Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 May 2016 to 30 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
OECD Guidelines for Testing of Chemicals 405 (2nd October 2012)
Deviations:
yes
Remarks:
see "Any other information" for details
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
Commission Regulation (EC) No 440/2008, B.5 (L 142, 30 May 2008)
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
OPPTS 870.2400 (EPA 712-C-98-195) August 1998
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Purity : 99%
Batch number: PCAF151028 Rs
CAS number : 21056-98-4
Chemical name : Phosphonic acid, calcium salt (1:1), Calcium hydrogen phosphonate
Manufacturing date : 28/10/2015
Shelf life or Expiry date : 28/10/2018
Date of analysis : 27/11/2015
Specific details on test material used for the study:
No further details specified in the study report.

Test animals / tissue source

Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Species and strain: New Zealand White rabbits
Source: S&K-LAP Kft., 2173 Kartal, Császár út 135, Hungary
Justification of strain: The New Zealand White rabbit is one of the standard strains used for acute irritation toxicity studies.
Animal health: Only animals in acceptable health condition were used for the test. Both eyes of each animal provisionally selected for testing were examined prior to starting the study. Animals showing eye irritation, ocular defects or pre-existing corneal injury were not used.
Number of animals: 3 animals
Age of animals at treatment: 11 weeks old
Sex: Male
Body weight range on the day of treatment: 2857 g – 3134 g before euthanasia: 2901 g – 3255 g
Date of receipt: 11 May 2016
Acclimatization time: 6-7 days
Animal identification: The animals were identified by engraved ear tags. The cages were marked with individual identity cards with information about study code, sex, cage number, dose and individual animal number.
HUSBANDRY
Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
Housing/Enrichment: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
Number of animal room: 610
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1 – 23.2 °C
Relative humidity: 25 – 69%
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity values were measured continuously. The measured range was checked at least daily during the acclimatisation and experimental phases.
FOOD AND FEEDING
Animals received UNI diet for rabbits produced by Cargill Takarmány Zrt., H-5300 Karcag, Madarasi út 0399, Hungary, ad libitum.
WATER SUPPLY
The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
An amount of 0.1 g of the test item was administered to the animals.
Duration of treatment / exposure:
As the solid test item remained in the eye sac in all animals at the one hour observation time point, the treated eye of test animals was rinsed with physiological saline solution.
Observation period (in vivo):
The eyes were examined at 1, 24, 48, 72 hours after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes.
Number of animals or in vitro replicates:
Initially only one rabbit was treated with test item. The local effects were non-irritant (all scores were zero) at 24 hours, then two further rabbits were treated with the test item.
Details on study design:
In vitro study results
An in vitro eye irritation study was performed by PHYCHER Bio développement (study code: ICE-PH-15/0794) in the Isolated Chicken Eye model with test item prior to this study.The results from the in vitro eye irritation study indicated that the test item was not predicted as causing serious eye damage. However the score obtained did not allow a prediction in terms of eye irritation. Thus, an additional testing was required to establish a definitive classification.

Identification of pH
The pH of the test item was measured as pH 5.5, permitting the test item to be used in the animal studies.

Pre-study examination
Three male animals in acceptable health condition were selected for the test. Care was taken to select only those animals that had a normal eye condition and any with ocular lesions were rejected.

Analgesic and anaesthetic treatment
Sixty minutes (60 ±10 min) prior to test substance application, a systemic opiate analgesic was administered by subcutaneous injection (SC) under direct Veterinary supervision.Five minutes (5 ±1.5 min) prior to test substance application, a topical ocular anaesthetic was applied to each eye (including the control eye to ensure direct comparison of any ocular observations). Eight hours (8 to 9 hr) after test substance application, a systemic opiate analgesic and a nonsteroidal anti-inflammatory drug (NSAID) were administered by subcutaneous injection (SC) under direct Veterinary supervision. The systemic opiate analgesic was again injected ~12 hours after the post-treatment analgesic.
Systemic opiate analgesic: Buprenorphine 0.01 mg/kg.
Topical ocular anaesthetic: Humacain (oxybuprocaine) one-two drops per eye.
Nonsteroidal anti-inflammatory drug: Meloxicam 0.5 mg/kg.

Application of the Test Item
The test substance was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for at least one second in order to prevent loss of the material.The test item was supplied as a fine dust therefore it was applied as supplied, without grinding.

OBSERVATIONS AND SCORING
Clinical Observations and Evaluation of Ocular Irritation
The eyes were examined at 1, 24, 48, 72 hours after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of ill-health during the study were recorded. At the end of the observation period, the animal was sacrificed by intramuscular injections of Ketamin 10% (Ketamidor) and Xylazin 2% (Primazin 2%) followed by i.v. pentobarbital sodium (Euthanimal 40%). Death was verified by checking pupil and corneal reflex and the absence of respiration.All rabbits were examined for distress at least twice daily, with observations at least 6 hours apart. Clinical observations or signs of ill-health were recorded.

Scoring and Assessment of Local Reaction
The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (02 October 2012). See “Any other information” for scoring system.

Measurement of Body Weight
Individual body weight was recorded on the day of treatment and at the end of observation period.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
8
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
6
Irritant / corrosive response data:
Examination of eye-irritancy
No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period.

Animal 1 (No: 960) clinical observation: At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

Animal 2 (No: 949) clinical observation: At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

Animal 3 (No: 954) clinical observation: At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.
Other effects:
MORTALITY
There was no mortality observed during the study.

BODY WEIGHTS
The body weight of the animals was considered to be within the normal range of variability.

CLINICAL OBSERVATION
General daily examinationThere were no clinical signs observed that could be related to treatment.As no clinical signs were observed, the study was terminated after 72 hours observation.During the study, the control eye of each animal was symptom-free.The general state and behaviour of animals were normal throughout the study period.The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) are as detailed above.

Any other information on results incl. tables

INDIVIDUAL SCORES FOR OCULAR IRRITATION

Abbreviations:

R

=

Redness

OD

=

Opacity degree of density

CH

=

Chemosis

OE

=

Extent of opaque area

D

=

Discharge

IPR/PR

=

Initial or any pain reaction

0

=

Normal (in case of control eye and other lesions)

 

Animal No.: 960

Time

Score of irritation

IPR/PR

Other sign

Conjunctivae

Cornea

Iris

Control eye

R

CH

D

OD

OE

R

Pre-treatment

0

0

0

0

0

0

0

0

0

Post-treatment (h = hour)

1 h

2

2

1

0

0

0

0

0

*

24 h

0

0

0

0

0

0

0

0

0

48 h

0

0

0

0

0

0

0

0

0

72 h

0

0

0

0

0

0

0

0

0

* Test item remained in the eye sac

 

Animal No.: 949

Time

Score of irritation

IPR/PR

Other sign

Conjunctivae

Cornea

Iris

Control eye

R

CH

D

OD

OE

R

Pre-treatment

0

0

0

0

0

0

0

0

0

Post-treatment (h = hour)

1 h

2

2

1

0

0

0

0

0

*

24 h

0

0

0

0

0

0

0

0

0

48 h

0

0

0

0

0

0

0

0

0

72 h

0

0

0

0

0

0

0

0

0

* Test item remained in the eye sac

 

Animal No.: 954

Time

Score of irritation

IPR/PR

Other sign

Conjunctivae

Cornea

Iris

Control eye

R

CH

D

OD

OE

R

Pre-treatment

0

0

0

0

0

0

0

0

0

Post-treatment (h = hour)

1 h

2

2

1

0

0

0

0

0

*

24 h

0

0

0

0

0

0

0

0

0

48 h

0

0

0

0

0

0

0

0

0

72 h

0

0

0

0

0

0

0

0

0

* Test item remained in the eye sac

 

MEAN VALUES OF EYE IRRITATION (24, 48, 72 hour reading)

Animal Number

Sex

Cornea Opacity

Iris

Conjunctive

Redness

Chemosis

Discharge

960

Male

0.00

0.00

0.00

0.00

0.00

949

Male

0.00

0.00

0.00

0.00

0.00

954

Male

0.00

0.00

0.00

0.00

0.00

 

BODY WEIGHT DATA

Animal Number

Before treatment (g)

Before euthanasia (g)

Body weight gain (g)

960

2857

2901

44

949

3007

2156

149

954

3134

3255

121

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Calcium hydrogen phosphonate, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.According to Regulation (EC) No 1272/2008, Calcium hydrogen phosphonate does not require classification as an eye irritant.According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, Calcium hydrogen phosphonatedoes not require classification as an eye irritant.
Executive summary:

An acute eye irritation study of the test item, Calcium hydrogen phosphonate was performed in New Zealand White rabbits. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 405, 2012). Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification.

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A single amount of 0.1 g of the test item was administered as a single dose.

 The eyes were examined at 1, 24, 48 and 72 hours after application.

 No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period.

 

Animal 1 (No: 960) clinical observation

At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

 

Animal 2 (No: 949) clinical observation

At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

 

Animal 3 (No: 954) clinical observation

At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

 

As no clinical signs were observed, the experiment was terminated after 72 hours observation.

 During the experiment, the control eye of each animal was symptom-free.

 The general state and behaviour of animals were normal throughout the study period.

 No mortality occurred during the study. The bodyweights of all rabbits were considered to be within the normal range of variability.

 The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

Animal 1             Animal 2             Animal 3

Chemosis          0.00                      0.00                      0.00

Discharge          0.00                      0.00                     0.00

Redness            0.00                      0.00                      0.00

Cornea              0.00                      0.00                      0.00

Iris                     0.00                      0.00                      0.00

 

The test item, Calcium hydrogen phosphonate, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.

 According to Regulation (EC) No 1272/2008, Calcium hydrogen phosphonate does not require classification as an eye irritant.

 According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, Calcium hydrogen phosphonate does not require classification as an eye irritant.