours after injection of the solution. The dorsal skin was dissected away and skin lesions were punched out with a standard steel punch (3-cm diameter). Each piece of skin containing the lesion was cut into small fragments and the dye was extracted upon immersion in 10 mL formamide for 72 hours at 45 °C. After filtration with glass filter, the optical density was measured at 620 μm in a spectrophotometer (Ultrospec 2000, Pharmacia Biotech). The total amount of dye extracted from the samples was calculated by means of a standard calibration curve. Data were analyzed statistically by two-way ANOVA and Tukey’s HSD test.
Remarks on result:
other: see "Remarks"
Remarks:
According to the authors, compared to saline (139.55 µg) a larger amount of dye was extracted after citric acid administration (329.81 µg), for all four periods evaluated. Furthermore, considering the time period, a significant difference (p<0.05) was observed between the 3 hour and 6 hour groups, but not between the 30-min and 1 hour groups. Also, the authors stated that there was an enhanced vascular permeability at the 6 hour experimental time. Although a statistically significant difference was seen between citric acid and saline, the means of dye extracted in these groups were numerically close to each other. Lastly, the authors stated that the results indicated that the citric acid solution yielded a continuous irritating potential even after 6 hours.
Irritant / corrosive response data:
Compared to saline (139.55 µg) a larger amount of dye was extracted after citric acid administration (329.81 µg), for all four periods evaluated.
Considering the time period, a significant difference (p<0.05) was observed between the 3 hour and 6 hour groups, but not between the 30-min and 1 hour groups. There was an enhanced vascular permeability at the 6 hour experimental time.
Although a statistically significant difference was seen between citric acid and saline, the means of dye extracted in these groups were numerically close to each other.
The results indicated that the citric acid solution yielded a continuous irritating potential even after 6 hours.

Please aslo refer to the field "Any other information on results incl. tables" below.

Table 1: Means (μg) and standard deviations (± SD) of the total amount of dye extracted in the control and experimental groups.

Time

Citric acid

Saline

30 minutes

280.92 ± 140.84

153.93 ± 47.15

1 hour

271.41 ± 135.09

115.68 ± 48.57

3 hours

286.43 ± 102.08

115.36 ± 58.60

6 hours

480.50 ± 212.34

173.23 ± 51.31

Conclusions:
According to the authors, compared to saline (139.55 µg) a larger amount of dye was extracted after citric acid administration (329.81 µg), for all four periods evaluated. Furthermore, considering the time period, a significant difference (p<0.05) was observed between the 3 hour and 6 hour groups, but not between the 30-min and 1 hour groups. Also, the authors stated that there was an enhanced vascular permeability at the 6 hour experimental time. Although a statistically significant difference was seen between citric acid and saline, the means of dye extracted in these groups were numerically close to each other. Lastly, the authors stated that the results indicated that the citric acid solution yielded a continuous irritating potential even after 6 hours.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Deviations to the OECD guideline 405 (1981): purity/stability of test item missing; 5 % solution was tested only; observation period lasted only 7 day instead of 21 days (conjunctivitis was not reversed after 7 days); individual animal data missing (the severity of the conjunctivitis could not be determined from the data provided in the publication, which makes it impossible to decide on classification or non-classification for eye irritation); eye washing schedule was not according to the guideline; housing conditions were not fully described (exception: water/food and acclimatisation period only)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1981-05-12
Deviations:
yes
Remarks:
please refer to the field "Rationale for reliability incl. deficiencies" above
GLP compliance:
not specified
Remarks:
not specified in the publication, but GLP was not compulsory at time of study conduct
Specific details on test material used for the study:
not applicable
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Zartman Farms, PA
- Weight at study initiation: 2.0 - 2.5 kg
- Diet (ad libitum)
- Water (ad libitum)
- Acclimation period: 7 days
Vehicle:
not specified
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration: 5.0 % (w/v)
Duration of treatment / exposure:
30 seconds (treatment group with washed eyes only)
Observation period (in vivo):
prior to administration as well as 1 hour and days 1, 2, 3 and 7 after instillation
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
6 rabbits/group (the eyes of one group were washed after exposure and the eyes of the second group were not washed after exposure)
Details on study design:
APPLICATION PROCEDURE:
The right eyes of the rabbits were used for testing. The lids of the eye were held apart and the test material was placed directly on the central portion of the cornea instead of placing it in a cup formed by the conjuctival sac. The lids were then gently brought together for approximately 1 second and released. The left eye served as an untreated control.
A 1.0-mL tuberculin syringe was used to instill 0.1 mL of the test material solution onto the central aspect of the cornea.

REMOVAL OF TEST SUBSTANCE
- Washing: the eyes of the one group were gently washed for 2 minutes with 300 mL of tap water
- Time after start of exposure: 30 seconds after exposure to the test material

SCORING SYSTEM:
The eyes were examined grossly and the grades of damage and irritation to the cornea, iris and conjunctiva were recorded at all observation times. All grading of eye irritation was performed by the method of Draize et al. (1944)*, prior to the instillation of fluorescein, at the specified times. The area of corneal damage, pannus and keratoconus were not included in the scoring system but were recorded separately. Each portion of the eye was considered independently without reference to the total score.

The following categories were established for both washed and unwashed rabbit eyes for the purpose of rating chemicals as to severity of ocular reactions.
(1) Severe: corneal opacity, iritis and conjunctivitis-positive at 24 hours, one or more of the treated eyes still exhibit opacity, iritis, and conjunctivitis at the 7th day.
(2) Moderate: corneal opacity and/or iritis and conjunctivitis-positive at 24 - 72 hours, conjunctivitis and iritis remaining at 7th day.
(3} Irritant: iritis and/or conjunctivitis-positive at 24 hours, eyes normal at 3rd day.
(4) Non-irritant: no positive responses in any of the test animals at 24 hours.

Thus, a corneal opacity of grade 1 or more, iritis of grade 1 or more, and/or conjunctival redness and chemosis each of grade 2 or more are considered positive.

TOOL USED TO ASSESS SCORE: fluorescein
Exposed eyes of the washed group were stained with 1 drop of a 2% fluorescein solution 1 hour after the initial instillation; exposed eyes of both groups were stained at 24 h and at 3 days. The stain was allowed to remain in the eye for 15--20 seconds and then was flushed out with approx. 5 -10 mL of sterile isotonic saline.

pH MEASUREMENTS:
The pH of all solutions was measured with the Corning Model 110 pH meter by using the Beckman 4049 standard calomel reference glass electrode with ceramic pin.

*Reference:
- J.H. Draize, G. Woodard and H.N. Calvery, J. Pharmacol. Exp. Ther., 82 (1944) 377, 389.
Remarks on result:
other: Acc. to the authors, no corneal opacity was observed in the washed & unwashed eyes. Conjunctivitis was present in all animals tested & lasted through day 7. Lastly, they stated that no pannus or keratoconus was observed in the washed & unwashed eyes.
Irritant / corrosive response data:
pH MEASUREMENT:
The pH of the test item concentration is 2.1.

EYE MEASUREMENTS:
- no corneal opacity was observed in the washed and unwashed eyes.
- conjunctivitis was present in all animals tested and lasted through day 7.
- no pannus or keratoconus was observed in the washed and unwashed eyes.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
According to the authors, no corneal opacity was observed in the washed and unwashed eyes. Conjunctivitis was present in all animals tested and lasted through day 7. Lastly, they stated that no pannus or keratoconus was observed in the washed and unwashed eyes.
According to the EC Regulation No. 1272/2008 and subsequent adaptations, the substance is classified as an eye irritant (Category 2; H319).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation:

Substance specific information is not available. Instead read-across to citric acid was performed although (in principle) read-across should be avoided to predict local effects. For the following reason read-across was used:

Depending on solution pH, citrate anions exist ascitrate, hydrogencitrate, dihydrogencitrate and citric acid  species inaqueous solutions under physiologically/environmentally relevant conditions as summarised in the following equations:

 

C6H8O7+ H2O <->(C6H7O7)- + H3O+                       [pka1: 3.13; citric acid             <->dihydrogencitrate]

(C6H7O7)- + H2O <-> H3O++(C6H6O7)2-                 [pKa2: 4.76;dihydrogencitrate <-> hydrogencitrate]

(C6H6O7)2-+ H2O <->(C6H5O7)3-+H3O+                [pKa3:6.40; hydrogencitrate    <->citrate]

Since an aqueous 0.1 molar solution of diammonium hydrogen citrate (which acts as a weak acid) has a measured pH value of ca. 4.3 local effects cannot be excluded. Hence, a worst case information was used to cover this endpoint.

A non-guideline study performed with citric acid (15% solution) afforded the following findings:

The authors stated that the results indicated that the citric acid solution yielded a continuous irritating potential even after 6 hours.

Eye irritation:

The citric acid monohydrate (5 % solution (w/v)) was observed to be irritating to the eyes in a reliable in vivo eye irritation study equivalent or similar to OECD 405. Furthermore, CIR (2012) stated that "citric acid was predicted to be a moderate/severe to severe/extreme ocular irritant in in vitro studies, and it was minimally irritating to rabbit eyes at a concentration of 10 % and mildly irritating at a concentration of 30 %".

Reference:

- CIR (2012) Final Report: On the safety assessment of citric acid, inorganic citrate salts, and alkyl citrate esters as used in cosmetics. 1 - 36.

Justification for classification or non-classification

Skin irritation

Considering available information of the corresponding acid (citric acid), diammonium hydrogen citrate it is assumed that the substance does possess a skin irritation potential. According to Regulation (EC) No 1272/2008 and its subsequent adaptations the substance should be classified as Category 2; H315.

Eye irritation

Considering available information of the corresponding acid (citric acid), diammonium hydrogen citrate it is assumed that the substance does possess an eye irritation potential. An in vivo eye irritation test (equivalent or similar to OECD 405) for citric acid is avaialble and proposes classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations (Category 2; H319).